Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.

ABSTRACT Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consistingmore » of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation. IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.« less

Overexpression of stress-inducible OsBURP16, the β subunit of polygalacturonase 1, decreases pectin content and cell adhesion and increases abiotic stress sensitivity in rice

PubMed Central

Liu, Huanhuan; Ma, Yan; Chen, Na; Guo, Siyi; Liu, Huili; Guo, Xiaoyu; Chong, Kang; Xu, Yunyuan

2014-01-01

Polygalacturonase (PG), one of the hydrolases responsible for cell wall pectin degradation, is involved in organ consenescence and biotic stress in plants. PG1 is composed of a catalytic subunit, PG2, and a non-catalytic PG1β subunit. OsBURP16 belongs to the PG1β-like subfamily of BURP-family genes and encodes one putative PG1β subunit precursor in rice (Oryza sativa L.). Transcription of OsBURP16 is induced by cold, salinity and drought stresses, as well as by abscisic acid (ABA) treatment. Analysis of plant survival rates, relative ion leakage rates, accumulation levels of H2O2 and water loss rates of leaves showed that overexpression of OsBURP16 enhanced sensitivity to cold, salinity and drought stresses compared with controls. Young leaves of Ubi::OsBURP16 transgenic plants showed reduced cell adhesion and increased cuticular transpiration rate. Mechanical strength measurement of Ubi::OsBURP16 plants showed that reduced force was required to break leaves as compared with wild type. Transgenic rice showed enhanced PG activity and reduced pectin content. All these results suggested that overexpression of OsBURP16 caused pectin degradation and affected cell wall integrity as well as transpiration rate, which decreased tolerance to abiotic stresses. The cell wall is a barrier against biotic and abiotic stresses. Overexpression of stress-inducible OsBURP16, the beta-subunit of polygalacturonase 1, decreases pectin contents and cell adhesion in rice. Analyses of plant survival, ion leakage, H2O2 levels, and leaf water loss showed that these effects of overexpression were accompanied by enhanced sensitivity to cold, salinity and drought compared to the wild-type. Our data therefore provide new information on links between polygalacturonase activity and abiotic stress resistance in rice. PMID:24237159

Simultaneous transgenic suppression of LePG and LeExp1 influences fruit texture and juice viscosity in a fresh market tomato variety.

PubMed

Powell, Ann L T; Kalamaki, Mary S; Kurien, Philip A; Gurrieri, Sergio; Bennett, Alan B

2003-12-03

Tomatoes are grown for fresh consumption or for processing of the fruit. Some ripening-associated processes of the fruit can either contribute to or degrade attributes associated with both fresh and processing quality. For example, cell wall disassembly is associated with loss of fresh fruit firmness as well as with loss of processed tomato product viscosity. Several enzymes contribute to cell wall polysaccharide disassembly. Polygalacturonase (PG, poly[1,4-alpha-d-galactouronide] glucanohydrolase, EC 3.2.1.15) is among the most abundant polysaccharide hydrolases in ripening tomato fruit and is the major contributor to pectin depolymerization. Expansin (LeExp1) is also abundant in ripening fruit and is proposed to contribute to cell wall disassembly by nonhydrolytic activity, possibly by increasing substrate accessibility to other enzymes. Suppression of either LePG or LeExp1 expression alone results in altered softening and/or shelf life characteristics. To test whether simultaneous suppression of both LePG and LeExp1 expression influences fruit texture in additive or synergistic ways, transgenic Lycopersicon esculentum var. Ailsa Craig lines with reduced expression of either LePG or LeExp1 were crossed. Fruits from the third generation of progeny, homozygous for both transgenic constructs, were analyzed for firmness and other quality traits during ripening on or off the vine. In field-grown transgenic tomato fruit, suppression of LeExp1 or LePG alone did not significantly increase fruit firmness. However, fruits suppressed for both LePG and LeExp1 expression were significantly firmer throughout ripening and were less susceptible to deterioration during long-term storage. Juice prepared from the transgenic tomato fruit with reduced LePG and LeExp1 expression was more viscous than juice prepared from control fruit.

Regulation of Cell Wall Plasticity by Nucleotide Metabolism in Lactococcus lactis*

PubMed Central

Solopova, Ana; Formosa-Dague, Cécile; Courtin, Pascal; Furlan, Sylviane; Veiga, Patrick; Péchoux, Christine; Armalyte, Julija; Sadauskas, Mikas; Kok, Jan; Hols, Pascal; Dufrêne, Yves F.; Kuipers, Oscar P.; Chapot-Chartier, Marie-Pierre; Kulakauskas, Saulius

2016-01-01

To ensure optimal cell growth and separation and to adapt to environmental parameters, bacteria have to maintain a balance between cell wall (CW) rigidity and flexibility. This can be achieved by a concerted action of peptidoglycan (PG) hydrolases and PG-synthesizing/modifying enzymes. In a search for new regulatory mechanisms responsible for the maintenance of this equilibrium in Lactococcus lactis, we isolated mutants that are resistant to the PG hydrolase lysozyme. We found that 14% of the causative mutations were mapped in the guaA gene, the product of which is involved in purine metabolism. Genetic and transcriptional analyses combined with PG structure determination of the guaA mutant enabled us to reveal the pivotal role of the pyrB gene in the regulation of CW rigidity. Our results indicate that conversion of l-aspartate (l-Asp) to N-carbamoyl-l-aspartate by PyrB may reduce the amount of l-Asp available for PG synthesis and thus cause the appearance of Asp/Asn-less stem peptides in PG. Such stem peptides do not form PG cross-bridges, resulting in a decrease in PG cross-linking and, consequently, reduced PG thickness and rigidity. We hypothesize that the concurrent utilization of l-Asp for pyrimidine and PG synthesis may be part of the regulatory scheme, ensuring CW flexibility during exponential growth and rigidity in stationary phase. The fact that l-Asp availability is dependent on nucleotide metabolism, which is tightly regulated in accordance with the growth rate, provides L. lactis cells the means to ensure optimal CW plasticity without the need to control the expression of PG synthesis genes. PMID:27022026

Overexpression of stress-inducible OsBURP16, the β subunit of polygalacturonase 1, decreases pectin content and cell adhesion and increases abiotic stress sensitivity in rice.

PubMed

Liu, Huanhuan; Ma, Yan; Chen, Na; Guo, Siyi; Liu, Huili; Guo, Xiaoyu; Chong, Kang; Xu, Yunyuan

2014-05-01

Polygalacturonase (PG), one of the hydrolases responsible for cell wall pectin degradation, is involved in organ consenescence and biotic stress in plants. PG1 is composed of a catalytic subunit, PG2, and a non-catalytic PG1β subunit. OsBURP16 belongs to the PG1β-like subfamily of BURP-family genes and encodes one putative PG1β subunit precursor in rice (Oryza sativa L.). Transcription of OsBURP16 is induced by cold, salinity and drought stresses, as well as by abscisic acid (ABA) treatment. Analysis of plant survival rates, relative ion leakage rates, accumulation levels of H2 O2 and water loss rates of leaves showed that overexpression of OsBURP16 enhanced sensitivity to cold, salinity and drought stresses compared with controls. Young leaves of Ubi::OsBURP16 transgenic plants showed reduced cell adhesion and increased cuticular transpiration rate. Mechanical strength measurement of Ubi::OsBURP16 plants showed that reduced force was required to break leaves as compared with wild type. Transgenic rice showed enhanced PG activity and reduced pectin content. All these results suggested that overexpression of OsBURP16 caused pectin degradation and affected cell wall integrity as well as transpiration rate, which decreased tolerance to abiotic stresses. © 2013 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.

PgTeL, the lectin found in Punica granatum juice, is an antifungal agent against Candida albicans and Candida krusei.

PubMed

da Silva, Pollyanna Michelle; de Moura, Maiara Celine; Gomes, Francis Soares; da Silva Trentin, Danielle; Silva de Oliveira, Ana Patrícia; de Mello, Gabriela Souto Vieira; da Rocha Pitta, Maira Galdino; de Melo Rego, Moacyr Jesus Barreto; Coelho, Luana Cassandra Breitenbach Barroso; Macedo, Alexandre José; de Figueiredo, Regina Celia Bressan Queiroz; Paiva, Patrícia Maria Guedes; Napoleão, Thiago Henrique

2018-03-01

The pomegranate (Punica granatum) sarcotesta contains a chitin-binding lectin (PgTeL) with antibacterial activity against human pathogenic species. In this work, the structural stability of PgTeL was evaluated by fluorimetric analysis and the lectin was evaluated for cytotoxicity to human peripheral blood mononuclear cells (PBMCs) and antifungal activity against Candida albicans and Candida krusei. PgTeL folding was impaired when lectin was incubated at pH≥6.0. On the other hand, the lectin did not undergo unfolding even when heated at 100°C. PgTeL (1, 10, and 100μg/mL) was not cytotoxic to PBMCs. Antifungal activity was detected for C. albicans (MIC: 25μg/mL; MFC: 50μg/mL) and C. krusei (MIC and MFC of 12.5μg/mL). Treatment of yeast cells with PgTeL resulted in decrease of intracellular ATP content even at sub-inhibitory concentrations (½MIC and ¼MIC) and induced lipid peroxidation. In addition, PgTeL damaged the integrity of fungal cell wall of both species, with more pronounced effects in C. krusei. The lectin showed significant antibiofilm activity on C. albicans at sub-inhibitory concentrations (0.195 and 0.39μg/mL). In conclusion, PgTeL is an anti-Candida agent whose action mechanism involves oxidative stress, energetic collapse, damage to the cell wall and rupture of yeast cells. Copyright © 2017 Elsevier B.V. All rights reserved.

A Single Amino-Acid Substitution Allows Endo-Polygalacturonase of Fusarium verticillioides to Acquire Recognition by PGIP2 from Phaseolus vulgaris

PubMed Central

Galantini, Luciano; Di Matteo, Adele; Pavel, Nicolae Viorel; De Lorenzo, Giulia; Cervone, Felice; Federici, Luca; Sicilia, Francesca

2013-01-01

Polygalacturonases (PGs) are secreted by phytopathogenic fungi to degrade the plant cell wall homogalacturonan during plant infection. To counteract Pgs, plants have evolved polygalacturonase-inhibiting proteins (PGIPs) that slow down fungal infection and defend cell wall integrity. PGIPs favour the accumulation of oligogalacturonides, which are homogalacturonan fragments that act as endogenous elicitors of plant defence responses. We have previously shown that PGIP2 from Phaseolus vulgaris (PvPGIP2) forms a complex with PG from Fusarium phyllophilum (FpPG), hindering the enzyme active site cleft from substrate. Here we analyse by small angle X-ray scattering (SAXS) the interaction between PvPGIP2 and a PG from Colletotrichum lupini (CluPG1). We show a different shape of the PG-PGIP complex, which allows substrate entry and provides a structural explanation for the different inhibition kinetics exhibited by PvPGIP2 towards the two isoenzymes. The analysis of SAXS structures allowed us to investigate the basis of the inability of PG from Fusarium verticilloides (FvPG) to be inhibited by PvPGIP2 or by any other known PGIP. FvPG is 92.5% identical to FpPG, and we show here, by both loss- and gain-of-function mutations, that a single amino acid site acts as a switch for FvPG recognition by PvPGIP2. PMID:24260434

Morphology, Growth, and Size Limit of Bacterial Cells

NASA Astrophysics Data System (ADS)

Jiang, Hongyuan; Sun, Sean X.

2010-07-01

Bacterial cells utilize a living peptidoglycan network (PG) to separate the cell interior from the surroundings. The shape of the cell is controlled by PG synthesis and cytoskeletal proteins that form bundles and filaments underneath the cell wall. The PG layer also resists turgor pressure and protects the cell from osmotic shock. We argue that mechanical influences alter the chemical equilibrium of the reversible PG assembly and determine the cell shape and cell size. Using a mechanochemical approach, we show that the cell shape can be regarded as a steady state of a growing network under the influence of turgor pressure and mechanical stress. Using simple elastic models, we predict the size of common spherical and rodlike bacteria. The influence of cytoskeletal bundles such as crescentin and MreB are discussed within the context of our model.

Rhizoctonia resistance conferred by a sugar beet polygalacturonase-inhibiting protein gene

USDA-ARS?s Scientific Manuscript database

Polygalacturonase-inhibiting proteins (PGIPs) are cell wall leucine-rich repeat (LRR) proteins recognized as having a role in plant defense. PGIPs inhibit fungal polygalacturonase (PG) enzymes that break down the polygalacturonate chain in plant cell walls to initiate disease development. The inte...

Identification and expression analysis of BoMF25, a novel polygalacturonase gene involved in pollen development of Brassica oleracea.

PubMed

Lyu, Meiling; Liang, Ying; Yu, Youjian; Ma, Zhiming; Song, Limin; Yue, Xiaoyan; Cao, Jiashu

2015-06-01

BoMF25 acts on pollen wall. Polygalacturonase (PG) is a pectin-digesting enzyme involved in numerous plant developmental processes and is described to be of critical importance for pollen wall development. In the present study, a PG gene, BoMF25, was isolated from Brassica oleracea. BoMF25 is the homologous gene of At4g35670, a PG gene in Arabidopsis thaliana with a high expression level at the tricellular pollen stage. Collinear analysis revealed that the orthologous gene of BoMF25 in Brassica campestris (syn. B. rapa) genome was probably lost because of genome deletion and reshuffling. Sequence analysis indicated that BoMF25 contained four classical conserved domains (I, II, III, and IV) of PG protein. Homology and phylogenetic analyses showed that BoMF25 was clustered in Clade F. The putative promoter sequence, containing classical cis-acting elements and pollen-specific motifs, could drive green fluorescence protein expression in onion epidermal cells. Quantitative RT-PCR analysis suggested that BoMF25 was mainly expressed in the anther at the late stage of pollen development. In situ hybridization analysis also indicated that the strong and specific expression signal of BoMF25 existed in pollen grains at the mature pollen stage. Subcellular localization showed that the fluorescence signal was observed in the cell wall of onion epidermal cells, which suggested that BoMF25 may be a secreted protein localized in the pollen wall.

Partial functional redundancy of MreB isoforms, MreB, Mbl and MreBH, in cell morphogenesis of Bacillus subtilis.

PubMed

Kawai, Yoshikazu; Asai, Kei; Errington, Jeffery

2009-08-01

MreB proteins are bacterial actin homologues thought to have a role in cell shape determination by positioning the cell wall synthetic machinery. Many bacteria, particularly Gram-positives, have more than one MreB isoform. Bacillus subtilis has three, MreB, Mbl and MreBH, which colocalize in a single helical structure. We now show that the helical pattern of peptidoglycan (PG) synthesis in the cylindrical part of the rod-shaped cell is governed by the redundant action of the three MreB isoforms. Single mutants for any one of mreB isoforms can still incorporate PG in a helical pattern and generate a rod shape. However, after depletion of MreB in an mbl mutant (or depletion of all three isoforms) lateral wall PG synthesis was impaired and the cells became spherical and lytic. Overexpression of any one of the MreB isoforms overcame the lethality as well as the defects in lateral PG synthesis and cell shape. Furthermore, MreB and Mbl can associate with the peptidoglycan biosynthetic machinery independently. However, no single MreB isoform was able to support normal growth under various stress conditions, suggesting that the multiple isoforms are used to allow cells to maintain proper growth and morphogenesis under changing and sometimes adverse conditions.

The secondary cell wall polysaccharide of Bacillus anthracis provides the specific binding ligand for the C-terminal cell wall-binding domain of two phage endolysins, PlyL and PlyG

PubMed Central

Ganguly, Jhuma; Low, Lieh Y; Kamal, Nazia; Saile, Elke; Forsberg, L Scott; Gutierrez-Sanchez, Gerardo; Hoffmaster, Alex R; Liddington, Robert; Quinn, Conrad P; Carlson, Russell W; Kannenberg, Elmar L

2013-01-01

Endolysins are bacteriophage enzymes that lyse their bacterial host for phage progeny release. They commonly contain an N-terminal catalytic domain that hydrolyzes bacterial peptidoglycan (PG) and a C-terminal cell wall-binding domain (CBD) that confers enzyme localization to the PG substrate. Two endolysins, phage lysin L (PlyL) and phage lysin G (PlyG), are specific for Bacillus anthracis. To date, the cell wall ligands for their C-terminal CBD have not been identified. We recently described structures for a number of secondary cell wall polysaccharides (SCWPs) from B. anthracis and B. cereus strains. They are covalently bound to the PG and are comprised of a -ManNAc-GlcNAc-HexNAc- backbone with various galactosyl or glucosyl substitutions. Surface plasmon resonance (SPR) showed that the endolysins PlyL and PlyG bind to the SCWP from B. anthracis (SCWPBa) with high affinity (i.e. in the μM range with dissociation constants ranging from 0.81 × 10−6 to 7.51 × 10−6 M). In addition, the PlyL and PlyG SCWPBa binding sites reside with their C-terminal domains. The dissociation constants for the interactions of these endolysins and their derived C-terminal domains with the SCWPBa were in the range reported for other protein–carbohydrate interactions. Our findings show that the SCWPBa is the ligand that confers PlyL and PlyG lysin binding and localization to the PG. PlyL and PlyG also bound the SCWP from B. cereus G9241 with comparable affinities to SCWPBa. No detectable binding was found to the SCWPs from B. cereus ATCC (American Type Culture Collection) 10987 and ATCC 14579, thus demonstrating specificity of lysin binding to SCWPs. PMID:23493680

Analysis of papaya cell wall-related genes during fruit ripening indicates a central role of polygalacturonases during pulp softening.

PubMed

Fabi, João Paulo; Broetto, Sabrina Garcia; da Silva, Sarah Lígia Garcia Leme; Zhong, Silin; Lajolo, Franco Maria; do Nascimento, João Roberto Oliveira

2014-01-01

Papaya (Carica papaya L.) is a climacteric fleshy fruit that undergoes dramatic changes during ripening, most noticeably a severe pulp softening. However, little is known regarding the genetics of the cell wall metabolism in papayas. The present work describes the identification and characterization of genes related to pulp softening. We used gene expression profiling to analyze the correlations and co-expression networks of cell wall-related genes, and the results suggest that papaya pulp softening is accomplished by the interactions of multiple glycoside hydrolases. The polygalacturonase cpPG1 appeared to play a central role in the network and was further studied. The transient expression of cpPG1 in papaya results in pulp softening and leaf necrosis in the absence of ethylene action and confirms its role in papaya fruit ripening.

Analysis of Papaya Cell Wall-Related Genes during Fruit Ripening Indicates a Central Role of Polygalacturonases during Pulp Softening

PubMed Central

Fabi, João Paulo; Broetto, Sabrina Garcia; da Silva, Sarah Lígia Garcia Leme; Zhong, Silin; Lajolo, Franco Maria; do Nascimento, João Roberto Oliveira

2014-01-01

Papaya (Carica papaya L.) is a climacteric fleshy fruit that undergoes dramatic changes during ripening, most noticeably a severe pulp softening. However, little is known regarding the genetics of the cell wall metabolism in papayas. The present work describes the identification and characterization of genes related to pulp softening. We used gene expression profiling to analyze the correlations and co-expression networks of cell wall-related genes, and the results suggest that papaya pulp softening is accomplished by the interactions of multiple glycoside hydrolases. The polygalacturonase cpPG1 appeared to play a central role in the network and was further studied. The transient expression of cpPG1 in papaya results in pulp softening and leaf necrosis in the absence of ethylene action and confirms its role in papaya fruit ripening. PMID:25162506

Xylella fastidiosa requires polygalacturonase for colonization and pathogenicity in Vitis vinifera grapevines.

PubMed

Roper, M Caroline; Greve, L Carl; Warren, Jeremy G; Labavitch, John M; Kirkpatrick, Bruce C

2007-04-01

Xylella fastidiosa is the causal agent of Pierce's disease of grape, an economically significant disease for the grape industry. X. fastidiosa systemically colonizes the xylem elements of grapevines and is able to breach the pit pore membranes separating xylem vessels by unknown mechanisms. We hypothesized that X. fastidiosa utilizes cell wall degrading enzymes to break down pit membranes, based on the presence of genes involved in plant cell wall degradation in the X. fastidiosa genome. These genes include several beta-1,4 endoglucanases, several xylanases, several xylosidases, and one polygalacturonase (PG). In this study, we demonstrated that the pglA gene encodes a functional PG. A mutant in pglA lost pathogenicity and was compromised in its ability to systemically colonize Vitis vinifera grapevines. The results indicate that PG is required for X. fastidiosa to successfully infect grapevines and is a critical virulence factor for X. fastidiosa pathogenesis in grapevine.

The SlFSR Gene Controls Fruit Shelf-Life in Tomato.

PubMed

Zhang, Lincheng; Zhu, Mingku; Ren, Lijun; Li, Anzhou; Chen, Guoping; Hu, Zongli

2018-04-04

Fruit ripening represents a process changing flavor and appearance and also a process dramatically increasing fruit softening. Fruit softening and textural variations are mainly resulted from the disrupted cell wall of fruit throughout ripening, whereas, the exact mechanisms and specific modifications of cell wall remain unclear. Plant-specific GRAS proteins play a critical role in development and growth. To date, few GRAS genes have been functionally categorized in tomato. The expression of a novel GRAS gene revealed herein is designated as SlFSR (fruitshelf-liferegulator), specifically increased during fruit ripening, whereas significantly decreased in tomato mutant rin (ripening inhibitor). RNAi repression of SlFSR resulted in reduced expression of multiple cell wall modification-related genes, decreased PG (polygalacturonase), TBG (tomato β-galactosidase), CEL (cellulase) and XYL (β-D-xylosidase) activities, and significantly prolonged fruit shelf-life. Furthermore, overexpression of SlFSR in mutant rin gave rise to up-regulated expression of multiple cell wall modification-related genes, such as PG, TBG4, CEL2, XYL1, PL, PE, MAN1, EXP1 and XTH5, and significantly shortened fruit shelf-life. Our findings make it possible to reveal the genetic mechanisms underlying fruit cell wall metabolisms and suggest that SlFSR gene is another biotechnological targeted control of tomato fruit shelf-life.

Estrogen receptor expression and vessel density in the vagina wall in postmenopausal women with prolapse.

PubMed

Lara, Lúcia Alves da Silva; Ribeiro da Silva, Alfredo; Rosa-e-Silva, Julio Cesar; Silva-de-Sá, Marcos Felipe; Rosa-e-Silva, Ana Carolina Japur de Sá

2014-04-01

After menopause, critically estrogen low levels result in modifications in vaginal wall. This cross-sectional study aims to determine whether there is a change in the number of vessels in the lamina propria of the vagina after menopause in parallel to the ER-alpha expression on the vaginal wall. Twelve women who underwent a genital surgery for genital prolapse up to grade II were selected. They were divided into two groups: a premenopausal group (PG) consisting of six women who were 18-40 years old with FSH levels =12 mIU/ml and regular cycles, and a menopausal group (MG) consisting of six women at least one year after menopause who were <65 years old with FSH levels =40 mIU/ml. Slides were stained for ER-alpha immunohistochemistry, and an endothelial cell marker CD3 was used to label vessels which were identified by using a system for morphometry. The number of vessels was significantly higher in the PG than in the MG both on the anterior wall (PG: 1.055 ± 145.8 vessels/mm(2), MG: 346.6 ± 209.9 vessels/mm(2), p<0.0001) and on the posterior wall (PG: 1064 ± 303.3 vessels/mm(2), MG: 348.6 ± 167.3 vessels/mm(2), p=0.0005). The ER-alpha score was significantly higher in the PG than the score for the MG on both the anterior and posterior walls (PG: 6.0 ± 0.52, MG: 2.5 ± 0.89, p=0.007; PG: 5.8 ± 0.79, MG: 2.7 ± 0.95, p=0.03, respectively). There was a positive correlation between the ER-alpha score and the vessel concentration on the anterior (r=0.6656, p=0.018) and posterior (r=0.6738, p=0.016) vaginal walls. Age was strongly negatively correlated with vessel concentration on the vaginal walls (respectively r=-0.9033, p<0.0001, r=-0.7440, p=0.0055). Therefore, postmenopausal women with genital prolapse have a smaller number of vessels on the vaginal wall compared to normoestrogenic controls with the same pathological condition. Hypoestrogenism and advancing age are factors that are associated to these changes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Roles of the bacterial cell wall and capsule in induction of tumor necrosis factor alpha by type III group B streptococci.

PubMed Central

Vallejo, J G; Baker, C J; Edwards, M S

1996-01-01

Group B streptococci (GBS) are the major cause of sepsis and fatal shock in neonates in the United States. The precise role of tumor necrosis factor alpha (TNF-alpha) in the development of human GBS sepsis has not been defined; however, whole GBS have been shown to induce the production of this inflammatory cytokine. We sought to determine which bacterial cell wall components of GBS are responsible for triggering TNF-alpha production. Human cord blood monocytes were stimulated with encapsulated (COH1) or unencapsulated (COH1-13) whole type III GBS or with purified bacterial components, including type III capsular polysaccharide (III-PS), group B polysaccharide (GB-PS), lipoteichoic acid (LTA), or peptidoglycan (PG). Lipopolysaccharide from Escherichia coli served as a control. Supernatants were harvested at specific timed intervals, and TNF-alpha levels were measured by enzyme-linked immunosorbent assay. Monocytes exposed to COH1 and COH1-13 induced similar amounts of TNF-alpha. III-PS, GB-PS, LTA, and PG each induced TNF-alpha in a time- and concentration-dependent manner. However, TNF-alpha release was significantly greater after stimulation by the GB-PS or PG than after stimulation by III-PS or LTA (P < 0.05). Our findings indicate that GB-PS and PG are the bacterial cell wall components primarily evoking TNF-alpha release. These, alone or in concert with other factors, may be responsible for septic shock accompanying GBS sepsis. PMID:8945544

The polygalacturonase FaPG1 gene plays a key role in strawberry fruit softening

PubMed Central

García-Gago, Juan A; Posé, Sara; Muñoz-Blanco, Juan; Quesada, Miguel A

2009-01-01

The loss of firm texture is one of the most characteristic physiological processes that occur during the ripening of fleshy fruits. It is generally accepted that the disassembly of primary cell wall and middle lamella is the main factor involved in fruit softening. In this process, polygalacturonase (PG) has been implicated in the degradation of the polyuronide network in several fruits. However, the minor effect of PG downregulation on tomato softening, reported during the nineties, minimized the role of this enzyme in softening. Further works in other fruits are challenging this general assumption, as is occurring in strawberry. The strawberry (Fragaria × ananassa) fruit undergoes an extensive and fast softening that limit its shelf life and postharvest. Traditionally, it has also been considered that PG plays a minor role on this process, due to the low PG activity found in ripened strawberry fruits. Transgenic strawberry plants expressing an antisense sequence of the ripening-specific PG gene FaPG1 have been generated to get an insight into the role of this gene in softening. Half of the transgenic lines analyzed yielded fruits significantly firmer than control, without being affected other fruit parameters such as weight, color or soluble solids. The increase on firmness was maintained after several days of posharvest. In these firmer lines, FaPG1 was silenced to 95%, but total PG activity was only minor reduced. At the cell wall level, transgenic fruits contained a higher amount of covalently bound pectins whereas the soluble fraction was diminished. A microarray analysis of genes expressed in ripened receptacle did not show any significant change between control and transgenic fruits. Thus, contrary to the most accepted view, it is concluded that PG plays a key role on pectin metabolism and softening of strawberry fruit. PMID:19820312

Antisense Down-Regulation of the FaPG1 Gene Reveals an Unexpected Central Role for Polygalacturonase in Strawberry Fruit Softening1[W

PubMed Central

Quesada, Miguel A.; Blanco-Portales, Rosario; Posé, Sara; García-Gago, Juan A.; Jiménez-Bermúdez, Silvia; Muñoz-Serrano, Andrés; Caballero, José L.; Pliego-Alfaro, Fernando; Mercado, José A.; Muñoz-Blanco, Juan

2009-01-01

The strawberry (Fragaria × ananassa ‘Chandler’) fruit undergoes a fast softening during ripening. Polygalacturonase (PG) activity is low during this process, but two ripening-related PG genes, FaPG1 and FaPG2, have been cloned. Both genes were up-regulated during fruit ripening and were also negatively regulated by auxin. To further assess the role of FaPG1 on strawberry softening, transgenic plants containing an antisense sequence of this gene under the control of the 35S promoter (APG lines) were obtained. Sixteen out of 30 independent transgenic lines showed fruit yields similar to those of the control. Several quality parameters were measured in ripe fruits from these 16 lines. Fruit weight was slightly reduced in four lines, and most of them showed an increase in soluble solid content. Half of these lines yielded fruits significantly firmer than did the control. Four APG lines were selected, their ripened fruits being on average 163% firmer than the control. The postharvest softening of APG fruits was also diminished. Ripened fruits from the four selected lines showed a 90% to 95% decrease in FaPG1 transcript abundance, whereas the level of FaPG2 was not significantly altered. Total PG activity was reduced in three of these lines when compared with control fruits. Cell wall extracts from APG fruits showed a reduction in pectin solubilization and an increase in pectins covalently bound to the cell wall. A comparative transcriptomic analysis of gene expression between the ripened receptacle of the control and those of the APG fruits (comprising 1,250 receptacle expressed sequence tags) did not show any statistically significant change. These results indicate that FaPG1 plays a central role in strawberry softening. PMID:19395408

The polygalacturonase FaPG1 gene plays a key role in strawberry fruit softening.

PubMed

García-Gago, Juan A; Posé, Sara; Muñoz-Blanco, Juan; Quesada, Miguel A; Mercado, José A

2009-08-01

The loss of firm texture is one of the most characteristic physiological processes that occur during the ripening of fleshy fruits. It is generally accepted that the disassembly of primary cell wall and middle lamella is the main factor involved in fruit softening. In this process, polygalacturonase (PG) has been implicated in the degradation of the polyuronide network in several fruits. However, the minor effect of PG downregulation on tomato softening, reported during the nineties, minimized the role of this enzyme in softening. Further works in other fruits are challenging this general assumption, as is occurring in strawberry. The strawberry (Fragaria x ananassa) fruit undergoes an extensive and fast softening that limit its shelf life and postharvest. Traditionally, it has also been considered that PG plays a minor role on this process, due to the low PG activity found in ripened strawberry fruits. Transgenic strawberry plants expressing an antisense sequence of the ripening-specific PG gene FaPG1 have been generated to get an insight into the role of this gene in softening. Half of the transgenic lines analyzed yielded fruits significantly firmer than control, without being affected other fruit parameters such as weight, color or soluble solids. The increase on firmness was maintained after several days of posharvest. In these firmer lines, FaPG1 was silenced to 95%, but total PG activity was only minor reduced. At the cell wall level, transgenic fruits contained a higher amount of covalently bound pectins whereas the soluble fraction was diminished. A microarray analysis of genes expressed in ripened receptacle did not show any significant change between control and transgenic fruits. Thus, contrary to the most accepted view, it is concluded that PG plays a key role on pectin metabolism and softening of strawberry fruit.

Structural requirements of endopolygalacturonase for the interaction with PGIP (polygalacturonase-inhibiting protein)

PubMed Central

Federici, L.; Caprari, C.; Mattei, B.; Savino, C.; Di Matteo, A.; De Lorenzo, G.; Cervone, F.; Tsernoglou, D.

2001-01-01

To invade a plant tissue, phytopathogenic fungi produce several cell wall-degrading enzymes; among them, endopolygalacturonase (PG) catalyzes the fragmentation and solubilization of homogalacturonan. Polygalacturonase-inhibiting proteins (PGIPs), found in the cell wall of many plants, counteract fungal PGs by forming specific complexes with them. We report the crystal structure at 1.73 Å resolution of PG from the phytopathogenic fungus Fusarium moniliforme (FmPG). The structure of FmPG was useful to study the mode of interaction of the enzyme with PGIP-2 from Phaseolus vulgaris. Several amino acids of FmPG were mutated, and their contribution to the formation of the complex with PGIP-2 was investigated by surface plasmon resonance. The residues Lys-269 and Arg-267, located inside the active site cleft, and His-188, at the edge of the active site cleft, are critical for the formation of the complex, which is consistent with the observed competitive inhibition of the enzyme played by PGIP-2. The replacement of His-188 with a proline or the insertion of a tryptophan after position 270, variations that both occur in plant PGs, interferes with the formation of the complex. We suggest that these variations are important structural requirements of plant PGs to prevent PGIP binding. PMID:11687632

The SlFSR gene controls fruit shelf-life in tomato

PubMed Central

Zhang, Lincheng; Zhu, Mingku; Ren, Lijun; Li, Anzhou; Chen, Guoping

2018-01-01

Abstract Fruit ripening represents a process that changes flavor and appearance and also a process that dramatically increases fruit softening. Fruit softening and textural variations mainly result from disruptions to the cell walls of the fruit throughout ripening, but the exact mechanisms and specific modifications of the cell wall remain unclear. Plant-specific GRAS proteins play a critical role in development and growth. To date, few GRAS genes have been functionally categorized in tomato. The expression of a novel GRAS gene described in this study and designated as SlFSR (fruit shelf-life regulator) specifically increased during fruit ripening, but was significantly decreased in the tomato mutant rin (ripening inhibitor). RNAi repression of SlFSR resulted in reduced expression of multiple cell wall modification-related genes, decreased the activities of PG (polygalacturonase), TBG (tomato β-galactosidase), CEL (cellulase), and XYL (β-D-xylosidase), and significantly prolonged fruit shelf-life. Furthermore, overexpression of SlFSR in mutant rin gave rise to up-regulated expression of multiple cell wall modification-related genes, such as PG, TBG4, CEL2, XYL1, PL, PE, MAN1, EXP1, and XTH5, and significantly shortened the fruit shelf-life. These findings reveal some of the genetic mechanisms underlying fruit cell wall metabolism and suggest that the SlFSR gene is another potential biotechnological target for the control of tomato fruit shelf-life. PMID:29635354

Changes in cell wall pectins and their relation to postharvest mesocarp softening of "Hass" avocados (Persea americana Mill.).

PubMed

Defilippi, Bruno G; Ejsmentewicz, Troy; Covarrubias, María Paz; Gudenschwager, Orianne; Campos-Vargas, Reinaldo

2018-05-17

The avocado is a climacteric fruit and begins a softening process after harvest. During ripening, the mesocarp changes in texture, and this affects fruit quality and cold storage capacity. Softening is commonly associated with cell wall disassembly in climacteric fruits. However, changes in the cell wall structure and composition during avocado softening are poorly understood. To understand this process, cell wall pectins in "Hass" avocado fruit were studied during ripening at 20 °C after harvest and after cold storage. Additionally, avocados were treated with 1-MCP to evaluate the delay in softening. Biochemical analysis showed a decrease in galacturonic acid (GalA) in alcohol-insoluble residues (AIR) and water-soluble pectin concomitant to softening, paralleled by an increase in polygalacturonase (PG) activity. In the same way, the β-galactosidase activity increased in soft avocado fruit, along with a reduction in galactose in cell wall material and the Na 2 CO 3 -soluble fraction. The arabinose content in the cell wall material did not change during softening. However, there was a change in arabinose ratios between the different fractions of pectin, mainly in the fractions soluble in water and in Na 2 CO 3 . The cold storage of avocado fruit did not induce softening of the fruit, but the content of GalA showed a substantial decrease, accompanied by an increase in PG activity. Thus, our work supports the hypothesis that the solubilization of neutral sugars such as arabinose and rhamnose, as well as the loss of galactose content mediated by the enzyme β-galactosidase, were the main factors that began the coordinated action of cell wall remodeling enzymes that resulted in the loss of firmness of avocado fruit. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Polygalacturonase Causes Lygus-like Damage on Plants: Cloning and Identification of Western Tarnished Plant Bug (Lygus hesperus) Polygalacturonases Secreted During Feeding

USDA-ARS?s Scientific Manuscript database

Abstract: Polygalacturonase (PG), an enzyme that degrades pectin within the plant tissue cell wall, has been postulated as the chemical cause of damage to plants by the mirid Lygus hesperus. Micro-injection of two pure recombinant Aspergillus niger PG II protein forms, the wild type enzymically acti...

Isolation and characterisation of a pod dehiscence zone-specific polygalacturonase from Brassica napus.

PubMed

Petersen, M; Sander, L; Child, R; van Onckelen, H; Ulvskov, P; Borkhardt, B

1996-06-01

Seven distinct partial cDNAs, similar in sequence to previously described polygalacturonases (PGs), were amplified from cDNA derived from rape pod wall, dehiscence zone and leaves by the polymerase chain reaction. Northern analysis showed that one clone, PG35-8, was expressed at low levels in the dehiscence zone during the first five weeks after anthesis but was very abundantly expressed at week 6. In contrast, no PG35-8-related RNA was detected in the pod wall. Our data suggest that there are temporal and spatial correlations between the breakdown of the middle lamella, of the dehiscence zone cells and the pattern of synthesis of PG35-8 transcripts which may indicate a role for this particular PG in rape pod dehiscence. PG35-8 was used to isolate five cDNA clones from a rape dehiscence zone cDNA library. Restriction enzyme analysis and partial sequencing revealed that they were derived from four highly homologous transcripts which are probably allelic forms of a single gene. One full-length clone, RDPG1, was completely sequenced. The predicted protein of RDPG1 showed its highest identity with PG from apple fruit with an identity of 52%.

Isolation and characterization of a tomato non-specific lipid transfer protein involved in polygalacturonase-mediated pectin degradation.

PubMed

Tomassen, Monic M M; Barrett, Diane M; van der Valk, Henry C P M; Woltering, Ernst J

2007-01-01

An important aspect of the ripening process of tomato fruit is softening. Softening is accompanied by hydrolysis of the pectin in the cell wall by pectinases, causing loss of cell adhesion in the middle lamella. One of the most significant pectin-degrading enzymes is polygalacturonase (PG). Previous reports have shown that PG in tomato may exist in different forms (PG1, PG2a, PG2b, and PGx) commonly referred to as PG isoenzymes. The gene product PG2 is differentially glycosylated and is thought to associate with other proteins to form PG1 and PGx. This association is thought to modulate its pectin-degrading activity in planta. An 8 kDa protein that is part of the tomato PG1 multiprotein complex has been isolated, purified, and functionally characterized. This protein, designated 'activator' (ACT), belongs to the class of non-specific lipid transfer proteins (nsLTPs). ACT is capable of 'converting' the gene product PG2 into a more active and heat-stable form, which increases PG-mediated pectin degradation in vitro and stimulates PG-mediated tissue breakdown in planta. This finding suggests a new, not previously identified, function for nsLTPs in the modification of hydrolytic enzyme activity. It is proposed that ACT plays a role in the modulation of PG activity during tomato fruit softening.

MreB: pilot or passenger of cell wall synthesis?

PubMed

White, Courtney L; Gober, James W

2012-02-01

The discovery that the bacterial cell shape determinant MreB is related to actin spurred new insights into bacterial morphogenesis and development. The trafficking and mechanical roles of the eukaryotic cytoskeleton were hypothesized to have a functional ancestor in MreB based on evidence implicating MreB as an organizer of cell wall synthesis. Genetic, biochemical and cytological studies implicate MreB as a coordinator of a large multi-protein peptidoglycan (PG) synthesizing holoenzyme. Recent advances in microscopy and new biochemical evidence, however, suggest that MreB may function differently than previously envisioned. This review summarizes our evolving knowledge of MreB and attempts to refine the generalized model of the proteins organizing PG synthesis in bacteria. This is generally thought to be conserved among eubacteria and the majority of the discussion will focus on studies from a few well-studied model organisms. Copyright © 2011 Elsevier Ltd. All rights reserved.

Bacteriophage virion-associated peptidoglycan hydrolases: potential new enzybiotics

USDA-ARS?s Scientific Manuscript database

Virion-associated peptidoglycan hydrolases (VAPGH) are phage-encoded lytic enzymes that locally degrade the peptidoglycan (PG) of the bacterial cell wall during infection. Their action usually generates a small hole through which the phage tail crosses the cell envelope to inject the phage genetic m...

Expression of a Polygalacturonase Associated with Tomato Seed Germination1

PubMed Central

Sitrit, Yaron; Hadfield, Kristen A.; Bennett, Alan B.; Bradford, Kent J.; Downie, A. Bruce

1999-01-01

Radicle protrusion from tomato (Lycopersicon esculentum Mill.) seeds to complete germination requires weakening of the endosperm tissue opposite the radicle tip. In common with other cell wall disassembly processes in plants, polygalacturonases (PGs) may be involved. Only calcium-dependent exo-PG activity was detected in tomato seed protein extracts. Chromatographic profiles of a partially acid-hydrolyzed fraction of polygalacturonic acid further digested with seed extract were consistent with the presence of only calcium-dependent exo-PG activity. In addition, a transcript encoding a previously unknown PG was detected prior to the completion of germination. The mRNA, produced from a gene (LeXPG1) estimated by Southern analysis to be represented once in the genome, was also present in flowers (anthers) and in lower amounts in roots and stems. LeXPG1 mRNA abundance was low during seed development, increased during imbibition, and was even greater in seeds that had completed germination. Expression of LeXPG1 during germination predominates in the endosperm cap and radicle tip, and in the radicle appears as a distinct band possibly associated with vascular tissue differentiation. We suggest that PG is involved in cell wall loosening of the endosperm necessary for radicle protrusion from tomato seeds and in subsequent embryo and seedling growth. PMID:10517833

Characterisation of ATP-dependent Mur ligases involved in the biogenesis of cell wall peptidoglycan in Mycobacterium tuberculosis.

PubMed

Munshi, Tulika; Gupta, Antima; Evangelopoulos, Dimitrios; Guzman, Juan David; Gibbons, Simon; Keep, Nicholas H; Bhakta, Sanjib

2013-01-01

ATP-dependent Mur ligases (Mur synthetases) play essential roles in the biosynthesis of cell wall peptidoglycan (PG) as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw) operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthetic pathway. In line with the previous findings on the regulation of mycobacterial MurD and corynebacterial MurC synthetases via phosphorylation, we found that all of the Mur synthetases interacted with the Ser/Thr protein kinases, PknA and PknB. In addition, we critically analyzed the interaction network of all of the Mur synthetases with proteins involved in cell division and cell wall PG biosynthesis to re-evaluate the importance of these key enzymes as novel therapeutic targets in anti-tubercular drug discovery.

Characterisation of ATP-Dependent Mur Ligases Involved in the Biogenesis of Cell Wall Peptidoglycan in Mycobacterium tuberculosis

PubMed Central

Munshi, Tulika; Gupta, Antima; Evangelopoulos, Dimitrios; Guzman, Juan David; Gibbons, Simon; Keep, Nicholas H.; Bhakta, Sanjib

2013-01-01

ATP-dependent Mur ligases (Mur synthetases) play essential roles in the biosynthesis of cell wall peptidoglycan (PG) as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw) operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthetic pathway. In line with the previous findings on the regulation of mycobacterial MurD and corynebacterial MurC synthetases via phosphorylation, we found that all of the Mur synthetases interacted with the Ser/Thr protein kinases, PknA and PknB. In addition, we critically analyzed the interaction network of all of the Mur synthetases with proteins involved in cell division and cell wall PG biosynthesis to re-evaluate the importance of these key enzymes as novel therapeutic targets in anti-tubercular drug discovery. PMID:23555903

The Redundancy of Peptidoglycan Carboxypeptidases Ensures Robust Cell Shape Maintenance in Escherichia coli

PubMed Central

Peters, Katharina; Kannan, Suresh; Rao, Vincenzo A.; Biboy, Jacob; Vollmer, Daniela; Erickson, Stephen W.; Lewis, Richard J.

2016-01-01

ABSTRACT Peptidoglycan (PG) is an essential structural component of the bacterial cell wall and maintains the integrity and shape of the cell by forming a continuous layer around the cytoplasmic membrane. The thin PG layer of Escherichia coli resides in the periplasm, a unique compartment whose composition and pH can vary depending on the local environment of the cell. Hence, the growth of the PG layer must be sufficiently robust to allow cell growth and division under different conditions. We have analyzed the PG composition of 28 mutants lacking multiple PG enzymes (penicillin-binding proteins [PBPs]) after growth in acidic or near-neutral-pH media. Statistical analysis of the muropeptide profiles identified dd-carboxypeptidases (DD-CPases) that were more active in cells grown at acidic pH. In particular, the absence of the DD-CPase PBP6b caused a significant increase in the pentapeptide content of PG as well as morphological defects when the cells were grown at acidic pH. Other DD-CPases (PBP4, PBP4b, PBP5, PBP6a, PBP7, and AmpH) and the PG synthase PBP1B made a smaller or null contribution to the pentapeptide-trimming activity at acidic pH. We solved the crystal structure of PBP6b and also demonstrated that the enzyme is more stable and has a lower Km at acidic pH, explaining why PBP6b is more active at low pH. Hence, PBP6b is a specialized DD-CPase that contributes to cell shape maintenance at low pH, and E. coli appears to utilize redundant DD-CPases for normal growth under different conditions. PMID:27329754

The ectopic expression of a pectin methyl esterase inhibitor increases pectin methyl esterification and limits fungal diseases in wheat.

PubMed

Volpi, Chiara; Janni, Michela; Lionetti, Vincenzo; Bellincampi, Daniela; Favaron, Francesco; D'Ovidio, Renato

2011-09-01

Cell wall pectin methyl esterification can influence plant resistance because highly methyl-esterified pectin can be less susceptible to the hydrolysis by pectic enzymes such as fungal endopolygalacturonases (PG). Pectin is secreted into the cell wall in a highly methyl-esterified form and, here, is de-methyl esterified by pectin methyl esterase (PME). The activity of PME is controlled by specific protein inhibitors called PMEI; consequently, an increased inhibition of PME by PMEI might modify the pectin methyl esterification. In order to test the possibility of improving wheat resistance by modifying the methyl esterification of pectin cell wall, we have produced durum wheat transgenic lines expressing the PMEI from Actinidia chinensis (AcPMEI). The expression of AcPMEI endows wheat with a reduced endogenous PME activity, and transgenic lines expressing a high level of the inhibitor showed a significant increase in the degree of methyl esterification. These lines showed a significant reduction of disease symptoms caused by the fungal pathogens Bipolaris sorokiniana or Fusarium graminearum. This increased resistance was related to the impaired ability of these fungal pathogens to grow on methyl-esterified pectin and to a reduced activity of the fungal PG to hydrolyze methyl-esterified pectin. In addition to their importance for wheat improvement, these results highlight the primary role of pectin despite its low content in the wheat cell wall.

Pectinases From Sphenophorus levis Vaurie, 1978 (Coleoptera: Curculionidae): Putative Accessory Digestive Enzymes

PubMed Central

Evangelista, Danilo Elton; de Paula, Fernando Fonseca Pereira; Rodrigues, André; Henrique-Silva, Flávio

2015-01-01

The cell wall in plants offers protection against invading organisms and is mainly composed of the polysaccharides pectin, cellulose, and hemicellulose, which can be degraded by plant cell wall degrading enzymes (PCWDEs). Such enzymes are often synthesized by free living microorganisms or endosymbionts that live in the gut of some animals, including certain phytophagous insects. Thus, the ability of an insect to degrade the cell wall was once thought to be related to endosymbiont enzyme activity. However, recent studies have revealed that some phytophagous insects are able to synthesize their own PCWDEs by endogenous genes, although questions regarding the origin of these genes remain unclear. This study describes two pectinases from the sugarcane weevil, Sphenophorus levis Vaurie, 1978 (Sl-pectinases), which is considered one of the most serious agricultural pests in Brazil. Two cDNA sequences identified in a cDNA library of the insect larvae coding for a pectin methylesterase (PME) and an endo-polygalacturonase (endo-PG)—denominated Sl-PME and Sl-endoPG, respectively—were isolated and characterized. The quantitative real-time reverse transcriptase polymerase chain reaction expression profile for both Sl-pectinases showed mRNA production mainly in the insect feeding stages and exclusively in midgut tissue of the larvae. This analysis, together Western blotting data, suggests that Sl-pectinases have a digestive role. Phylogenetic analyses indicate that Sl-PME and Sl-endoPG sequences are closely related to bacteria and fungi, respectively. Moreover, the partial genomic sequences of the pectinases were amplified from insect fat body DNA, which was certified to be free of endosymbiotic DNA. The analysis of genomic sequences revealed the existence of two small introns with 53 and 166 bp in Sl-endoPG, which is similar to the common pattern in fungal introns. In contrast, no intron was identified in the Sl-PME genomic sequence, as generally observed in bacteria. These data support the theory of horizontal gene transfer proposed for the origin of insect pectinases, reinforcing the acquisition of PME genes from bacteria and endo-PG genes from fungi. PMID:25673050

The Absence of a Mature Cell Wall Sacculus in Stable Listeria monocytogenes L-Form Cells Is Independent of Peptidoglycan Synthesis.

PubMed

Studer, Patrick; Borisova, Marina; Schneider, Alexander; Ayala, Juan A; Mayer, Christoph; Schuppler, Markus; Loessner, Martin J; Briers, Yves

2016-01-01

L-forms are cell wall-deficient variants of otherwise walled bacteria that maintain the ability to survive and proliferate in absence of the surrounding peptidoglycan sacculus. While transient or unstable L-forms can revert to the walled state and may still rely on residual peptidoglycan synthesis for multiplication, stable L-forms cannot revert to the walled form and are believed to propagate in the complete absence of peptidoglycan. L-forms are increasingly studied as a fundamental biological model system for cell wall synthesis. Here, we show that a stable L-form of the intracellular pathogen Listeria monocytogenes features a surprisingly intact peptidoglycan synthesis pathway including glycosyl transfer, in spite of the accumulation of multiple mutations during prolonged passage in the cell wall-deficient state. Microscopic and biochemical analysis revealed the presence of peptidoglycan precursors and functional glycosyl transferases, resulting in the formation of peptidoglycan polymers but without the synthesis of a mature cell wall sacculus. In conclusion, we found that stable, non-reverting L-forms, which do not require active PG synthesis for proliferation, may still continue to produce aberrant peptidoglycan.

Expression patterns of cell wall-modifying genes from banana during fruit ripening and in relationship with finger drop

PubMed Central

Mbéguié-A-Mbéguié, D.; Hubert, O.; Baurens, F. C.; Matsumoto, T.; Chillet, M.; Fils-Lycaon, B.; Sidibé-Bocs, S.

2009-01-01

Few molecular studies have been devoted to the finger drop process that occurs during banana fruit ripening. Recent studies revealed the involvement of changes in the properties of cell wall polysaccharides in the pedicel rupture area. In this study, the expression of cell-wall modifying genes was monitored in peel tissue during post-harvest ripening of Cavendish banana fruit, at median area (control zone) and compared with that in the pedicel rupture area (drop zone). To this end, three pectin methylesterase (PME) and seven xyloglucan endotransglycosylase/hydrolase (XTH) genes were isolated. The accumulation of their mRNAs and those of polygalaturonase, expansin, and pectate lyase genes already isolated from banana were examined. During post-harvest ripening, transcripts of all genes were detected in both zones, but accumulated differentially. MaPME1, MaPG1, and MaXTH4 mRNA levels did not change in either zone. Levels of MaPME3 and MaPG3 mRNAs increased greatly only in the control zone and at the late ripening stages. For other genes, the main molecular changes occurred 1–4 d after ripening induction. MaPME2, MaPEL1, MaPEL2, MaPG4, MaXTH6, MaXTH8, MaXTH9, MaEXP1, MaEXP4, and MaEXP5 accumulated highly in the drop zone, contrary to MaXTH3 and MaXTH5, and MaEXP2 throughout ripening. For MaPG2, MaXET1, and MaXET2 genes, high accumulation in the drop zone was transient. The transcriptional data obtained from all genes examined suggested that finger drop and peel softening involved similar mechanisms. These findings also led to the proposal of a sequence of molecular events leading to finger drop and to suggest some candidates. PMID:19357434

A novel type of peptidoglycan-binding domain highly specific for amidated D-Asp cross-bridge, identified in Lactobacillus casei bacteriophage endolysins.

PubMed

Regulski, Krzysztof; Courtin, Pascal; Kulakauskas, Saulius; Chapot-Chartier, Marie-Pierre

2013-07-12

Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-L-alanine amidase, whereas Lc-Lys-2 is a γ-D-glutamyl-L-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with D-Ala(4)→D-Asx-L-Lys(3) in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting D-Ala(4)→L-Ala-(L-Ala/L-Ser)-L-Lys(3); moreover, they do not lyse the L. lactis mutant containing only the nonamidated D-Asp cross-bridge, i.e. D-Ala(4)→D-Asp-L-Lys(3). In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 L-Lys(3)-D-Asn-L-Lys(3) bridges replacing the wild-type 4→3 D-Ala(4)-D-Asn-L-Lys(3) bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly D-Asn but not PG with only the nonamidated D-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the D-Asn interpeptide bridge of PG.

Cell wall-degrading enzymes of Didymella bryoniae in relation to fungal growth and virulence in cantaloupe fruit

PubMed Central

Zhang, J.; Bruton, B. D.; Biles, C. L.

2014-01-01

Didymella bryoniae is an important pathogen of cucurbits worldwide. Virulence factors of D. bryoniae were investigated in regard to fungal growth and the production of cell wall-degrading enzymes, polygalacturonase (PG), pectate lyase (PL), pectin lyase (PNL), β-galactosidase (β-Gal) and cellulase (Cx). Virulence levels of five D. bryoniae isolates were determined by the severity of inoculated cantaloupe fruit decay. The highly virulent isolates had more mycelial growth than the moderately virulent isolates in different media. PG activities produced by the highly virulent isolates in shake cultures and in decayed fruit were greater than those of the moderately virulent isolates. PNL, but not PL, in decayed fruit was higher with the highly virulent isolates compared to the moderately virulent ones. The highly virulent isolates showed higher Cx activity than the moderately virulent ones in decayed fruit and in fruit tissue shake culture. β-Gal activities of the highly virulent isolates in pectin shake culture and in decayed fruit were greater than those of the two moderately virulent isolates although fruit also produced β-Gal. Protein analysis showed two fungal β-Gal isozymes in decayed fruit compared to those of healthy fruit. Correlation analysis indicated that the activities of PG, PNL, β-Gal and Cx in cultures and in decayed fruit positively correlated with fungal growth and fruit decay severity. The results of this study suggest that PG, PNL, β-Gal, and Cx appear to be virulence factors of D. bryoniae in cantaloupe decay with PG and β-Gal as the most predominant fruit decay enzymes. PMID:25364138

Characterization of Mutants Deficient in the l,d-Carboxypeptidase (DacB) and WalRK (VicRK) Regulon, Involved in Peptidoglycan Maturation of Streptococcus pneumoniae Serotype 2 Strain D39▿†

PubMed Central

Barendt, Skye M.; Sham, Lok-To; Winkler, Malcolm E.

2011-01-01

Peptidoglycan (PG) hydrolases play critical roles in the remodeling of bacterial cell walls during division. PG hydrolases have been studied extensively in several bacillus species, such as Escherichia coli and Bacillus subtilis, but remain relatively uncharacterized in ovococcus species, such as Streptococcus pneumoniae (pneumococcus). In this work, we identified genes that encode proteins with putative PG hydrolytic domains in the genome of S. pneumoniae strain D39. Knockout mutations in these genes were constructed, and the resulting mutants were characterized in comparison with the parent strain for growth, cell morphology, PG peptide incorporation, and in some cases, PG peptide composition. In addition, we characterized deletion mutations in nonessential genes of unknown function in the WalRKSpn two-component system regulon, which also contains the essential pcsB cell division gene. Several mutants did not show overt phenotypes, which is perhaps indicative of redundancy. In contrast, two new mutants showed distinct defects in PG biosynthesis. One mutation was in a gene designated dacB (spd_0549), which we showed encodes an l,d-carboxypeptidase involved in PG maturation. Notably, dacB mutants, similar to dacA (d,d-carboxypeptidase) mutants, exhibited defects in cell shape and septation, consistent with the idea that the availability of PG peptide precursors is important for proper PG biosynthesis. Epistasis analysis indicated that DacA functions before DacB in d-Ala removal, and immunofluorescence microscopy showed that DacA and DacB are located over the entire surface of pneumococcal cells. The other mutation was in WalRKSpn regulon gene spd_0703, which encodes a putative membrane protein that may function as a type of conserved streptococcal shape, elongation, division, and sporulation (SEDS) protein. PMID:21378199

Co-Inactivation of GlnR and CodY Regulators Impacts Pneumococcal Cell Wall Physiology.

PubMed

Johnston, Calum; Bootsma, Hester J; Aldridge, Christine; Manuse, Sylvie; Gisch, Nicolas; Schwudke, Dominik; Hermans, Peter W M; Grangeasse, Christophe; Polard, Patrice; Vollmer, Waldemar; Claverys, Jean-Pierre

2015-01-01

CodY, a nutritional regulator highly conserved in low G+C Gram-positive bacteria, is essential in Streptococcus pneumoniae (the pneumococcus). A published codY mutant possessed suppressing mutations inactivating the fatC and amiC genes, respectively belonging to iron (Fat/Fec) and oligopeptide (Ami) ABC permease operons, which are directly repressed by CodY. Here we analyzed two additional published codY mutants to further explore the essentiality of CodY. We show that one, in which the regulator of glutamine/glutamate metabolism glnR had been inactivated by design, had only a suppressor in fecE (a gene in the fat/fec operon), while the other possessed both fecE and amiC mutations. Independent isolation of three different fat/fec suppressors thus establishes that reduction of iron import is crucial for survival without CodY. We refer to these as primary suppressors, while inactivation of ami, which is not essential for survival of codY mutants and acquired after initial fat/fec inactivation, can be regarded as a secondary suppressor. The availability of codY- ami+ cells allowed us to establish that CodY activates competence for genetic transformation indirectly, presumably by repressing ami which is known to antagonize competence. The glnR codY fecE mutant was then found to be only partially viable on solid medium and hypersensitive to peptidoglycan (PG) targeting agents such as the antibiotic cefotaxime and the muramidase lysozyme. While analysis of PG and teichoic acid composition uncovered no alteration in the glnR codY fecE mutant compared to wildtype, electron microscopy revealed altered ultrastructure of the cell wall in the mutant, establishing that co-inactivation of GlnR and CodY regulators impacts pneumococcal cell wall physiology. In light of rising levels of resistance to PG-targeting antibiotics of natural pneumococcal isolates, GlnR and CodY constitute potential alternative therapeutic targets to combat this debilitating pathogen, as co-inactivation of these regulators renders pneumococci sensitive to iron and PG-targeting agents.

Accumulation of Peptidoglycan O-Acetylation Leads to Altered Cell Wall Biochemistry and Negatively Impacts Pathogenesis Factors of Campylobacter jejuni.

PubMed

Ha, Reuben; Frirdich, Emilisa; Sychantha, David; Biboy, Jacob; Taveirne, Michael E; Johnson, Jeremiah G; DiRita, Victor J; Vollmer, Waldemar; Clarke, Anthony J; Gaynor, Erin C

2016-10-21

Campylobacter jejuni is a leading cause of bacterial gastroenteritis in the developed world. Despite its prevalence, its mechanisms of pathogenesis are poorly understood. Peptidoglycan (PG) is important for helical shape, colonization, and host-pathogen interactions in C. jejuni Therefore, changes in PG greatly impact the physiology of this organism. O-acetylation of peptidoglycan (OAP) is a bacterial phenomenon proposed to be important for proper cell growth, characterized by acetylation of the C6 hydroxyl group of N-acetylmuramic acid in the PG glycan backbone. The OAP gene cluster consists of a PG O-acetyltransferase A (patA) for translocation of acetate into the periplasm, a PG O-acetyltransferase B (patB) for O-acetylation, and an O-acetylpeptidoglycan esterase (ape1) for de-O-acetylation. In this study, reduced OAP in ΔpatA and ΔpatB had minimal impact on C. jejuni growth and fitness under the conditions tested. However, accumulation of OAP in Δape1 resulted in marked differences in PG biochemistry, including O-acetylation, anhydromuropeptide levels, and changes not expected to result directly from Ape1 activity. This suggests that OAP may be a form of substrate level regulation in PG biosynthesis. Ape1 acetylesterase activity was confirmed in vitro using p-nitrophenyl acetate and O-acetylated PG as substrates. In addition, Δape1 exhibited defects in pathogenesis-associated phenotypes, including cell shape, motility, biofilm formation, cell surface hydrophobicity, and sodium deoxycholate sensitivity. Δape1 was also impaired for chick colonization and adhesion, invasion, intracellular survival, and induction of IL-8 production in INT407 cells in vitro The importance of Ape1 in C. jejuni biology makes it a good candidate as an antimicrobial target. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Engineering the cell wall by reducing de-methyl-esterified homogalacturonan improves saccharification of plant tissues for bioconversion.

PubMed

Lionetti, Vincenzo; Francocci, Fedra; Ferrari, Simone; Volpi, Chiara; Bellincampi, Daniela; Galletti, Roberta; D'Ovidio, Renato; De Lorenzo, Giulia; Cervone, Felice

2010-01-12

Plant cell walls represent an abundant, renewable source of biofuel and other useful products. The major bottleneck for the industrial scale-up of their conversion to simple sugars (saccharification), to be subsequently converted by microorganisms into ethanol or other products, is their recalcitrance to enzymatic saccharification. We investigated whether the structure of pectin that embeds the cellulose-hemicellulose network affects the exposure of cellulose to enzymes and consequently the process of saccharification. Reduction of de-methyl-esterified homogalacturonan (HGA) in Arabidopsis plants through the expression of a fungal polygalacturonase (PG) or an inhibitor of pectin methylesterase (PMEI) increased the efficiency of enzymatic saccharification. The improved enzymatic saccharification efficiency observed in transformed plants could also reduce the need for acid pretreatment. Similar results were obtained in PG-expressing tobacco plants and in PMEI-expressing wheat plants, indicating that reduction of de-methyl-esterified HGA may be used in crop species to facilitate the process of biomass saccharification.

Molecular modeling of Gram-positive bacteria peptidoglycan layer, selected glycopeptide antibiotics and vancomycin derivatives modified with sugar moieties.

PubMed

Ślusarz, Rafał; Szulc, Monika; Madaj, Janusz

2014-05-07

Proper understanding of the mechanisms of binding to Gram-positive bacteria cell wall layers-especially to the peptidoglycan (PG) layer, seems to be crucial for proper development of new drug candidates which are effective against these bacteria. In this work we have constructed two different models of the Gram-positive bacteria PG layer: the layered and the scaffold models. PG conformational changes during geometry optimization, models relaxation, and molecular dynamics were described and discussed. We have found that the border surface of both PG layer models differs from the surface located away from the edge of models and the chains formed by disaccharide units prefer helix-like conformation. This curling of PG chains significantly affects the shape of antibiotic-accessible surface and the process is thus crucial for new drug development. Glycopeptide antibiotics effective against Gram-positive bacteria, such as vancomycin and its semisynthetic derivatives-oritavancin and telavancin, bind to d-alanyl-d-alanine stem termini on the peptidoglycan precursors of the cell wall. This binding inhibits cross-linking between the peptides and subsequently prevents cell wall synthesis. In this study some of the aspects of conformational freedom of vancomycin and restrictions from the modifications of vancomycin structure introduced into oritavancin and telavancin and five other vancomycin derivatives (with addition of 2-acetamido-2-deoxy-β-d-galactopyranosylamine, 2-acetamido-2-deoxy-β-d-glucopyranosylamine, 1-amine-1-deoxy-d-glucitol, 2-amino-2-deoxy-d-galactitol, or 2-amino-2-deoxy-d-glucitol to the C-terminal amino acid group in the vancomycin) are presented and discussed. The resulting molecular dynamics trajectories, root mean square deviation changes of aglycon and saccharide moieties as well as a comparative study of possible interactions with cyclic and chain forms of modified groups have been carried out, measured, and analyzed. Energetically advantageous conformations show close similarity to the structures known from the experimental data, but the diversity of others suggest very high conformational freedom of all modeled antibiotics and vancomycin derivatives. Alditol derivatives move closer to the peptidoglycan chain more easily but they also form intramolecular interactions more frequently than their homologous cyclic forms. One of the proposed derivatives seems to be a promising agent which is efficient in treatment of infections caused by Gram-positive bacteria. Copyright © 2014 Elsevier Ltd. All rights reserved.

Surfaceome and Proteosurfaceome in Parietal Monoderm Bacteria: Focus on Protein Cell-Surface Display

PubMed Central

Desvaux, Mickaël; Candela, Thomas; Serror, Pascale

2018-01-01

The cell envelope of parietal monoderm bacteria (archetypal Gram-positive bacteria) is formed of a cytoplasmic membrane (CM) and a cell wall (CW). While the CM is composed of phospholipids, the CW is composed at least of peptidoglycan (PG) covalently linked to other biopolymers, such as teichoic acids, polysaccharides, and/or polyglutamate. Considering the CW is a porous structure with low selective permeability contrary to the CM, the bacterial cell surface hugs the molecular figure of the CW components as a well of the external side of the CM. While the surfaceome corresponds to the totality of the molecules found at the bacterial cell surface, the proteinaceous complement of the surfaceome is the proteosurfaceome. Once translocated across the CM, secreted proteins can either be released in the extracellular milieu or exposed at the cell surface by associating to the CM or the CW. Following the gene ontology (GO) for cellular components, cell-surface proteins at the CM can either be integral (GO: 0031226), i.e., the integral membrane proteins, or anchored to the membrane (GO: 0046658), i.e., the lipoproteins. At the CW (GO: 0009275), cell-surface proteins can be covalently bound, i.e., the LPXTG-proteins, or bound through weak interactions to the PG or wall polysaccharides, i.e., the cell wall binding proteins. Besides monopolypeptides, some proteins can associate to each other to form supramolecular protein structures of high molecular weight, namely the S-layer, pili, flagella, and cellulosomes. After reviewing the cell envelope components and the different molecular mechanisms involved in protein attachment to the cell envelope, perspectives in investigating the proteosurfaceome in parietal monoderm bacteria are further discussed. PMID:29491848

Impact of the antifungal protein PgAFP from Penicillium chrysogenum on the protein profile in Aspergillus flavus.

PubMed

Delgado, Josué; Owens, Rebecca A; Doyle, Sean; Asensio, Miguel A; Núñez, Félix

2015-10-01

Antifungal proteins produced by molds are generally small, highly basic, and cysteine-rich. The best known effects of these proteins include morphological changes, metabolic inactivation, and membrane perturbation on sensitive fungi. Reactive oxygen species (ROS) generation leads to apoptosis, with G -protein playing a key role in transduction of cell death signals. The antifungal protein PgAFP from Penicillium chrysogenum inhibits growth of some toxigenic molds. Here we analyzed the effect of the antifungal protein PgAFP on the growth of Aspergillus flavus. For this, comparative proteomic analysis was used to identify the whole protein profile and protein change in abundance after PgAFP treatment. PgAFP provoked metabolic changes related to reduced energy metabolism, cell wall integrity alteration, and increased stress response due to higher levels of ROS. The observed changes in protein abundance, favoring a higher glutathione concentration as well as the increased abundance in heat shock proteins, do not seem to be enough to avoid necrosis. The decreased chitin deposition observed in PgAFP-treated A. flavus is attributed to a lower relative quantity of Rho1. The reduced relative abundance of a β subunit of G -protein seems to be the underlying reason for modulation of apoptosis in PgAFP-treated A. flavus hyphae. We propose Rho1 and G -protein subunit β CpcB to be the main factors in the mode of action of PgAFP in A. flavus. Additionally, enzymes essential for the biosynthesis of aflatoxin were no longer detectable in A. flavus hyphae at 24 h, following treatment with PgAFP. This presents a promising effect of PgAFP, which may prevent A. flavus from producing mycotoxins. However, the impact of PgAFP on actual aflatoxin production requires further study.

Serum pepsinogen-A, canine pancreatic lipase immunoreactivity, and C-reactive protein as prognostic markers in dogs with gastric dilatation-volvulus.

PubMed

Israeli, I; Steiner, J; Segev, G; Kass, P H; Suchodolski, J S; Sattasathuchana, P; Bruchim, Y; Yudelevitch, S; Aroch, I

2012-01-01

Pepsinogens are proenzymes secreted by gastric chief cells. In humans, their serum concentrations reflect gastric mucosal morphological and functional status. To evaluate serum canine pepsinogen-A (cPG-A), C-reactive protein (CRP), and canine pancreatic lipase immunoreactivity (cPLI) concentrations in dogs with gastric dilatation-volvulus (GDV). Sixty-six dogs presented with GDV and 79 healthy controls. Blood was collected prospectively, and records retrospectively reviewed. Median cPG-A concentration was higher in GDV dogs (median, 397 μg/L; range, 37-5,410) compared to controls (median, cPG-A 304 μg/L; range, 18-848; P = .07). Mortality rate in GDV dogs was 22.7%. In nonsurvivors of GDV, median cPG-A was higher compared to survivors (median, 746 μg/L; range, 128-5,409 versus median, 346; range, 36-1,575, respectively; P = .003). The proportion of dogs with increased cPG-A increased with gastric wall damage score (P = .007). An ROC analysis of cPG-A as a predictor of death showed an area under the curve (AUC) of 0.75, higher than lactate (AUC 0.66), and corresponded to a sensitivity and specificity of 53% and 88%, respectively. CRP was increased in 48 dogs (75%), cPLI was >200 μg/L in 26 dogs (39.4%) and >400 μg/L in 12 dogs (18.2%) but both analytes had no association with outcome. Presurgical cPG-A concentration was positively and significantly associated with gastric wall lesion severity, but, based on ROC analysis, it was only a moderate outcome predictor. CRP and cPLI were commonly increased in dogs with GDV. Copyright © 2012 by the American College of Veterinary Internal Medicine.

Temporal and spatial expression of polygalacturonase gene family members reveals divergent regulation during fleshy fruit ripening and abscission in the monocot species oil palm.

PubMed

Roongsattham, Peerapat; Morcillo, Fabienne; Jantasuriyarat, Chatchawan; Pizot, Maxime; Moussu, Steven; Jayaweera, Dasuni; Collin, Myriam; Gonzalez-Carranza, Zinnia H; Amblard, Philippe; Tregear, James W; Tragoonrung, Somvong; Verdeil, Jean-Luc; Tranbarger, Timothy J

2012-08-25

Cell separation that occurs during fleshy fruit abscission and dry fruit dehiscence facilitates seed dispersal, the final stage of plant reproductive development. While our understanding of the evolutionary context of cell separation is limited mainly to the eudicot model systems tomato and Arabidopsis, less is known about the mechanisms underlying fruit abscission in crop species, monocots in particular. The polygalacturonase (PG) multigene family encodes enzymes involved in the depolymerisation of pectin homogalacturonan within the primary cell wall and middle lamella. PG activity is commonly found in the separation layers during organ abscission and dehiscence, however, little is known about how this gene family has diverged since the separation of monocot and eudicots and the consequence of this divergence on the abscission process. The objective of the current study was to identify PGs responsible for the high activity previously observed in the abscission zone (AZ) during fruit shedding of the tropical monocot oil palm, and to analyze PG gene expression during oil palm fruit ripening and abscission. We identified 14 transcripts that encode PGs, all of which are expressed in the base of the oil palm fruit. The accumulation of five PG transcripts increase, four decrease and five do not change during ethylene treatments that induce cell separation. One PG transcript (EgPG4) is the most highly induced in the fruit base, with a 700-5000 fold increase during the ethylene treatment. In situ hybridization experiments indicate that the EgPG4 transcript increases preferentially in the AZ cell layers in the base of the fruit in response to ethylene prior to cell separation. The expression pattern of EgPG4 is consistent with the temporal and spatial requirements for cell separation to occur during oil palm fruit shedding. The sequence diversity of PGs and the complexity of their expression in the oil palm fruit tissues contrast with data from tomato, suggesting functional divergence underlying the ripening and abscission processes has occurred between these two fruit species. Furthermore, phylogenetic analysis of EgPG4 with PGs from other species suggests some conservation, but also diversification has occurred between monocots and eudicots, in particular between dry and fleshy fruit species.

Temporal and spatial expression of polygalacturonase gene family members reveals divergent regulation during fleshy fruit ripening and abscission in the monocot species oil palm

PubMed Central

2012-01-01

Background Cell separation that occurs during fleshy fruit abscission and dry fruit dehiscence facilitates seed dispersal, the final stage of plant reproductive development. While our understanding of the evolutionary context of cell separation is limited mainly to the eudicot model systems tomato and Arabidopsis, less is known about the mechanisms underlying fruit abscission in crop species, monocots in particular. The polygalacturonase (PG) multigene family encodes enzymes involved in the depolymerisation of pectin homogalacturonan within the primary cell wall and middle lamella. PG activity is commonly found in the separation layers during organ abscission and dehiscence, however, little is known about how this gene family has diverged since the separation of monocot and eudicots and the consequence of this divergence on the abscission process. Results The objective of the current study was to identify PGs responsible for the high activity previously observed in the abscission zone (AZ) during fruit shedding of the tropical monocot oil palm, and to analyze PG gene expression during oil palm fruit ripening and abscission. We identified 14 transcripts that encode PGs, all of which are expressed in the base of the oil palm fruit. The accumulation of five PG transcripts increase, four decrease and five do not change during ethylene treatments that induce cell separation. One PG transcript (EgPG4) is the most highly induced in the fruit base, with a 700–5000 fold increase during the ethylene treatment. In situ hybridization experiments indicate that the EgPG4 transcript increases preferentially in the AZ cell layers in the base of the fruit in response to ethylene prior to cell separation. Conclusions The expression pattern of EgPG4 is consistent with the temporal and spatial requirements for cell separation to occur during oil palm fruit shedding. The sequence diversity of PGs and the complexity of their expression in the oil palm fruit tissues contrast with data from tomato, suggesting functional divergence underlying the ripening and abscission processes has occurred between these two fruit species. Furthermore, phylogenetic analysis of EgPG4 with PGs from other species suggests some conservation, but also diversification has occurred between monocots and eudicots, in particular between dry and fleshy fruit species. PMID:22920238

Development of an experimental model of cholestasis induced by hypoxic/ischemic damage to the bile duct and liver tissues in infantile rats.

PubMed

Toki, Fumiaki; Takahashi, Atsushi; Suzuki, Makoto; Ootake, Sayaka; Hirato, Junko; Kuwano, Hiroyuki

2011-05-01

We aimed to develop experimental models of hypoxia/ischemia-induced cholestasis using neonatal and infantile rats. Hypoxia/ischemia was induced in the bile duct (BD) by injecting prostaglandin (PG) at birth and/or by coagulation of the hepatic artery (CHA) at about 3 weeks after birth. The rats were divided into 6 groups: control; PG-injected; sham-operated with or without PG; CHA; and CHA + PG. CHA was also performed in adult rats. Liver specimens and blood samples were obtained at 5 weeks after birth, and immunohistochemical and biochemical examinations were performed. (1) BD proliferation with fibrosis (BDPF) was found in the intrahepatic portal tract in the CHA and CHA + PG groups. Low-grade BDPF was observed in the PG group. (2) Cyst formation in the extrahepatic BD (EBD) was observed in the porta hepatis of some rats in the CHA and CHA + PG groups. In these groups, the number of peribiliary vascular plexuses (PVPs) decreased. BD proliferation and infiltration of inflammatory cells were observed in the EBD wall in the CHA + PG group. (3) Ki-67 was expressed in BD and EBD cells in the CHA + PG group. (4) BDPF was not detected in adult rats with CHA. (5) Serum liver function tests indicated obstructive changes in the EBD in the CHA and CHA + PG groups. Reduced blood flow in the EBD during infancy induced BDPF and obstructive changes in the EBD, which may, along with immature PVP and inflammatory changes in the EBD, contribute to hypoxia/ischemia of the EBD.

Production of recombinant protein G through high-density fermentation of engineered bacteria as well as purification.

PubMed

Zhang, Hu-Cheng; Yang, Jun; Yang, Guo-Wei; Wang, Xiao-Jie; Fan, Hai-Tao

2015-08-01

Recombinant Streptococcus Protein G (PG) is a cell wall protein, which, when combined with mammal immunoglobulin, is used in separating antibody technology. High-density fermentation technologies using an engineered recombinant PG-producing bacteria as well as PG separation and purification technologies have a direct impact on the availability and application of PG. Through primary and secondary seed cultivation, a recombinant E. coli strain was subjected to high-density fermentation with controlled feed supplement concentration under stimulation with isopropyl β-D-1-thiogalactopyranoside. The present study investigated the effect of factors including inoculum size, oxygen levels, pH and the cultivating method on the fermentation process, as well as the effect of the separation and purification technologies, including ultrasonication, nickel column affinity chromatography, Sephadex G-25 gel filtration chromatography and diethylaminoethanol-sepharose fast flow ion exchange chromatography on the yield and purity of PG. The efficiency of extraction was detected using SDS-PAGE. High-density fermentation yielded 80-150 g/l of bacteria and 1 g PG was obtained from one liter broth. The present study delivered a highly efficient novel method via which PG can be obtained at a high concentration and a purity >95%.

Detection of antistaphylococcal and toxic compounds by biological assay systems developed with a reporter Staphylococcus aureus strain harboring a heat inducible promoter - lacZ transcriptional fusion.

PubMed

Chanda, Palas Kumar; Ganguly, Tridib; Das, Malabika; Lee, Chia Yen; Luong, Thanh T; Sau, Subrata

2007-11-30

Previously it was reported that promoter of groES-groEL operon of Staphylococcus aureus is induced by various cell-wall active antibiotics. In order to exploit the above promoter for identifying novel antistaphylococcal drugs, we have cloned the promoter containing region (P(g)) of groES-groEL operon of S. aureus Newman and found that the above promoter is induced by sublethal concentrations of many antibiotics including cell-wall active antibiotics. A reporter S. aureus RN4220 strain (designated SAU006) was constructed by inserting the P(g)-lacZ transcriptional fusion into its chromosome. Agarose-based assay developed with SAU006 shows that P(g) in single-copy is also induced distinctly by different classes of antibiotics. Data indicate that ciprofloxacin, rifampicin, ampicillin, and cephalothin are strong inducers, whereas, tetracycline, streptomycin and vancomycin induce the above promoter weakly. Sublethal concentrations of ciprofloxacin and ampicilin even have induced P(g) efficiently in microtiter plate grown SAU006. Additional studies show for the first time that above promoter is also induced weakly by arsenate salt and hydrogen peroxide. Taken together, we suggest that our simple and sensitive assay systems with SAU006 could be utilized for screening and detecting not only novel antistaphylococcal compounds but also different toxic chemicals.

Sugar beet polygalacturonase-inhibiting proteins with 11 LRRs confer Rhizoctonia, Fusarium and Botrytis resistance in Nicotiana plants

USDA-ARS?s Scientific Manuscript database

Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) proteins that inhibit polygalacturonase (PG) enzymes secreted by pathogens to break down plant cell walls during early stage of disease development. Sugar beet (Beta vulgaris L.) PGIP genes (BvPGIPs) have 11 LRR domains as ...

Molecular cloning and functional analysis of three genes encoding polygalacturonase-inhibiting proteins from Capsicum annuum, and their relation to increased resistance to two fungal pathogens

USDA-ARS?s Scientific Manuscript database

Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall glycoproteins that can inhibit fungal endopolygalacturonases (PGs). Inhibiting by PGIPs directly reduces potential PG activity in specific plant pathogenic fungi, reducing their aggressiveness. Here, we isolated and functionally chara...

Diagnosis and treatment of acute phlegmonous gastritis: A case report.

PubMed

Yang, Hongxin; Yan, Zhiqiang; Chen, Jiaju; Xie, Haitao; Wang, Haibin; Wang, Qian

2018-05-01

Acute phlegmonous gastritis (PG) is a rare and often fatal condition mainly characterized by severe bacterial infection of the gastric wall. Case reports of PG over the past century average about 1 per year. Early diagnosis and immediate treatment are crucial to achieve positive outcomes. A 47-year-old man was referred to our hospital because of abdominal pain, high fever, and vomiting for 4 days, with aggravation for 24 hours. Physical examination revealed epigastric abdominal pain, rebound pain, and abdominal wall tightness. Abdominal CT showed thickening of the stomach wall with edema and gas. On the basis of symptoms and CT imaging findings, the patient was diagnosed with acute PG. Antibiotic therapy and operation. The patient immediately underwent an operation after conservative treatment using antibiotics proved ineffective. The whole stomach was obviously swollen, and the anterior side and posterior wall of the stomach were nigrescent necrotic. Hence, total gastrectomy was performed followed by reconstruction (roux-en-y), and pus that accumulated in the stomach wall was cultured. At postoperative broad-spectrum antibiotic coverage, the patient finally recovered. Acute PG is a rare infection of the gastric wall especially after antibiotic treatment. Given the fast progression of this disease, early recognition and immediate action are crucial to achieve positive outcomes.

Structure and Inhibitor Specificity of L,D-Transpeptidase (LdtMt2) from Mycobacterium tuberculosis and Antibiotic Resistance: Calcium Binding Promotes Dimer Formation.

PubMed

Gokulan, Kuppan; Khare, Sangeeta; Cerniglia, Carl E; Foley, Steven L; Varughese, Kottayil I

2018-03-09

The final step of peptidoglycan (PG) synthesis in all bacteria is the formation of cross-linkage between PG-stems. The cross-linking between amino acids in different PG chains gives the peptidoglycan cell wall a 3-dimensional structure and adds strength and rigidity to it. There are two distinct types of cross-linkages in bacterial cell walls. D,D-transpeptidase (D,D-TPs) generate the classical 4➔3 cross-linkages and the L,D-transpeptidase (L,D-TPs) generate the 3➔3 non-classical peptide cross-linkages. The present study is aimed at understanding the nature of drug resistance associated with L,D-TP and gaining insights for designing novel antibiotics against multi-drug resistant bacteria. Penicillin and cephalosporin classes of β-lactams cannot inhibit L,D-TP function; however, carbapenems inactivate its function. We analyzed the structure of L,D-TP of Mycobacterium tuberculosis in the apo form and in complex with meropenem and imipenem. The periplasmic region of L,D-TP folds into three domains. The catalytic residues are situated in the C-terminal domain. The acylation reaction occurs between carbapenem antibiotics and the catalytic Cys-354 forming a covalent complex. This adduct formation mimics the acylation of L,D-TP with the donor PG-stem. A novel aspect of this study is that in the crystal structures of the apo and the carbapenem complexes, the N-terminal domain has a muropeptide unit non-covalently bound to it. Another interesting observation is that the calcium complex crystallized as a dimer through head and tail interactions between the monomers.

A candidate gene based approach validates Md-PG1 as the main responsible for a QTL impacting fruit texture in apple (Malus x domestica Borkh).

PubMed

Longhi, Sara; Hamblin, Martha T; Trainotti, Livio; Peace, Cameron P; Velasco, Riccardo; Costa, Fabrizio

2013-03-04

Apple is a widely cultivated fruit crop for its quality properties and extended storability. Among the several quality factors, texture is the most important and appreciated, and within the apple variety panorama the cortex texture shows a broad range of variability. Anatomically these variations depend on degradation events occurring in both fruit primary cell wall and middle lamella. This physiological process is regulated by an enzymatic network generally encoded by large gene families, among which polygalacturonase is devoted to the depolymerization of pectin. In apple, Md-PG1, a key gene belonging to the polygalacturonase gene family, was mapped on chromosome 10 and co-localized within the statistical interval of a major hot spot QTL associated to several fruit texture sub-phenotypes. In this work, a QTL corresponding to the position of Md-PG1 was validated and new functional alleles associated to the fruit texture properties in 77 apple cultivars were discovered. 38 SNPs genotyped by gene full length resequencing and 2 SSR markers ad hoc targeted in the gene metacontig were employed. Out of this SNP set, eleven were used to define three significant haplotypes statistically associated to several texture components. The impact of Md-PG1 in the fruit cell wall disassembly was further confirmed by the cortex structure electron microscope scanning in two apple varieties characterized by opposite texture performance, such as 'Golden Delicious' and 'Granny Smith'. The results here presented step forward into the genetic dissection of fruit texture in apple. This new set of haplotypes, and microsatellite alleles, can represent a valuable toolbox for a more efficient parental selection as well as the identification of new apple accessions distinguished by superior fruit quality features.

Role of Melatonin in Cell-Wall Disassembly and Chilling Tolerance in Cold-Stored Peach Fruit.

PubMed

Cao, Shifeng; Bian, Kun; Shi, Liyu; Chung, Hsiao-Hang; Chen, Wei; Yang, Zhenfeng

2018-06-06

Melatonin reportedly increases chilling tolerance in postharvest peach fruit during cold storage, but information on its effects on cell-wall disassembly in chilling-injured peaches is limited. In this study, we investigated the role of cell-wall depolymerization in chilling-tolerance induction in melatonin-treated peaches. Treatment with 100 μM melatonin alleviated chilling symptoms (mealiness) characterized by a decrease in fruit firmness and increase in juice extractability in treated peaches during storage. The loss of neutral sugars, such as arabinose and galactose, in both the 1,2-cyclohexylenedinitrilotetraacetic acid (CDTA)- and Na 2 CO 3 -soluble fractions was observed at 7 days in treated peaches, but the contents increased after 28 days of storage. Atomic-force-microscopy (AFM) analysis revealed that the polysaccharide widths in the CDTA- and Na 2 CO 3 -soluble fractions in the treated fruit were mainly distributed in a shorter range, as compared with those in the control fruit. In addition, the expression profiles of a series of cell-wall-related genes showed that melatonin treatment maintained the balance between transcripts of PpPME and PpPG, which accompany the up-regulation of several other genes involved in cell-wall disassembly. Taken together, our results suggested that the reduced mealiness by melatonin was probably associated with its positive regulation of numerous cell-wall-modifying enzymes and proteins; thus, the depolymerization of the cell-wall polysaccharides in the peaches treated with melatonin was maintained, and the treated fruit could soften gradually during cold storage.

A polygalacturonase localized in the Golgi apparatus in Pisum sativum.

PubMed

Ohashi, Takao; Jinno, Jun; Inoue, Yoshiyuki; Ito, Shoko; Fujiyama, Kazuhito; Ishimizu, Takeshi

2017-09-01

Pectin is a plant cell wall constituent that is mainly composed of polygalacturonic acid (PGA), a linear α1,4-d-galacturonic acid (GalUA) backbone. Polygalacturonase (PG) hydrolyzes the α1,4-linkages in PGA. Nearly all plant PGs identified thus far are secreted as soluble proteins. Here we describe the microsomal PG activity in pea (Pisum sativum) epicotyls and present biochemical evidence that it was localized to the Golgi apparatus, where pectins are biosynthesized. The microsomal PG was purified, and it was enzymatically characterized. The purified enzyme showed maximum activity towards pyridylaminated oligogalacturonic acids with six degrees of polymerization (PA-GalUA6), with a Km value of 11 μM for PA-GalUA6. The substrate preference of the enzyme was complementary to that of PGA synthase. The main PG activity in microsomes was detected in the Golgi fraction by sucrose density gradient ultracentrifugation. The activity of the microsomal PG was lower in rapidly growing epicotyls, in contrast to the high expression of PGA synthase. The role of this PG in the regulation of pectin biosynthesis or plant growth is discussed. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Roles of tRNA in cell wall biosynthesis

PubMed Central

Dare, Kiley; Ibba, Michael

2013-01-01

Recent research into various aspects of bacterial metabolism such as cell wall and antibiotic synthesis, degradation pathways, cellular stress, and amino acid biosynthesis has elucidated roles of aminoacyl-transfer ribonucleic acid (aa-tRNA) outside of translation. Although the two enzyme families responsible for cell wall modifications, aminoacyl-phosphatidylglycerol synthases (aaPGSs) and Fem, were discovered some time ago, they have recently become of intense interest for their roles in the antimicrobial resistance of pathogenic microorganisms. The addition of positively charged amino acids to phosphatidylglycerol (PG) by aaPGSs neutralizes the lipid bilayer making the bacteria less susceptible to positively charged antimicrobial agents. Fem transferases utilize aa-tRNA to form peptide bridges that link strands of peptidoglycan. These bridges vary among the bacterial species in which they are present and play a role in resistance to antibiotics that target the cell wall. Additionally, the formation of truncated peptides results in shorter peptide bridges and loss of branched linkages which makes bacteria more susceptible to antimicrobials. A greater understanding of the structure and substrate specificity of this diverse enzymatic family is necessary to aid current efforts in designing potential bactericidal agents. These two enzyme families are linked only by the substrate with which they modify the cell wall, aa-tRNA; their structure, cell wall modification processes and the physiological changes they impart on the bacterium differ greatly. PMID:22262511

Double mutation of cell wall proteins CspB and PBP1a increases secretion of the antibody Fab fragment from Corynebacterium glutamicum

PubMed Central

2014-01-01

Background Among other advantages, recombinant antibody-binding fragments (Fabs) hold great clinical and commercial potential, owing to their efficient tissue penetration compared to that of full-length IgGs. Although production of recombinant Fab using microbial expression systems has been reported, yields of active Fab have not been satisfactory. We recently developed the Corynebacterium glutamicum protein expression system (CORYNEX®) and demonstrated improved yield and purity for some applications, although the system has not been applied to Fab production. Results The Fab fragment of human anti-HER2 was successfully secreted by the CORYNEX® system using the conventional C. glutamicum strain YDK010, but the productivity was very low. To improve the secretion efficiency, we investigated the effects of deleting cell wall-related genes. Fab secretion was increased 5.2 times by deletion of pbp1a, encoding one of the penicillin-binding proteins (PBP1a), mediating cell wall peptidoglycan (PG) synthesis. However, this Δpbp1a mutation did not improve Fab secretion in the wild-type ATCC13869 strain. Because YDK010 carries a mutation in the cspB gene encoding a surface (S)-layer protein, we evaluated the effect of ΔcspB mutation on Fab secretion from ATCC13869. The Δpbp1a mutation showed a positive effect on Fab secretion only in combination with the ΔcspB mutation. The ΔcspBΔpbp1a double mutant showed much greater sensitivity to lysozyme than either single mutant or the wild-type strain, suggesting that these mutations reduced cell wall resistance to protein secretion. Conclusion There are at least two crucial permeability barriers to Fab secretion in the cell surface structure of C. glutamicum, the PG layer, and the S-layer. The ΔcspBΔpbp1a double mutant allows efficient Fab production using the CORYNEX® system. PMID:24731213

Suppression of 9-cis-epoxycarotenoid dioxygenase, which encodes a key enzyme in abscisic acid biosynthesis, alters fruit texture in transgenic tomato.

PubMed

Sun, Liang; Sun, Yufei; Zhang, Mei; Wang, Ling; Ren, Jie; Cui, Mengmeng; Wang, Yanping; Ji, Kai; Li, Ping; Li, Qian; Chen, Pei; Dai, Shengjie; Duan, Chaorui; Wu, Yan; Leng, Ping

2012-01-01

Cell wall catabolism during fruit ripening is under complex control and is key for fruit quality and shelf life. To examine the role of abscisic acid (ABA) in tomato (Solanum lycopersicum) fruit ripening, we suppressed SlNCED1, which encodes 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme in the biosynthesis of ABA. To suppress SlNCED1 specifically in tomato fruits, and thus avoid the pleiotropic phenotypes associated with ABA deficiency, we used an RNA interference construct driven by the fruit-specific E8 promoter. ABA accumulation and SlNCED1 transcript levels in the transgenic fruit were down-regulated to between 20% and 50% of the levels measured in the control fruit. This significant reduction in NCED activity led to a down-regulation in the transcription of genes encoding major cell wall catabolic enzymes, specifically polygalacturonase (SlPG), pectin methyl esterase (SlPME), β-galactosidase precursor mRNA (SlTBG), xyloglucan endotransglycosylase (SlXET), endo-1,4-β-cellulose (SlCels), and expansin (SlExp). This resulted in an increased accumulation of pectin during ripening. In turn, this led to a significant extension of the shelf life to 15 to 29 d compared with a shelf life of only 7 d for the control fruit and an enhancement of fruit firmness at the mature stage by 30% to 45%. In conclusion, ABA affects cell wall catabolism during tomato fruit ripening via down-regulation of the expression of major catabolic genes (SlPG, SlPME, SlTBG, SlXET, SlCels, and SlExp).

The Surface Layer Homology Domain-Containing Proteins of Alkaliphilic Bacillus pseudofirmus OF4 Play an Important Role in Alkaline Adaptation via Peptidoglycan Synthesis.

PubMed

Fujinami, Shun; Ito, Masahiro

2018-01-01

It is well known that the Na + cycle and the cell wall are essential for alkaline adaptation of Na + -dependent alkaliphilic Bacillus species. In Bacillus pseudofirmus OF4, surface layer protein A (SlpA), the most abundant protein in the surface layer (S-layer) of the cell wall, is involved in alkaline adaptation, especially under low Na + concentrations. The presence of a large number of genes that encode S-layer homology (SLH) domain-containing proteins has been suggested from the genome sequence of B. pseudofirmus OF4. However, other than SlpA, the functions of SLH domain-containing proteins are not well known. Therefore, a deletion mutant of the csaB gene, required for the retention of SLH domain-containing proteins on the cell wall, was constructed to investigate its physiological properties. The csaB mutant strain of B. pseudofirmus OF4 had a chained morphology and alkaline sensitivity even under a 230 mM Na + concentration at which there is no growth difference between the parental strain and the slpA mutant strain. Ultra-thin section transmission electron microscopy showed that a csaB mutant strain lacked an S-layer part, and its peptidoglycan (PG) layer was disturbed. The slpA mutant strain also lacked an S-layer part, although its PG layer was not disturbed. These results suggested that the surface layer homology domain-containing proteins of B. pseudofirmus OF4 play an important role in alkaline adaptation via peptidoglycan synthesis.

Adhesive force between graphene nanoscale flakes and living biological cells.

PubMed

Al Faouri, Radwan; Henry, Ralph; Biris, Alexandru S; Sleezer, Rob; Salamo, Gregory J

2017-11-01

We report on a measurement technique that quantifies the adhesive force between multi-layers of graphene flakes and the cell wall of live Escherichia coli cells using atomic force microscopy (AFM) in-fluid Peak Force- Quantitative Nanomechanical Mapping mode. To measure the adhesive force, we made use of the negative charge of E. coli cells to allow them to stick to positively charged surfaces, such as glass or silicon, that were covered by poly-L-Lysine. With this approach, cells were held in place for AFM characterization. Both pristine graphene (PG) flakes and functionalized graphene (FG) flakes were put on the E. coli cells and measurements of lateral size, flake thickness, and adhesion were made. Using this approach, the measured values of the adhesive force between multi-layers of graphene flakes (total thickness of 50 nm) and E. coli was determined to be equal or greater than 431 ± 65pN for (PG) and 694 ± 98pN for the (FG). More interestingly, the adhesive force of a graphene flake (thickness 1.3 nm) with the cell is determined to be equal or greater than 38.2 ± 16.4pN for the (PG) and 34.8 ± 15.3pN for the (FG). These interaction values can play an important role in determining and understanding the possible toxicity of graphene flakes. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Genes Sufficient for Synthesizing Peptidoglycan are Retained in Gymnosperm Genomes, and MurE from Larix gmelinii can Rescue the Albino Phenotype of Arabidopsis MurE Mutation.

PubMed

Lin, Xiaofei; Li, Ningning; Kudo, Hiromi; Zhang, Zhe; Li, Jinyu; Wang, Li; Zhang, Wenbo; Takechi, Katsuaki; Takano, Hiroyoshi

2017-03-01

The endosymbiotic theory states that plastids are derived from a single cyanobacterial ancestor that possessed a cell wall. Peptidoglycan (PG), the main component of the bacteria cell wall, gradually degraded during plastid evolution. PG-synthesizing Mur genes have been found to be retained in the genomes of basal streptophyte plants, although many of them have been lost from the genomes of angiosperms. The enzyme encoded by bacterial MurE genes catalyzes the formation of the UDP-N-acetylmuramic acid (UDP-MurNAc) tripeptide in bacterial PG biosynthesis. Knockout of the MurE gene in the moss Physcomitrella patens resulted in defects of chloroplast division, whereas T-DNA-tagged mutants of Arabidopsis thaliana for MurE revealed inhibition of chloroplast development but not of plastid division, suggesting that AtMurE is functionally divergent from the bacterial and moss MurE proteins. Here, we could identify 10 homologs of bacterial Mur genes, including MurE, in the recently sequenced genomes of Picea abies and Pinus taeda, suggesting the retention of the plastid PG system in gymnosperms. To investigate the function of gymnosperm MurE, we isolated an ortholog of MurE from the larch, Larix gmelinii (LgMurE) and confirmed its presence as a single copy per genome, as well as its abundant expression in the leaves of larch seedlings. Analysis with a fusion protein combining green fluorescent protein and LgMurE suggested that it localizes in chloroplasts. Cross-species complementation assay with MurE mutants of A. thaliana and P. patens showed that the expression of LgMurE cDNA completely rescued the albefaction defects in A. thaliana but did not rescue the macrochloroplast phenotype in P. patens. The evolution of plastid PG and the mechanism behind the functional divergence of MurE genes are discussed in the context of information about plant genomes at different evolutionary stages. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Phlegmonous gastritis associated with group A streptococcal toxic shock syndrome.

PubMed

Morimoto, Masaya; Tamura, Shinobu; Hayakawa, Takahiro; Yamanishi, Hirofumi; Nakamoto, Chiaki; Nakamoto, Hiromichi; Ikebe, Tadayoshi; Nakano, Yoshio; Fujimoto, Tokuzo

2014-01-01

Phlegmonous gastritis (PG) is a rare, acute, severe infectious disease of the gastric wall that is often fatal due to Streptococcus spp. A 77-year-old man with diabetes and a gastric ulcer was urgently admitted due to prolonged nausea and vomiting. Computed tomography revealed widespread diffuse thickening of the gastric wall, and PG was suspected. The patient expired less than 9 hours after admission despite intensive treatments. Later, an analysis of the blood and gastric juice revealed group A streptococcus (GAS) and virulence factors associated with toxic shock syndrome (TSS). We herein diagnosed a patient with an extremely aggressive course of PG caused by GAS TSS.

A candidate gene based approach validates Md-PG1 as the main responsible for a QTL impacting fruit texture in apple (Malus x domestica Borkh)

PubMed Central

2013-01-01

Background Apple is a widely cultivated fruit crop for its quality properties and extended storability. Among the several quality factors, texture is the most important and appreciated, and within the apple variety panorama the cortex texture shows a broad range of variability. Anatomically these variations depend on degradation events occurring in both fruit primary cell wall and middle lamella. This physiological process is regulated by an enzymatic network generally encoded by large gene families, among which polygalacturonase is devoted to the depolymerization of pectin. In apple, Md-PG1, a key gene belonging to the polygalacturonase gene family, was mapped on chromosome 10 and co-localized within the statistical interval of a major hot spot QTL associated to several fruit texture sub-phenotypes. Results In this work, a QTL corresponding to the position of Md-PG1 was validated and new functional alleles associated to the fruit texture properties in 77 apple cultivars were discovered. 38 SNPs genotyped by gene full length resequencing and 2 SSR markers ad hoc targeted in the gene metacontig were employed. Out of this SNP set, eleven were used to define three significant haplotypes statistically associated to several texture components. The impact of Md-PG1 in the fruit cell wall disassembly was further confirmed by the cortex structure electron microscope scanning in two apple varieties characterized by opposite texture performance, such as ‘Golden Delicious’ and ‘Granny Smith’. Conclusions The results here presented step forward into the genetic dissection of fruit texture in apple. This new set of haplotypes, and microsatellite alleles, can represent a valuable toolbox for a more efficient parental selection as well as the identification of new apple accessions distinguished by superior fruit quality features. PMID:23496960

Differential diagnosis of periapical cyst using collagen birefringence pattern of the cyst wall.

PubMed

Ji, Hyo Jin; Park, Se-Hee; Cho, Kyung-Mo; Lee, Suk Keun; Kim, Jin Woo

2017-05-01

Periapical lesions, including periapical cyst (PC), periapical granuloma (PG), and periapical abscess (PA), are frequently affected by chemical/physical damage during root canal treatment or severe bacterial infection, and thus, the differential diagnosis of periapical lesions may be difficult due to the presence of severe inflammatory reaction. The aim of this study was to make differential diagnosis among PC, PG, and PA under polarizing microscope. The collagen birefringence patterns of 319 cases of PC ( n = 122), PG ( n = 158), and PA ( n = 39) obtained using a polarizing microscope were compared. In addition, 6 cases of periodontal fibroma (PF) were used as positive controls. Collagen birefringence was condensed with a thick, linear band-like pattern in PC, but was short and irregularly scattered in PG, and scarce or absent in PA. PF showed intense collagen birefringence with a short, palisading pattern but no continuous band-like pattern. The linear band-like birefringence in PC was ascribed to pre-existing expansile tensile stress of the cyst wall. In this study all PCs ( n = 122) were distinguishable from PGs and PAs by their characteristic birefringence, despite the absence of lining epithelium ( n = 20). Therefore, the authors suggest that the presence of linear band-like collagen birefringence of the cyst wall aids the diagnostic differentiation of PC from PG and PA.

Genome-wide identification and characterization of polygalacturonase genes in Cucumis sativus and Citrullus lanatus.

PubMed

Yu, Youjian; Liang, Ying; Lv, Meiling; Wu, Jian; Lu, Gang; Cao, Jiashu

2014-01-01

Polygalacturonase (PG, EC3.2.1.15), one of the hydrolytic enzymes associated with the modification of pectin network in plant cell wall, has an important role in various cell-separation processes that are essential for plant development. PGs are encoded by a large gene family in plants. However, information on this gene family in plant development remains limited. In the present study, 53 and 62 putative members of the PG gene family in cucumber and watermelon genomes, respectively, were identified by genome-wide search to explore the composition, structure, and evolution of the PG family in Cucurbitaceae crops. The results showed that tandem duplication could be an important factor that contributes to the expansion of the PG genes in the two crops. The phylogenetic and evolutionary analyses suggested that PGs could be classified into seven clades, and that the exon/intron structures and intron phases were conserved within but divergent between clades. At least 24 ancestral PGs were detected in the common ancestor of Arabidopsis and Cucumis sativus. Expression profile analysis by quantitative real-time polymerase chain reaction demonstrated that most CsPGs exhibit specific or high expression pattern in one of the organs/tissues. The 16 CsPGs associated with fruit development could be divided into three subsets based on their specific expression patterns and the cis-elements of fruit-specific, endosperm/seed-specific, and ethylene-responsive exhibited in their promoter regions. Our comparative analysis provided some basic information on the PG gene family, which would be valuable for further functional analysis of the PG genes during plant development. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Constitutive expression of a grapevine polygalacturonase-inhibiting protein affects gene expression and cell wall properties in uninfected tobacco

PubMed Central

2011-01-01

Background Polygalacturonase-inhibiting proteins (PGIPs) directly limit the effective ingress of fungal pathogens by inhibiting cell wall-degrading endopolygalacturonases (ePGs). Transgenic tobacco plants over-expressing grapevine (Vitis vinifera) Vvpgip1 have previously been shown to be resistant to Botrytis infection. In this study we characterized two of these PGIP over-expressing lines with known resistance phenotypes by gene expression and hormone profiling in the absence of pathogen infection. Results Global gene expression was performed by a cross-species microarray approach using a potato cDNA microarray. The degree of potential cross-hybridization between probes was modeled by a novel computational workflow designed in-house. Probe annotations were updated by predicting probe-to-transcript hybridizations and combining information derived from other plant species. Comparing uninfected Vvpgip1-overexpressing lines to wild-type (WT), 318 probes showed significant change in expression. Functional groups of genes involved in metabolism and associated to the cell wall were identified and consequent cell wall analysis revealed increased lignin-levels in the transgenic lines, but no major differences in cell wall-derived polysaccharides. GO enrichment analysis also identified genes responsive to auxin, which was supported by elevated indole-acetic acid (IAA) levels in the transgenic lines. Finally, a down-regulation of xyloglucan endotransglycosylase/hydrolases (XTHs), which are important in cell wall remodeling, was linked to a decrease in total XTH activity. Conclusions This evaluation of PGIP over-expressing plants performed under pathogen-free conditions to exclude the classical PGIP-ePG inhibition interaction indicates additional roles for PGIPs beyond the inhibition of ePGs. PMID:22078230

Characterization of a major 31-kilodalton peptidoglycan-bound protein of Legionella pneumophila

DOE Office of Scientific and Technical Information (OSTI.GOV)

Butler, C.A.; Hoffman, P.S.

1990-05-01

A 31-kilodalton (kDa) protein was solubilized from the peptidoglycan (PG) fraction of Legionella pneumophila after treatment with either N-acetylmuramidase from the fungus Chalaropsis sp. or with mutanolysin from Streptomyces globisporus. The protein exhibited a ladderlike banding pattern by autoradiography when radiolabeled ((35S)cysteine or (35S)methionine) PG material was extensively treated with hen lysozyme. The banding patterns ranging between 31 and 45 kDa and between 55 and 60 kDa resolved as a single 31-kDa protein when the material was subsequently treated with N-acetylmuramidase. Analysis of the purified 31-kDa protein for diaminopimelic acid by gas chromatography revealed 1 mol of diaminopimelic acid permore » mol of protein. When outer membrane PG material containing the major outer membrane porin protein was treated with N-acetylmuramidase or mutanolysin, both the 28.5-kDa major outer membrane protein and the 31-kDa protein were solubilized from the PG material under reducing conditions. In the absence of 2-mercaptoethanol, a high-molecular-mass complex (100 kDa) was resolved. The results of this study indicate that a 31-kDa PG-bound protein is a major component of the cell wall of L. pneumophila whose function may be to anchor the major outer membrane protein to PG. Finally, a survey of other Legionella species and other serogroups of L. pneumophila suggested that PG-bound proteins may be a common feature of this genus.« less

Suppression of 9-cis-Epoxycarotenoid Dioxygenase, Which Encodes a Key Enzyme in Abscisic Acid Biosynthesis, Alters Fruit Texture in Transgenic Tomato1[W][OA

PubMed Central

Sun, Liang; Sun, Yufei; Zhang, Mei; Wang, Ling; Ren, Jie; Cui, Mengmeng; Wang, Yanping; Ji, Kai; Li, Ping; Li, Qian; Chen, Pei; Dai, Shengjie; Duan, Chaorui; Wu, Yan; Leng, Ping

2012-01-01

Cell wall catabolism during fruit ripening is under complex control and is key for fruit quality and shelf life. To examine the role of abscisic acid (ABA) in tomato (Solanum lycopersicum) fruit ripening, we suppressed SlNCED1, which encodes 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme in the biosynthesis of ABA. To suppress SlNCED1 specifically in tomato fruits, and thus avoid the pleiotropic phenotypes associated with ABA deficiency, we used an RNA interference construct driven by the fruit-specific E8 promoter. ABA accumulation and SlNCED1 transcript levels in the transgenic fruit were down-regulated to between 20% and 50% of the levels measured in the control fruit. This significant reduction in NCED activity led to a down-regulation in the transcription of genes encoding major cell wall catabolic enzymes, specifically polygalacturonase (SlPG), pectin methyl esterase (SlPME), β-galactosidase precursor mRNA (SlTBG), xyloglucan endotransglycosylase (SlXET), endo-1,4-β-cellulose (SlCels), and expansin (SlExp). This resulted in an increased accumulation of pectin during ripening. In turn, this led to a significant extension of the shelf life to 15 to 29 d compared with a shelf life of only 7 d for the control fruit and an enhancement of fruit firmness at the mature stage by 30% to 45%. In conclusion, ABA affects cell wall catabolism during tomato fruit ripening via down-regulation of the expression of major catabolic genes (SlPG, SlPME, SlTBG, SlXET, SlCels, and SlExp). PMID:22108525

Increased chitin biosynthesis contributes to the resistance of Penicillium polonicum against the antifungal protein PgAFP.

PubMed

Delgado, Josué; Owens, Rebecca A; Doyle, Sean; Asensio, Miguel A; Núñez, Félix

2016-01-01

Antifungal proteins from molds have been proposed as a valuable tool against unwanted molds, but the resistance of some fungi limits their use. Resistance to antimicrobial peptides has been suggested to be due to lack of interaction with the mold or to a successful response. The antifungal protein PgAFP produced by Penicillium chrysogenum inhibits the growth of various ascomycetes, but not Penicillium polonicum. To study the basis for resistance to this antifungal protein, localization of PgAFP and metabolic, structural, and morphological changes were investigated in P. polonicum. PgAFP bound the outer layer of P. polonicum but not regenerated chitin, suggesting an interaction with specific molecules. Comparative two-dimensional gel electrophoresis (2D-PAGE) and comparative quantitative proteomics revealed changes in the relative abundance of several proteins from ribosome, spliceosome, metabolic, and biosynthesis of secondary metabolite pathways. The proteome changes and an altered permeability reveal an active reaction of P. polonicum to PgAFP. The successful response of the resistant mold seems to be based on the higher abundance of protein Rho GTPase Rho1 that would lead to the increased chitin deposition via cell wall integrity (CWI) signaling pathway. Thus, combined treatment with chitinases could provide a complementary means to combat resistance to antifungal proteins.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ito, Gen; Kobayashi, Takeshi; Takeda, Yoshie

Highlights: • Proteoglycan from salmon nasal cartridge (SNC-PG) promoted wound healing in fibroblast monolayers. • SNC-PG stimulated both cell proliferation and cell migration. • Interaction between chondroitin sulfate-units and CD44 is responsible for the effect. - Abstract: Proteoglycans (PGs) are involved in various cellular functions including cell growth, adhesion, and differentiation; however, their physiological roles are not fully understood. In this study, we examined the effect of PG purified from salmon nasal cartilage (SNC-PG) on wound closure using tissue-cultured cell monolayers, an in vitro wound-healing assay. The results indicated that SNC-PG significantly promoted wound closure in NIH/3T3 cell monolayers bymore » stimulating both cell proliferation and cell migration. SNC-PG was effective in concentrations from 0.1 to 10 μg/ml, but showed much less effect at higher concentrations (100–1000 μg/ml). The effect of SNC-PG was abolished by chondroitinase ABC, indicating that chondroitin sulfates (CSs), a major component of glycosaminoglycans (GAGs) in SNC-PG, are crucial for the SNC-PG effect. Furthermore, chondroitin 6-sulfate (C-6-S), a major CS of SNC-PG GAGs, could partially reproduce the SNC-PG effect and partially inhibit the binding of SNC-PG to cells, suggesting that SNC-PG exerts its effect through an interaction between the GAGs in SNC-PG and the cell surface. Neutralization by anti-CD44 antibodies or CD44 knockdown abolished SNC-PG binding to the cells and the SNC-PG effect on wound closure. These results suggest that interactions between CS-rich GAG-chains of SNC-PG and CD44 on the cell surface are responsible for the SNC-PG effect on wound closure.« less

Differential diagnosis of periapical cyst using collagen birefringence pattern of the cyst wall

PubMed Central

2017-01-01

Objectives Periapical lesions, including periapical cyst (PC), periapical granuloma (PG), and periapical abscess (PA), are frequently affected by chemical/physical damage during root canal treatment or severe bacterial infection, and thus, the differential diagnosis of periapical lesions may be difficult due to the presence of severe inflammatory reaction. The aim of this study was to make differential diagnosis among PC, PG, and PA under polarizing microscope. Materials and Methods The collagen birefringence patterns of 319 cases of PC (n = 122), PG (n = 158), and PA (n = 39) obtained using a polarizing microscope were compared. In addition, 6 cases of periodontal fibroma (PF) were used as positive controls. Results Collagen birefringence was condensed with a thick, linear band-like pattern in PC, but was short and irregularly scattered in PG, and scarce or absent in PA. PF showed intense collagen birefringence with a short, palisading pattern but no continuous band-like pattern. The linear band-like birefringence in PC was ascribed to pre-existing expansile tensile stress of the cyst wall. Conclusions In this study all PCs (n = 122) were distinguishable from PGs and PAs by their characteristic birefringence, despite the absence of lining epithelium (n = 20). Therefore, the authors suggest that the presence of linear band-like collagen birefringence of the cyst wall aids the diagnostic differentiation of PC from PG and PA. PMID:28503476

Cell-wall preparation containing poly-γ-D-glutamate covalently linked to peptidoglycan, a straightforward extractable molecule, protects mice against experimental anthrax infection.

PubMed

Candela, Thomas; Dumetz, Fabien; Tosi-Couture, Evelyne; Mock, Michèle; Goossens, Pierre L; Fouet, Agnès

2012-12-17

Bacillus anthracis is the causative agent of anthrax that is characterized by septicemia and toxemia. Many vaccine strategies were described to counteract anthrax infection. In contrast with veterinary live vaccines, currently human vaccines are acellular with the protective antigen, a toxin component, as the main constituent. However, in animal models this vaccine is less efficient than the live vaccine. In this study, we analyzed the protection afforded by a single extractable surface element. The poly-γ-D-glutamate capsule is covalently linked to the peptidoglycan. A preparation of peptidoglycan-linked poly-γ-D-glutamate (GluPG) was tested for its immunogenicity and its protective effect. GluPG injection, in mice, elicited the production of specific antibodies directed against poly-glutamate and partially protected the animals against lethal challenges with a non-toxinogenic strain. When combined to protective antigen, GluPG immunization conferred full protection against cutaneous anthrax induced with a fully virulent strain. Copyright © 2012 Elsevier Ltd. All rights reserved.

BcMF26a and BcMF26b Are Duplicated Polygalacturonase Genes with Divergent Expression Patterns and Functions in Pollen Development and Pollen Tube Formation in Brassica campestris

PubMed Central

Lyu, Meiling; Yu, Youjian; Jiang, Jingjing; Song, Limin; Liang, Ying; Ma, Zhiming; Xiong, Xingpeng; Cao, Jiashu

2015-01-01

Polygalacturonase (PG) is one of the cell wall hydrolytic enzymes involving in pectin degradation. A comparison of two highly conserved duplicated PG genes, namely, Brassica campestris Male Fertility 26a (BcMF26a) and BcMF26b, revealed the different features of their expression patterns and functions. We found that these two genes were orthologous genes of At4g33440, and they originated from a chromosomal segmental duplication. Although structurally similar, their regulatory and intron sequences largely diverged. QRT-PCR analysis showed that the expression level of BcMF26b was higher than that of BcMF26a in almost all the tested organs and tissues in Brassica campestris. Promoter activity analysis showed that, at reproductive development stages, BcMF26b promoter was active in tapetum, pollen grains, and pistils, whereas BcMF26a promoter was only active in pistils. In the subcellular localization experiment, BcMF26a and BcMF26b proteins could be localized to the cell wall. When the two genes were co-inhibited, pollen intine was formed abnormally and pollen tubes could not grow or stretch. Moreover, the knockout mutants of At4g33440 delayed the growth of pollen tubes. Therefore, BcMF26a/b can participate in the construction of pollen wall by modulating intine information and BcMF26b may play a major role in co-inhibiting transformed plants. PMID:26153985

Cloning and characterisation of a putative pollen-specific polygalacturonase gene (CpPG1) differentially regulated during pollen development in zucchini (Cucurbita pepo L.).

PubMed

Carvajal, F; Garrido, D; Jamilena, M; Rosales, R

2014-03-01

Studies in zucchini (Cucurbita pepo L. spp. pepo) pollen have been limited to the viability and morphology of the mature pollen grain. The enzyme polygalacturonase (PG) is involved in pollen development and pollination in many species. In this work, we study anther and pollen development of C. pepo and present the cloning and characterisation of a putative PG CpPG1 (Accession no. HQ232488) from pollen cDNA in C. pepo. The predicted protein for CpPG1 has 416 amino acids, with a high homology to other pollen PGs, such as P22 from Oenothera organensis (76%) and PGA3 from Arabidopsis thaliana (73%). CpPG1 belongs to clade C, which comprises PGs expressed in pollen, and presents a 34 amino acid signal peptide for secretion towards the cell wall. DNA-blot analysis revealed that there are at least another two genes that code for PGs in C. pepo. The spatial and temporal accumulation of CpPG1 was studied by semi-quantitative- and qRT-PCR. In addition, mRNA was detected only in anthers, pollen and the rudimentary anthers of bisexual flowers (only present in some zucchini cultivars under certain environmental conditions that trigger anther development in the third whorl of female flowers). However, no expression was detected in cotyledons, stem or fruit. Furthermore, CpPG1 mRNA was accumulated throughout anther development, with the highest expression found in mature pollen. Similarly, exo-PG activity increased from immature anther stages to mature anthers and mature pollen. Overall, these data support the pollen specificity of this gene and suggest an involvement of CpPG1 in pollen development in C. pepo. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

Mechanism for longitudinal growth of rod-shaped bacteria

NASA Astrophysics Data System (ADS)

Taneja, Swadhin; Levitan, Ben; Rutenberg, Andrew

2013-03-01

The peptidoglycan (PG) cell wall along with MreB proteins are major determinants of shape in rod-shaped bacteria. However the mechanism guiding the growth of this elastic network of cross-linked PG (sacculus) that maintains the integrity and shape of the rod-shaped cell remains elusive. We propose that the known anisotropic elasticity and anisotropic loading, due to the shape and turgor pressure, of the sacculus is sufficient to direct small gaps in the sacculus to elongate around the cell, and that subsequent repair leads to longitudinal growth without radial growth. We computationally show in our anisotropically stressed anisotropic elasticity model small gaps can extend stably in the circumferential direction for the known elasticity of the sacculus. We suggest that MreB patches that normally propagate circumferentially, are associated with these gaps and are steered with this common mechanism. This basic picture is unchanged in Gram positive and Gram negative bacteria. We also show that small changes of elastic properties can in fact lead to bi-stable propagation of gaps, both longitudinal and circumferential, that can explain the bi-stability in patch movement observed in ΔmblΔmreb mutants.

Chitosan-immobilized pectinolytics with novel catalytic features and fruit juice clarification potentialities.

PubMed

Irshad, Muhammad; Murtza, Aimen; Zafar, Muddassar; Bhatti, Khizar Hayat; Rehman, Abdul; Anwar, Zahid

2017-11-01

Biological macromolecules are primarily composed of complex polysaccharides that strengthen microbial growth for the production of industrially relevant enzymes. The presence of polysaccharides in the form of the disrupted cell wall and cell materials are among major challenges in the fruit juice industry. The breakdown of such biological macromolecules including cellulose and pectin is vital for the juices processing. In this background, pectinolytic enzymes including polygalacturonase (PG), pectin lyase (PL), and pectin methylesterase (PME) were isolated from Aspergillus ornatus, statistically optimized and purified via ammonium sulfate fractionation (ASF), dialysis, and Sephadex G-100 gel permeation chromatography. After passing through Sephadex G-100 column, PG, PL, and PME were 2.60-fold, 3.30-fold, and 4.52-fold purified with specific activities of 475.2U/mg, 557.1U/mg, and 205.7U/mg. The active PG, PL, and PME, each separately, were surface immobilized using various concentrations of chitosan and dextran polyaldehyde as a macromolecular crosslinking agent. Prior to exploit for juice clarification purposes, various parameters including pH, thermal and Michaelis-Menten kinetic constants of purified and chitosan-immobilized fractions were investigated. A considerable improvement in the pH and thermal profiles was recorded after immobilization. However, the negligible difference between the K m and V max values of purified free and chitosan-immobilized fractions revealed that the conformational flexibility of pectinolytics was retained as such. A significant color and turbidity reductions were recorded after 60min treatment with CTS-PG, followed by CTS-PME, and CTS-PL. It can be concluded that the clarification of apples, mango, peach, and apricot juices was greatly affected by CTS-PG, CTS-PME, and CTS-PL treatments rendering them as potential candidatures for food industry applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Luteinizing hormone induces ovulation via tumor necrosis factor α-dependent increases in prostaglandin F2α in a nonmammalian vertebrate

PubMed Central

Crespo, Diego; Goetz, Frederick W.; Planas, Josep V.

2015-01-01

Ovulation is induced by the preovulatory surge of luteinizing hormone (LH) that acts on the ovary and triggers the rupture of the preovulatory ovarian follicle by stimulating proteolysis and apoptosis in the follicle wall, causing the release of the mature oocyte. The pro-inflammatory cytokine tumor necrosis factor α (TNFα) and prostaglandin (PG) F2α (PGF2α) are involved in the control of ovulation but their role mediating the pro-ovulatory actions of LH is not well established. Here we show that Lh induces PGF2α synthesis through its stimulation of Tnfα production in trout, a primitive teleost fish. Recombinant trout Tnfα (rTnfα) and PGF2α recapitulate the stimulatory in vitro effects of salmon Lh (sLh) on contraction, proteolysis and loss of cell viability in the preovulatory follicle wall and, finally, ovulation. Furthermore, all pro-ovulatory actions of sLh are blocked by inhibition of Tnfα secretion or PG synthesis and all actions of rTnfα are blocked by PG synthesis inhibitors. Therefore, we provide evidence that the Tnfα–dependent increase in PGF2α production is necessary for the pro-ovulatory actions of Lh. The results from this study shed light onto the mechanisms underlying the pro-ovulatory actions of LH in vertebrates and may prove important in clinical assessments of female infertility. PMID:26374476

An epitope-specific DerG-PG70 LEAPS vaccine modulates T cell responses and suppresses arthritis progression in two related murine models of rheumatoid arthritis

PubMed Central

Mikecz, Katalin; Glant, Tibor T.; Markovics, Adrienn; Rosenthal, Kenneth S.; Kurko, Julia; Carambula, Roy E.; Cress, Steve; Steiner, Harold L.; Zimmerman, Daniel H.

2017-01-01

Rheumatoid arthritis (RA) is an autoimmune joint disease maintained by aberrant immune responses involving CD4+ T helper (Th)1 and Th17 cells. In this study, we tested the therapeutic efficacy of Ligand Epitope Antigen Presentation System (LEAPS™) vaccines in two Th1 cell-driven mouse models of RA, cartilage proteoglycan (PG)-induced arthritis (PGIA) and PG G1-domain-induced arthritis (GIA). The immunodominant PG peptide PG70 was attached to a DerG or J immune cell binding peptide, and the DerG-PG70 and J-PG70 LEAPS vaccines were administered to the mice after the onset of PGIA or GIA symptoms. As indicated by significant decreases in visual and histopathological scores of arthritis, the DerG-PG70 vaccine inhibited disease progression in both PGIA and GIA, while the J-PG70 vaccine was ineffective. Splenic CD4+ cells from DerG-PG70-treated mice were diminished in Th1 and Th17 populations but enriched in Th2 and regulatory T (Treg) cells. In vitro spleen cell-secreted and serum cytokines from DerG-PG70-treated mice demonstrated a shift from a pro-inflammatory to an anti-inflammatory/regulatory profile. DerG-PG70 peptide tetramers preferentially bound to CD4+ T-cells of GIA spleen cells. We conclude that the DerG-PG70 vaccine (now designated CEL-4000) exerts its therapeutic effect by interacting with CD4+ cells, which results in an antigen-specific down-modulation of pathogenic T-cell responses in both the PGIA and GIA models of RA. Future studies will need to determine the potential of LEAPS vaccination to provide disease suppression in patients with RA. PMID:28583308

Interaction of graphene family materials with Listeria monocytogenes and Salmonella enterica

NASA Astrophysics Data System (ADS)

Kurantowicz, Natalia; Sawosz, Ewa; Jaworski, Sławomir; Kutwin, Marta; Strojny, Barbara; Wierzbicki, Mateusz; Szeliga, Jacek; Hotowy, Anna; Lipińska, Ludwika; Koziński, Rafał; Jagiełło, Joanna; Chwalibog, André

2015-01-01

Graphene family materials have unique properties, which make them valuable for a range of applications. The antibacterial properties of graphene have been reported; however, findings have been contradictory. This study reports on the antimicrobial proprieties of three different graphene materials (pristine graphene (pG), graphene oxide (GO), and reduced graphene oxide (rGO)) against the food-borne bacterial pathogens Listeria monocytogenes and Salmonella enterica. A high concentration (250 μg/mL) of all the analyzed graphenes completely inhibited the growth of both pathogens, despite their difference in bacterial cell wall structure. At a lower concentration (25 μg/mL), similar effects were only observed with GO, as growth inhibition decreased with pG and rGO at the lower concentration. Interaction of the nanoparticles with the pathogenic bacteria was found to differ depending on the form of graphene. Microscopic imaging demonstrated that bacteria were arranged at the edges of pG and rGO, while with GO, they adhered to the nanoparticle surface. GO was found to have the highest antibacterial activity.

Precision and sensitivity of the measurement of 15N enrichment in D-alanine from bacterial cell walls using positive/negative ion mass spectrometry

NASA Technical Reports Server (NTRS)

Tunlid, A.; Odham, G.; Findlay, R. H.; White, D. C.

1985-01-01

Sensitive detection of cellular components from specific groups of microbes can be utilized as 'signatures' in the examination of microbial consortia from soils, sediments or biofilms. Utilizing capillary gas chromatography/mass spectrometry and stereospecific derivatizing agents, D-alanine, a component localized in the prokaryotic (bacterial) cell wall, can be detected reproducibly. Enrichments of D-[15N]alanine determined in E. coli grown with [15N]ammonia can be determined with precision at 1.0 atom%. Chemical ionization with methane gas and the detection of negative ions (M - HF)- and (M - F or M + H - HF)- formed from the heptafluorobutyryl D-2 butanol ester of D-alanine allowed as little as 8 pg (90 fmol) to be detected reproducibly. This method can be utilized to define the metabolic activity in terms of 15N incorporation at the level of 10(3)-10(4) cells, as a function of the 15N-14N ratio.

Pectin methylesterase activity in vivo differs from activity in vitro and enhances polygalacturonase-mediated pectin degradation in tabasco pepper.

PubMed

Arancibia, Ramón A; Motsenbocker, Carl E

2006-03-01

Polygalacturonase (PG) and pectin methylesterase (PME) activities were analyzed in ripening fruits of two tabasco pepper (Capsicum frutescens) lines that differ in the extent of pectin degradation (depolymerization and dissolution). Ripe 'Easy Pick' fruit is characterized by pectin ultra-degradation and easy fruit detachment from the calyx (deciduous trait), while pectin depolymerization and dissolution in ripe 'Hard Pick' fruit is limited. PG activity in protein extracts increased similarly in both lines during fruit ripening. PME activity in vivo assessed by methanol production, however, was detected only in fruit of the 'Easy Pick' line and was associated with decreased pectin methyl-esterification. In contrast, methanol production in vivo was not detected in fruits of the 'Hard Pick' line and the degree of pectin esterification remained the same throughout ripening. Consequently, a ripening specific PME that is active in vivo appears to enhance PG-mediated pectin ultra-degradation resulting in cell wall dissolution and the deciduous fruit trait. PME activity in vitro, however, was detected in protein extracts from both lines at all ripening stages. This indicates that some PME isozymes are apparently inactive in vivo, particularly in green fruit and throughout ripening in the 'Hard Pick' line, limiting PG-mediated pectin depolymerization which results in moderately difficult fruit separation from the calyx.

T lymphocyte activation and cytokine expression in periapical granulomas and radicular cysts.

PubMed

Ihan Hren, N; Ihan, A

2009-02-01

Radicular cysts (RCs) are periapical lesions resulting in jaw bone destruction. The inflammatory dental periapical granuloma (PG) is considered to be the origin of RC formation; however the mechanism of RC development remains unclear. Cell suspension from the surgically extirpated tissue of 27 RCs and 25 PGs was obtained. Bacteriological analysis of the PG tissue samples was performed in order to define two major groups of PG according to the prevailing causative bacterial infection: the streptococcal PG (PG-S, n=10) and the anaerobe PG (PG-A, n=9) group. The inflammatory response of tissue infiltrating lymphocytes was assessed by following T lymphocyte activation (HLA-DR expression) as well as interferon gamma (IFN-gamma) and interleukin 4 (IL-4) production which were evaluated by the flow cytometry. In comparison to RC both types of PG contained a higher proportion of activated T cells (HLA-DR) and lower proportion of IL-4 producing cells. PG-A tissue contained increased percentage of CD3 cells and increased percentage of T helper 1 (Th1) cells in comparison with PG-S. In RC the IFN-gamma production is higher than in streptococcal PG-S but similar as in PG-A. Tissue infiltration by Th2 cells and IL-4 production is likely to play an etiopathogenic role in RC formation.

Hyperpolarized [U-(2) H, U-(13) C]Glucose reports on glycolytic and pentose phosphate pathway activity in EL4 tumors and glycolytic activity in yeast cells.

PubMed

Timm, Kerstin N; Hartl, Johannes; Keller, Markus A; Hu, De-En; Kettunen, Mikko I; Rodrigues, Tiago B; Ralser, Markus; Brindle, Kevin M

2015-12-01

A resonance at ∼181 ppm in the (13) C spectra of tumors injected with hyperpolarized [U-(2) H, U-(13) C]glucose was assigned to 6-phosphogluconate (6PG), as in previous studies in yeast, whereas in breast cancer cells in vitro this resonance was assigned to 3-phosphoglycerate (3PG). These peak assignments were investigated here using measurements of 6PG and 3PG (13) C-labeling using liquid chromatography tandem mass spectrometry (LC-MS/MS) METHODS: Tumor-bearing mice were injected with (13) C6 glucose and the (13) C-labeled and total 6PG and 3PG concentrations measured. (13) C MR spectra of glucose-6-phosphate dehydrogenase deficient (zwf1Δ) and wild-type yeast were acquired following addition of hyperpolarized [U-(2) H, U-(13) C]glucose and again (13) C-labeled and total 6PG and 3PG were measured by LC-MS/MS RESULTS: Tumor (13) C-6PG was more abundant than (13) C-2PG/3PG and the resonance at ∼181 ppm matched more closely that of 6PG. (13) C MR spectra of wild-type and zwf1Δ yeast cells showed a resonance at ∼181 ppm after labeling with hyperpolarized [U-(2) H, U-(13) C]glucose, however, there was no 6PG in zwf1Δ cells. In the wild-type cells 3PG was approximately four-fold more abundant than 6PG CONCLUSION: The resonance at ∼181 ppm in (13) C MR spectra following injection of hyperpolarized [U-(2) H, U-(13) C]glucose originates predominantly from 6PG in EL4 tumors and 3PG in yeast cells. © 2014 Wiley Periodicals, Inc.

Alterations in Peptidoglycan Cross-Linking Suppress the Secretin Assembly Defect Caused by Mutation of GspA in the Type II Secretion System.

PubMed

Vanderlinde, Elizabeth M; Strozen, Timothy G; Hernández, Sara B; Cava, Felipe; Howard, S Peter

2017-04-15

In Gram-negative bacteria, the peptidoglycan (PG) cell wall is a significant structural barrier for outer membrane protein assembly. In Aeromonas hydrophila , outer membrane multimerization of the type II secretion system (T2SS) secretin ExeD requires the function of the inner membrane assembly factor complex ExeAB. The putative mechanism of the complex involves the reorganization of PG and localization of ExeD, whereby ExeA functions by interacting with PG to form a site for secretin assembly and ExeB forms an interaction with ExeD. This mechanism led us to hypothesize that increasing the pore size of PG would circumvent the requirement for ExeA in the assembly of the ExeD secretin. Growth of A. hydrophila in 270 mM Gly reduced PG cross-links by approximately 30% and led to the suppression of secretin assembly defects in exeA strains and in those expressing ExeA mutants by enabling localization of the secretin in the outer membrane. We also established a heterologous ExeD assembly system in Escherichia coli and showed that ExeAB and ExeC are the only A. hydrophila proteins required for the assembly of the ExeD secretin in E. coli and that ExeAB-independent assembly of ExeD can occur upon overexpression of the d,d-carboxypeptidase PBP 5. These results support an assembly model in which, upon binding to PG, ExeA induces multimerization and pore formation in the sacculus, which enables ExeD monomers to interact with ExeB and assemble into a secretin that both is inserted in the outer membrane and crosses the PG layer to interact with the inner membrane platform of the T2SS. IMPORTANCE The PG layer imposes a strict structural impediment for the assembly of macromolecular structures that span the cell envelope and serve as virulence factors in Gram-negative species. This work revealed that by decreasing PG cross-linking by growth in Gly, the absolute requirement for the PG-binding activity of ExeA in the assembly of the ExeD secretin was alleviated in A. hydrophila In a heterologous assembly model in E. coli , expression of the carboxypeptidase PBP 5 could relieve the requirement for ExeAB in the assembly of the ExeD secretin. These results provide some mechanistic details of the ExeAB assembly complex function, in which the PG-binding and oligomerization functions of ExeAB are used to create a pore in the PG that is required for secretin assembly. Copyright © 2017 American Society for Microbiology.

Temporal and Spatial Expression of a Polygalacturonase during Leaf and Flower Abscission in Oilseed Rape and Arabidopsis1

PubMed Central

González-Carranza, Zinnia Haydé; Whitelaw, Catherine Ann; Swarup, Ranjan; Roberts, Jeremy Alan

2002-01-01

During leaf abscission in oilseed rape (Brassica napus), cell wall degradation is brought about by the action of several hydrolytic enzymes. One of these is thought to be polygalacturonase (PG). Degenerate primers were used to isolate a PG cDNA fragment by reverse transcriptase-polymerase chain reaction from RNA extracted from ethylene-promoted leaf abscission zones (AZs), and in turn a full-length clone (CAW471) from an oilseed rape AZ cDNA library. The highest homology of this cDNA (82%) was to an Arabidopsis sequence that was predicted to encode a PG protein. Analysis of expression revealed that CAW471 mRNA accumulated in the AZ of leaves and reached a peak 24 h after ethylene treatment. Ethylene-promoted leaf abscission in oilseed rape was not apparent until 42 h after exposure to the gas, reaching 50% at 48 h and 100% by 56 h. In floral organ abscission, expression of CAW471 correlated with cell separation. Genomic libraries from oilseed rape and Arabidopsis were screened with CAW471 and the respective genomic clones PGAZBRAN and PGAZAT isolated. Characterization of these PG genes revealed that they had substantial homology within both the coding regions and in the 5′-upstream sequences. Fusion of a 1,476-bp 5′-upstream sequence of PGAZAT to β-glucuronidase or green fluorescent protein and transformation of Arabidopsis revealed that this fragment was sufficient to drive expression of these reporter genes in the AZs at the base of the anther filaments, petals, and sepals. PMID:11842157

An epitope-specific DerG-PG70 LEAPS vaccine modulates T cell responses and suppresses arthritis progression in two related murine models of rheumatoid arthritis.

PubMed

Mikecz, Katalin; Glant, Tibor T; Markovics, Adrienn; Rosenthal, Kenneth S; Kurko, Julia; Carambula, Roy E; Cress, Steve; Steiner, Harold L; Zimmerman, Daniel H

2017-07-13

Rheumatoid arthritis (RA) is an autoimmune joint disease maintained by aberrant immune responses involving CD4+ T helper (Th)1 and Th17 cells. In this study, we tested the therapeutic efficacy of Ligand Epitope Antigen Presentation System (LEAPS™) vaccines in two Th1 cell-driven mouse models of RA, cartilage proteoglycan (PG)-induced arthritis (PGIA) and PG G1-domain-induced arthritis (GIA). The immunodominant PG peptide PG70 was attached to a DerG or J immune cell binding peptide, and the DerG-PG70 and J-PG70 LEAPS vaccines were administered to the mice after the onset of PGIA or GIA symptoms. As indicated by significant decreases in visual and histopathological scores of arthritis, the DerG-PG70 vaccine inhibited disease progression in both PGIA and GIA, while the J-PG70 vaccine was ineffective. Splenic CD4+ cells from DerG-PG70-treated mice were diminished in Th1 and Th17 populations but enriched in Th2 and regulatory T (Treg) cells. In vitro spleen cell-secreted and serum cytokines from DerG-PG70-treated mice demonstrated a shift from a pro-inflammatory to an anti-inflammatory/regulatory profile. DerG-PG70 peptide tetramers preferentially bound to CD4+ T-cells of GIA spleen cells. We conclude that the DerG-PG70 vaccine (now designated CEL-4000) exerts its therapeutic effect by interacting with CD4+ cells, which results in an antigen-specific down-modulation of pathogenic T-cell responses in both the PGIA and GIA models of RA. Future studies will need to determine the potential of LEAPS vaccination to provide disease suppression in patients with RA. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Phytophthora ×stagnum nothosp. nov., a New Hybrid from Irrigation Reservoirs at Ornamental Plant Nurseries in Virginia

PubMed Central

Yang, Xiao; Richardson, Patricia A.; Hong, Chuanxue

2014-01-01

A novel Phytophthora species was frequently recovered from irrigation reservoirs at several ornamental plant production facilities in eastern Virginia. Initial sequencing of the internal transcribed spacer (ITS) region of this species generated unreadable sequences due to continual polymorphic positions. Cloning and sequencing the ITS region as well as sequencing the mitochondrially encoded cytochrome c oxidase 1 and beta-tubulin genes revealed that it is a hybrid between P. taxon PgChlamydo as its paternal parent and an unknown species genetically close to P. mississippiae as its maternal parent. This hybrid has some diagnostic morphological features of P. taxon PgChlamydo and P. mississippiae. It produces catenulate hyphal swellings, characteristic of P. mississippiae, and chlamydospores, typical of P. taxon PgChlamydo. It also produces both ornamented and relatively smooth-walled oogonia. Ornamented oogonia are another important diagnostic character of P. mississippiae. The relatively smooth-walled oogonia may be indicative of oogonial character of P. taxon PgChlamydo. The new hybrid is described here as Phytophthora ×stagnum. PMID:25072374

Ethylene-induced gene expression, enzyme activities, and water soaking in immature and ripe watermelon (Citrullus lanatus) fruit.

PubMed

Karakurt, Yasar; Huber, Donald J

2004-04-01

Watermelon fruit exhibit acute softening and placental-tissue water soaking following short exposure to exogenous ethylene. Experiments were performed to address transcript abundance and activities of cell wall and membrane hydrolases in placental tissue in response to treatment of watermelon fruit with ethylene. Watermelon fruit were harvested at immature and full-ripe stages and exposed to 50 microL L(-1) ethylene for 6 days at 20 degrees C. Ethylene affected the abundance of transcripts for PME (EC 3.2.1.11), and alpha-(EC 3.2.1.22) and beta-GAL (EC 3.2.1.23) but these effects were dependent on fruit maturity and appeared not to be associated with the water-soaking syndrome. PG (EC 3.2.1.15) and EXP mRNAs accumulated significantly in response to ethylene exposure. Additionally, the levels of mRNA and activities of LOX (EC 1.13.11.12), PLC (EC 3.1.4.3) and PLD (EC 3.1.4.4) were elevated in fruit of both maturity classes exposed to ethylene and were temporally associated with the visible symptoms of water soaking. The activity trends and transcript abundance in ethylene- compared with air-treated fruit indicate that PG, EXP, LOX, PLC and PLD levels increase with the onset and development of the water-soaking disorder and support the view that catabolic reactions targeting the membranes and cell-walls contribute to the disorder.

Embryos aggregation improves development and imprinting gene expression in mouse parthenogenesis.

PubMed

Bai, Guang-Yu; Song, Si-Hang; Wang, Zhen-Dong; Shan, Zhi-Yan; Sun, Rui-Zhen; Liu, Chun-Jia; Wu, Yan-Shuang; Li, Tong; Lei, Lei

2016-04-01

Mouse parthenogenetic embryonic stem cells (PgESCs) could be applied to study imprinting genes and are used in cell therapy. Our previous study found that stem cells established by aggregation of two parthenogenetic embryos at 8-cell stage (named as a2 PgESCs) had a higher efficiency than that of PgESCs, and the paternal expressed imprinting genes were observably upregulated. Therefore, we propose that increasing the number of parthenogenetic embryos in aggregation may improve the development of parthenogenetic mouse and imprinting gene expression of PgESCs. To verify this hypothesis, we aggregated four embryos together at the 4-cell stage and cultured to the blastocyst stage (named as 4aPgB). qPCR detection showed that the expression of imprinting genes Igf2, Mest, Snrpn, Igf2r, H19, Gtl2 in 4aPgB were more similar to that of fertilized blastocyst (named as fB) compared to 2aPgB (derived from two 4-cell stage parthenogenetic embryos aggregation) or PgB (single parthenogenetic blastocyst). Post-implantation development of 4aPgB extended to 11 days of gestation. The establishment efficiency of GFP-a4 PgESCs which derived from GFP-4aPgB is 62.5%. Moreover, expression of imprinting genes Igf2, Mest, Snrpn, notably downregulated and approached the level of that in fertilized embryonic stem cells (fESCs). In addition, we acquired a 13.5-day fetus totally derived from GFP-a4 PgESCs with germline contribution by 8-cell under zona pellucida (ZP) injection. In conclusion, four embryos aggregation improves parthenogenetic development, and compensates imprinting genes expression in PgESCs. It implied that a4 PgESCs could serve as a better scientific model applied in translational medicine and imprinting gene study. © 2016 Japanese Society of Developmental Biologists.

Long-pulse production of high current negative ion beam by using actively temperature controlled plasma grid for JT-60SA negative ion source

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kojima, A.; Hanada, M.; Yoshida, M.

2015-04-08

The temperature control system of the large-size plasma grid has been developed to realize the long pulse production of high-current negative ions for JT-60SA. By using this prototype system for the JT-60SA ion source, 15 A negative ions has been sustained for 100 s for the first time, which is three times longer than that obtained in JT-60U. In this system, a high-temperature fluorinated fluid with a high boiling point of 270 degree Celsius is circulated in the cooling channels of the plasma grids (PG) where a cesium (Cs) coverage is formed to enhance the negative ion production. Because themore » PG temperature control had been applied to only 10% of the extraction area previously, the prototype PG with the full extraction area (110 cm × 45 cm) was developed to increase the negative ion current in this time. In the preliminary results of long pulse productions of high-current negative ions at a Cs conditioning phase, the negative ion production was gradually degraded in the last half of 100 s pulse where the temperature of an arc chamber wall was not saturated. From the spectroscopic measurements, it was found that the Cs flux released from the wall might affect to the negative ion production, which implied the wall temperature should be kept low to control the Cs flux to the PG for the long-pulse high-current production. The obtained results of long-pulse production and the PG temperature control method contributes the design of the ITER ion source.« less

Invasive mucinous adenocarcinoma with lepidic-predominant pattern coexisted with tuberculosis: a case report.

PubMed

Xu, Xinxin; Guo, Yinshi; Li, Qiuying; Yang, Ling; Kang, Jianqiang

2018-06-01

We observed a rare case of invasive mucinous adenocarcinoma (IMA) with a lepidic-predominant pattern accompanied by pulmonary tuberculosis. An 85-year-old man with repeated cough and sputum was admitted to Xinhua Hospital. T-SPOT test result was 212 pg/ml (reference value of negative is < 14 pg/ml), Mycobacterium tuberculosis culture was positive, and tuberculin skin test (PPD) was negative (skin induration < 5 mm). The patient was treated with several courses of antibiotics and anti-tuberculosis treatments. Repeated chest CT scans showed disease progression. Bronchoscopy yielded negative results. PET-CT scans showed negative results. A percutaneous lung biopsy revealed mucin-secreting cells lining the alveolar walls. IMA with a lepidic-predominant pattern was diagnosed after invasiveness was found after experimental treatments. Simultaneous occurrence of pulmonary tuberculosis and lung cancer are common; however, the present case of IMA having a lepidic-predominant pattern and coexisting with active tuberculosis has not been reported yet.

Genomic characterization of plant cell wall degrading enzymes and in silico analysis of xylanases and polygalacturonases of Fusarium virguliforme.

PubMed

Chang, Hao-Xun; Yendrek, Craig R; Caetano-Anolles, Gustavo; Hartman, Glen L

2016-07-12

Plant cell wall degrading enzymes (PCWDEs) are a subset of carbohydrate-active enzymes (CAZy) produced by plant pathogens to degrade plant cell walls. To counteract PCWDEs, plants release PCWDEs inhibitor proteins (PIPs) to reduce their impact. Several transgenic plants expressing exogenous PIPs that interact with fungal glycoside hydrolase (GH)11-type xylanases or GH28-type polygalacturonase (PG) have been shown to enhance disease resistance. However, many plant pathogenic Fusarium species were reported to escape PIPs inhibition. Fusarium virguliforme is a soilborne pathogen that causes soybean sudden death syndrome (SDS). Although the genome of F. virguliforme was sequenced, there were limited studies focused on the PCWDEs of F. virguliforme. Our goal was to understand the genomic CAZy structure of F. viguliforme, and determine if exogenous PIPs could be theoretically used in soybean to enhance resistance against F. virguliforme. F. virguliforme produces diverse CAZy to degrade cellulose and pectin, similar to other necrotorphic and hemibiotrophic plant pathogenic fungi. However, some common CAZy of plant pathogenic fungi that catalyze hemicellulose, such as GH29, GH30, GH44, GH54, GH62, and GH67, were deficient in F. virguliforme. While the absence of these CAZy families might be complemented by other hemicellulases, F. virguliforme contained unique families including GH131, polysaccharide lyase (PL) 9, PL20, and PL22 that were not reported in other plant pathogenic fungi or oomycetes. Sequence analysis revealed two GH11 xylanases of F. virguliforme, FvXyn11A and FvXyn11B, have conserved residues that allow xylanase inhibitor protein I (XIP-I) binding. Structural modeling suggested that FvXyn11A and FvXyn11B could be blocked by XIP-I that serves as good candidate for developing transgenic soybeans. In contrast, one GH28 PG, FvPG2, contains an amino acid substitution that is potentially incompatible with the bean polygalacturonase-inhibitor protein II (PvPGIP2). Identification and annotation of CAZy provided advanced understanding of genomic composition of PCWDEs in F. virguliforme. Sequence and structural analyses of FvXyn11A and FvXyn11B suggested both xylanases were conserved in residues that allow XIP-I inhibition, and expression of both xylanases were detected during soybean roots infection. We postulate that a transgenic soybean expressing wheat XIP-I may be useful for developing root rot resistance to F. virguliforme.

Structure-Function Analysis of MurJ Reveals a Solvent-Exposed Cavity Containing Residues Essential for Peptidoglycan Biogenesis in Escherichia coli

PubMed Central

Butler, Emily K.; Davis, Rebecca M.; Bari, Vase; Nicholson, Paul A.

2013-01-01

Gram-negative bacteria such as Escherichia coli build a peptidoglycan (PG) cell wall in their periplasm using the precursor known as lipid II. Lipid II is a large amphipathic molecule composed of undecaprenyl diphosphate and a disaccharide-pentapeptide that PG-synthesizing enzymes use to build the PG sacculus. During PG biosynthesis, lipid II is synthesized at the cytoplasmic face of the inner membrane and then flipped across the membrane. This translocation of lipid II must be assisted by flippases thought to shield the disaccharide-pentapeptide as it crosses the hydrophobic core of the membrane. The inner membrane protein MurJ is essential for PG biogenesis and homologous to known and putative flippases of the MOP (multidrug/oligo-saccharidyl-lipid/polysaccharide) exporter superfamily, which includes flippases that translocate undecaprenyl diphosphate-linked oligosaccharides across the cytoplasmic membranes of bacteria. Consequently, MurJ has been proposed to function as the lipid II flippase in E. coli. Here, we present a three-dimensional structural model of MurJ generated by the I-TASSER server that suggests that MurJ contains a solvent-exposed cavity within the plane of the membrane. Using in vivo topological studies, we demonstrate that MurJ has 14 transmembrane domains and validate features of the MurJ structural model, including the presence of a solvent-exposed cavity within its transmembrane region. Furthermore, we present functional studies demonstrating that specific charged residues localized in the central cavity are essential for function. Together, our studies support the structural homology of MurJ to MOP exporter proteins, suggesting that MurJ might function as an essential transporter in PG biosynthesis. PMID:23935042

Phosphorylation of the Peptidoglycan Synthase PonA1 Governs the Rate of Polar Elongation in Mycobacteria

PubMed Central

Kieser, Karen J.; Baer, Christina E.; Barczak, Amy K.; Meniche, Xavier; Chao, Michael C.; Rego, E. Hesper; Sassetti, Christopher M.; Fortune, Sarah M.; Rubin, Eric J.

2015-01-01

Cell growth and division are required for the progression of bacterial infections. Most rod-shaped bacteria grow by inserting new cell wall along their mid-section. However, mycobacteria, including the human pathogen Mycobacterium tuberculosis, produce new cell wall material at their poles. How mycobacteria control this different mode of growth is incompletely understood. Here we find that PonA1, a penicillin binding protein (PBP) capable of transglycosylation and transpeptidation of cell wall peptidoglycan (PG), is a major governor of polar growth in mycobacteria. PonA1 is required for growth of Mycobacterium smegmatis and is critical for M. tuberculosis during infection. In both cases, PonA1’s catalytic activities are both required for normal cell length, though loss of transglycosylase activity has a more pronounced effect than transpeptidation. Mutations that alter the amount or the activity of PonA1 result in abnormal formation of cell poles and changes in cell length. Moreover, altered PonA1 activity results in dramatic differences in antibiotic susceptibility, suggesting that a balance between the two enzymatic activities of PonA1 is critical for survival. We also find that phosphorylation of a cytoplasmic region of PonA1 is required for normal activity. Mutations in a critical phosphorylated residue affect transglycosylase activity and result in abnormal rates of cell elongation. Together, our data indicate that PonA1 is a central determinant of polar growth in mycobacteria, and its governance of cell elongation is required for robust cell fitness during both host-induced and antibiotic stress. PMID:26114871

Cell wall structures leading to cultivar differences in softening rates develop early during apple (Malus x domestica) fruit growth.

PubMed

Ng, Jovyn K T; Schröder, Roswitha; Sutherland, Paul W; Hallett, Ian C; Hall, Miriam I; Prakash, Roneel; Smith, Bronwen G; Melton, Laurence D; Johnston, Jason W

2013-11-19

There is a paucity of information regarding development of fruit tissue microstructure and changes in the cell walls during fruit growth, and how these developmental processes differ between cultivars with contrasting softening behaviour. In this study we compare two apple cultivars that show different softening rates during fruit development and ripening. We investigate whether these different softening behaviours manifest themselves late during ethylene-induced softening in the ripening phase, or early during fruit expansion and maturation. 'Scifresh' (slow softening) and 'Royal Gala' (rapid softening) apples show differences in cortical microstructure and cell adhesion as early as the cell expansion phase. 'Scifresh' apples showed reduced loss of firmness and greater dry matter accumulation compared with 'Royal Gala' during early fruit development, suggesting differences in resource allocation that influence tissue structural properties. Tricellular junctions in 'Scifresh' were rich in highly-esterified pectin, contributing to stronger cell adhesion and an increased resistance to the development of large airspaces during cell expansion. Consequently, mature fruit of 'Scifresh' showed larger, more angular shaped cells than 'Royal Gala', with less airspaces and denser tissue. Stronger cell adhesion in ripe 'Scifresh' resulted in tissue fracture by cell rupture rather than by cell-to-cell-separation as seen in 'Royal Gala'. CDTA-soluble pectin differed in both cultivars during development, implicating its involvement in cell adhesion. Low pectin methylesterase activity during early stages of fruit development coupled with the lack of immuno-detectable PG was associated with increased cell adhesion in 'Scifresh'. Our results indicate that cell wall structures leading to differences in softening rates of apple fruit develop early during fruit growth and well before the induction of the ripening process.

Cell wall structures leading to cultivar differences in softening rates develop early during apple (Malus x domestica) fruit growth

PubMed Central

2013-01-01

Background There is a paucity of information regarding development of fruit tissue microstructure and changes in the cell walls during fruit growth, and how these developmental processes differ between cultivars with contrasting softening behaviour. In this study we compare two apple cultivars that show different softening rates during fruit development and ripening. We investigate whether these different softening behaviours manifest themselves late during ethylene-induced softening in the ripening phase, or early during fruit expansion and maturation. Results ‘Scifresh’ (slow softening) and ‘Royal Gala’ (rapid softening) apples show differences in cortical microstructure and cell adhesion as early as the cell expansion phase. ‘Scifresh’ apples showed reduced loss of firmness and greater dry matter accumulation compared with ‘Royal Gala’ during early fruit development, suggesting differences in resource allocation that influence tissue structural properties. Tricellular junctions in ‘Scifresh’ were rich in highly-esterified pectin, contributing to stronger cell adhesion and an increased resistance to the development of large airspaces during cell expansion. Consequently, mature fruit of ‘Scifresh’ showed larger, more angular shaped cells than ‘Royal Gala’, with less airspaces and denser tissue. Stronger cell adhesion in ripe ‘Scifresh’ resulted in tissue fracture by cell rupture rather than by cell-to-cell-separation as seen in ‘Royal Gala’. CDTA-soluble pectin differed in both cultivars during development, implicating its involvement in cell adhesion. Low pectin methylesterase activity during early stages of fruit development coupled with the lack of immuno-detectable PG was associated with increased cell adhesion in ‘Scifresh’. Conclusions Our results indicate that cell wall structures leading to differences in softening rates of apple fruit develop early during fruit growth and well before the induction of the ripening process. PMID:24252512

Micro-Computed Tomography Evaluation of ProTaper Next and BioRace Shaping Outcomes in Maxillary First Molar Curved Canals.

PubMed

Pasqualini, Damiano; Alovisi, Mario; Cemenasco, Andrea; Mancini, Lucia; Paolino, Davide Salvatore; Bianchi, Caterina Chiara; Roggia, Andrea; Scotti, Nicola; Berutti, Elio

2015-10-01

The aim of this micro-computed tomography study was to describe the shaping properties of ProGlider/ProTaper Next (PG/PTN) and ScoutRace/BioRace (SR/BR) nickel-titanium rotary systems. Thirty maxillary first permanent molars were selected. Mesiobuccal canals were randomly assigned (n = 15) to PG/PTN or SR/BR groups. Irrigation was performed with 5% NaOCl and 10% EDTA. Specimens were scanned (voxel size, 9.1 μm) for matching volumes and surface areas and post-treatment analyses. Root canal centering ability, canal geometry enlargement, and thickness of dentinal wall at inner curvature were assessed at apical level and point of maximum curvature. Results were analyzed with 4 one-way analyses of variance. Canal centering ability was superior in PG/PTN (P = .006 at apical level, P = .025 at point of maximum curvature). PG/PTN demonstrated a more conservative increase of canal areas (P = .027 at apical level, P = .038 at point of maximum curvature). Centrifugal increase in canal diameters did not significantly differ between groups (P = .65 at apical level, P = .61 at point of maximum curvature). Inner dentinal wall thickness was less reduced with PG/PTN compared with SR/BR, with no statistical differences (P = .23 at point of maximum curvature, P = .89 at apical level). PG/PTN shaping taper ranged between 6% and 7%. Neither system produced significant shaping errors in curved canals. PG/PTN system showed better preservation of canal anatomy. PTN offset section did not influence final preparation taper. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

The Ser/Thr Protein Kinase Protein-Protein Interaction Map of M. tuberculosis.

PubMed

Wu, Fan-Lin; Liu, Yin; Jiang, He-Wei; Luan, Yi-Zhao; Zhang, Hai-Nan; He, Xiang; Xu, Zhao-Wei; Hou, Jing-Li; Ji, Li-Yun; Xie, Zhi; Czajkowsky, Daniel M; Yan, Wei; Deng, Jiao-Yu; Bi, Li-Jun; Zhang, Xian-En; Tao, Sheng-Ce

2017-08-01

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, the leading cause of death among all infectious diseases. There are 11 eukaryotic-like serine/threonine protein kinases (STPKs) in Mtb, which are thought to play pivotal roles in cell growth, signal transduction and pathogenesis. However, their underlying mechanisms of action remain largely uncharacterized. In this study, using a Mtb proteome microarray, we have globally identified the binding proteins in Mtb for all of the STPKs, and constructed the first STPK protein interaction (KPI) map that includes 492 binding proteins and 1,027 interactions. Bioinformatics analysis showed that the interacting proteins reflect diverse functions, including roles in two-component system, transcription, protein degradation, and cell wall integrity. Functional investigations confirmed that PknG regulates cell wall integrity through key components of peptidoglycan (PG) biosynthesis, e.g. MurC. The global STPK-KPIs network constructed here is expected to serve as a rich resource for understanding the key signaling pathways in Mtb, thus facilitating drug development and effective control of Mtb. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

In vitro degradation and cell response of calcium carbonate composite ceramic in comparison with other synthetic bone substitute materials.

PubMed

He, Fupo; Zhang, Jing; Yang, Fanwen; Zhu, Jixiang; Tian, Xiumei; Chen, Xiaoming

2015-05-01

The robust calcium carbonate composite ceramics (CC/PG) can be acquired by fast sintering calcium carbonate at a low temperature (650 °C) using a biocompatible, degradable phosphate-based glass (PG) as sintering agent. In the present study, the in vitro degradation and cell response of CC/PG were assessed and compared with 4 synthetic bone substitute materials, calcium carbonate ceramic (CC), PG, hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) ceramics. The degradation rates in decreasing order were as follows: PG, CC, CC/PG, β-TCP, and HA. The proliferation of rat bone mesenchymal stem cells (rMSCs) cultured on the CC/PG was comparable with that on CC and PG, but inferior to HA and β-TCP. The alkaline phosphatase (ALP) activity of rMSCs on CC/PG was lower than PG, comparable with β-TCP, but higher than HA. The rMSCs on CC/PG and PG had enhanced gene expression in specific osteogenic markers, respectively. Compared to HA and β-TCP, the rMSCs on the CC/PG expressed relatively lower level of collagen I and runt-related transcription factor 2, but showed more considerable expression of osteopontin. Although CC, PG, HA, and β-TCP possessed impressive performances in some specific aspects, they faced extant intrinsic drawbacks in either degradation rate or mechanical strength. Based on considerable compressive strength, moderate degradation rate, good cell response, and being free of obvious shortcoming, the CC/PG is promising as another choice for bone substitute materials. Copyright © 2015 Elsevier B.V. All rights reserved.

Transcriptome Analysis of Cell Wall and NAC Domain Transcription Factor Genes during Elaeis guineensis Fruit Ripening: Evidence for Widespread Conservation within Monocot and Eudicot Lineages

PubMed Central

Tranbarger, Timothy J.; Fooyontphanich, Kim; Roongsattham, Peerapat; Pizot, Maxime; Collin, Myriam; Jantasuriyarat, Chatchawan; Suraninpong, Potjamarn; Tragoonrung, Somvong; Dussert, Stéphane; Verdeil, Jean-Luc; Morcillo, Fabienne

2017-01-01

The oil palm (Elaeis guineensis), a monocotyledonous species in the family Arecaceae, has an extraordinarily oil rich fleshy mesocarp, and presents an original model to examine the ripening processes and regulation in this particular monocot fruit. Histochemical analysis and cell parameter measurements revealed cell wall and middle lamella expansion and degradation during ripening and in response to ethylene. Cell wall related transcript profiles suggest a transition from synthesis to degradation is under transcriptional control during ripening, in particular a switch from cellulose, hemicellulose, and pectin synthesis to hydrolysis and degradation. The data provide evidence for the transcriptional activation of expansin, polygalacturonase, mannosidase, beta-galactosidase, and xyloglucan endotransglucosylase/hydrolase proteins in the ripening oil palm mesocarp, suggesting widespread conservation of these activities during ripening for monocotyledonous and eudicotyledonous fruit types. Profiling of the most abundant oil palm polygalacturonase (EgPG4) and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) transcripts during development and in response to ethylene demonstrated both are sensitive markers of ethylene production and inducible gene expression during mesocarp ripening, and provide evidence for a conserved regulatory module between ethylene and cell wall pectin degradation. A comprehensive analysis of NAC transcription factors confirmed at least 10 transcripts from diverse NAC domain clades are expressed in the mesocarp during ripening, four of which are induced by ethylene treatment, with the two most inducible (EgNAC6 and EgNAC7) phylogenetically similar to the tomato NAC-NOR master-ripening regulator. Overall, the results provide evidence that despite the phylogenetic distance of the oil palm within the family Arecaceae from the most extensively studied monocot banana fruit, it appears ripening of divergent monocot and eudicot fruit lineages are regulated by evolutionarily conserved molecular physiological processes. PMID:28487710

Senescent intervertebral disc cells exhibit perturbed matrix homeostasis phenotype.

PubMed

Ngo, Kevin; Patil, Prashanti; McGowan, Sara J; Niedernhofer, Laura J; Robbins, Paul D; Kang, James; Sowa, Gwendolyn; Vo, Nam

2017-09-01

Aging greatly increases the risk for intervertebral disc degeneration (IDD) as a result of proteoglycan loss due to reduced synthesis and enhanced degradation of the disc matrix proteoglycan (PG). How disc matrix PG homeostasis becomes perturbed with age is not known. The goal of this study is to determine whether cellular senescence is a source of this perturbation. We demonstrated that disc cellular senescence is dramatically increased in the DNA repair-deficient Ercc1 -/Δ mouse model of human progeria. In these accelerated aging mice, increased disc cellular senescence is closely associated with the rapid loss of disc PG. We also directly examine PG homeostasis in oxidative damage-induced senescent human cells using an in vitro cell culture model system. Senescence of human disc cells treated with hydrogen peroxide was confirmed by growth arrest, senescence-associated β-galactosidase activity, γH2AX foci, and acquisition of senescence-associated secretory phenotype. Senescent human disc cells also exhibited perturbed matrix PG homeostasis as evidenced by their decreased capacity to synthesize new matrix PG and enhanced degradation of aggrecan, a major matrix PG. of the disc. Our in vivo and in vitro findings altogether suggest that disc cellular senescence is an important driver of PG matrix homeostatic perturbation and PG loss. Published by Elsevier B.V.

Mast cell activation by group A streptococcal polysaccharide in the rat and its role in experimental arthritis.

PubMed Central

Dalldorf, F. G.; Anderle, S. K.; Brown, R. R.; Schwab, J. H.

1988-01-01

Acute edematous responses were induced in Sprague-Dawley rats by the intravenous injection of group-specific polysaccharide (PS) isolated from group A streptococci. Thirty minutes after the intravenous injection of PS there was marked degranulation of subcutaneous and periarticular mast cells in all 4 feet, carbon particle labeling of adjacent venules, and an 8-fold increase in Evans blue dye content of the extremities. This acute reaction to PS was completely blocked by pretreatment with compound 48/80, but the polyarticular relapsing arthritis following the systemic injection of an arthropathic dose of streptococcal cell wall fragments containing large, covalently bound peptidoglycan-polysaccharide (PG-PS) was not blocked. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:3041843

The effect of harvesting strategy of grass silage on digestion and nutrient supply in dairy cows.

PubMed

Kuoppala, K; Rinne, M; Ahvenjärvi, S; Nousiainen, J; Huhtanen, P

2010-07-01

This study examined the effects of primary growth (PG) and regrowth (RG) timothy-meadow fescue silages harvested at 2 stages of growth on feed intake, cell wall digestion and ruminal passage kinetics in lactating dairy cows. Four dairy cows equipped with rumen cannulas were used in a study designed as a 4 x 4 Latin square with 21-d periods. The experimental silages were offered ad libitum with 8 kg/d of concentrate. Ruminal digestion and passage kinetics were assessed by the rumen evacuation technique. Silages of PG were on average more digestible than RG silages. The concentration of neutral detergent fiber (NDF) and indigestible NDF (iNDF) increased and the concentration of digestible organic matter in dry matter (DM) of silages decreased with advancing maturity in PG and RG. Cows consumed more feed DM, energy, and protein and produced more milk when fed PG diets rather than RG diets. Delaying the harvest decreased DM intake and milk production in PG and RG. There were no differences between PG and RG in rumen pH, ammonia N, or total volatile fatty acid concentrations. The intake of N, omasal canal flow of total nonammonia N and microbial N, excretion of N in feces, and ruminal true digestibility of N were higher for PG than for RG diets. The efficiency of microbial N synthesis was not different between PG and RG. Intake and omasal canal flow of organic matter, NDF, and potentially digestible NDF (pdNDF) were higher in PG than in RG. Whole-diet digestibility of organic matter, NDF, or pdNDF in the rumen or in the total tract was not different between PG and RG despite the higher digestibility of PG silages measured in sheep. Rumen pool sizes of crude protein and iNDF were lower for PG diets, whereas the pool size of pdNDF was higher for PG diets than for RG diets. The rate of passage of iNDF was higher for PG diets than for RG diets, with no difference between them in rate of digestion or passage of pdNDF. The lower milk production in cows fed regrowth grass silages compared with primary growth silages could be attributed to the lower silage DM intake potential. Chemical composition of the silages, rumen fill, digestion and passage kinetics of NDF, or the ratio of protein to energy in absorbed nutrients could not explain the differences in DM intake between silages made from primary and regrowth grass. Copyright (c) 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Renal Sympathetic Denervation System via Intraluminal Ultrasonic Ablation: Therapeutic Intravascular Ultrasound Design and Preclinical Evaluation.

PubMed

Chernin, Gil; Szwarcfiter, Iris; Bausback, Yvonne; Jonas, Michael

2017-05-01

To assess the safety and performance of a nonfocused and nonballooned ultrasonic (US) catheter-based renal sympathetic denervation (RDN) system in normotensive swine. RDN with the therapeutic intravascular US catheter was evaluated in 3 experiments: (i) therapeutic intravascular US RDN vs a control group of untreated animals with follow-up of 30, 45, and 90 days (n = 6; n = 12 renal arteries for each group); (ii) therapeutic intravascular US RDN vs radiofrequency (RF) RDN in the contralateral artery in the same animal (n = 2; n = 4 renal arteries); and (iii) therapeutic intravascular US RDN in a recently stent-implanted renal artery (n = 2; n = 4 renal arteries). In the first experiment, therapeutic intravascular US RDN was safe, without angiographic evidence of dissection or renal artery stenosis. Neuronal tissue vacuolization, nuclei pyknosis, and perineuronal inflammation were evident after RDN, without renal artery wall damage. Norepinephrine levels were significantly lower after therapeutic intravascular US RDN after 30, 45, and 90 days compared with the control group (200.17 pg/mg ± 63.35, 184.75 pg/mg ± 44.51, and 203.43 pg/mg ± 58.54, respectively, vs 342.42 pg/mg ± 79.97). In the second experiment, deeper neuronal ablation penetrance was found with therapeutic intravascular US RDN vs RF RDN (maximal penetrance from endothelium of 7.0 mm vs 3.5 mm, respectively). There was less damage to the artery wall after therapeutic intravascular US RDN than with RF RDN, after which edema and injured endothelium were seen. In the third experiment, denervation inside the stent-implanted segments was feasible without damage to the renal artery wall or stent. The therapeutic intravascular US system performed safely and reduced norepinephrine levels. Deeper penetrance and better preservation of vessel wall were observed with therapeutic intravascular US RDN vs RF RDN. Neuronal ablations were observed in stent-implanted renal arteries. Copyright © 2017 SIR. Published by Elsevier Inc. All rights reserved.

Using inositol as a biocompatible ligand for efficient transgene expression

PubMed Central

Zhang, Lei; Bellis, Susan L; Fan, Yiwen; Wu, Yunkun

2015-01-01

Transgene transfection techniques using cationic polymers such as polyethylenimines (PEIs) and PEI derivatives as gene vectors have shown efficacy, although they also have shortcomings. PEIs have decent DNA-binding capability and good cell internalization performance, but they cannot deliver gene payloads very efficiently to cell nuclei. In this study, three hyperbranched polyglycerol-polyethylenimine (PG6-PEI) polymers conjugated with myo-inositol (INO) molecules were developed. The three resulting PG6-PEI-INO polymers have an increased number of INO ligands per molecule. PG6-PEI-INO 1 had only 14 carboxymethyl INO (CMINO) units per molecule. PG6-PEI-INO 2 had approximately 130 CMINO units per molecule. PG6-PEI-INO 3 had as high as 415 CMINO units approximately. Mixing PG6-PEI-INO polymers with DNA produced compact nanocomposites. We then performed localization studies using fluorescent microscopy. As the number of conjugated inositol ligands increased in PG6-PEI-INO polymers, there was a corresponding increase in accumulation of the polymers within 293T cell nuclei. Transfection performed with spherical 293T cells yielded 82% of EGFP-positive cells when using PG6-PEI-INO 3 as the vehicle. Studies further revealed that extracellular adenosine triphosphate (eATP) can inhibit the transgene efficiency of PG6-PEI-INO polymers, as compared with PEI and PG6-PEI that were not conjugated with inositol. Our work unveiled the possibility of using inositol as an effective ligand for transgene expression. PMID:25926732

Interferon-Gamma and Fas Are Involved in Porphyromonas gingivalis-Induced Apoptosis of Human Extravillous Trophoblast-Derived HTR8/SVneo Cells via Extracellular Signal-Regulated Kinase 1/2 Pathway.

PubMed

Ren, Hongyu; Li, Yuhong; Jiang, Han; Du, Minquan

2016-11-01

A number of studies recently revealed a link between periodontal disease and preterm birth (PTB). PTB can be induced by dental infection with Porphyromonas gingivalis (Pg), a periodontopathic bacterium. This study aims to investigate responses of human extravillous trophoblast-derived HTR8/SVneo cells to Pg infection. Cell apoptosis, cell viability, protein expression, and cytokine production in HTR8 cells were measured via: 1) flow cytometry, 2) CCK-8 assay, 3) western blot, and 4) enzyme-linked immunosorbent assay methods, respectively. Pg decreased cell viability and increased cell apoptosis, active caspase-3 and Fas expression, and interferon-gamma (IFN-γ) secretion in HTR8 cells. Extracellular signal-regulated kinase (ERK) 1/2 inhibitor U0126 and FasL neutralizing antibody NOK1 that blocks FasL/Fas interaction both significantly suppressed Pg-induced apoptosis. U0126 also inhibited IFN-γ secretion and Fas expression close to control levels. Moreover, treatment with recombinant IFN-γ also significantly decreased number of viable HTR8 cells and increased Fas expression, suggesting IFN-γ may play an important role in Pg-induced apoptosis of HTR8 cells, at least partially through regulation of Fas expression. To the best of the authors' knowledge, this is the first study to demonstrate Pg induces IFN-γ secretion, Fas expression, and apoptosis in human extravillous trophoblast-derived HTR8/SVneo cells in an ERK1/2-dependent manner, and IFN-γ (explored by recombinant IFN-γ) and Fas are involved in Pg-induced apoptosis. The finding that Pg infection abnormally regulates inflammation and apoptosis of human trophoblasts may give new insights into the possible link of PTB with maternal periodontal disease and periodontal pathogens.

Digestion of peptidoglycan near the cross-link is necessary for the growth of Bacillus subtilis.

PubMed

Hashimoto, Masayuki; Matsushima, Hiroaki; Suparthana, I Putu; Ogasawara, Hiroshi; Yamamoto, Hiroki; Teng, ChingHao; Sekiguchi, Junichi

2018-03-01

Bacterial cells are covered with peptidoglycan (PG) layer(s), serving as the cellular exoskeleton. The PG sacculus changes its shape during cell growth, and thus both the synthesis and disassembly of PG are important for cell proliferation. In Bacillus subtilis, four dl-endopeptidases (DLEPases; LytE, LytF, CwlO and CwlS) are involved in the maintenance of cell morphology. The lytE cwlO double mutant exhibits synthetic lethality and defective cell elongation, while the lytE lytF cwlS triple mutant exhibits defective cell separation, albeit with septum formation. LytE is involved in both cell separation and elongation. We propose that DLEPases have varied roles in cell separation and elongation. To determine these roles, the catalytic domain of LytE was substituted with another catalytic domain that digests the other bonds in PG. By using the chimeric enzymes, we assessed the suppression of the synthetic lethality by the cell elongation defect and the disruption of chain morphology by the cell separation defect. All the constructed chimeric enzymes suppressed the cell separation defect, restoring the chain morphology. Digestion at any position of PG broke the linkage between two daughter cells, releasing them from each other. However, only d,d-endopeptidases suppressed the lack of DLEPase in the lytE cwlO double mutant. This indicated that the release of tension on the expanding PG sacculus is not the sole essential function of DLEPases. Considering that the structure of the digested PG is important for cell elongation, the digested product might be reused in the growth process in some way.

Influence of prostaglandin analogues on epithelial cell proliferation and xenograft growth.

PubMed Central

Tutton, P. J.; Barkla, D. H.

1980-01-01

The influence of two prostaglandin (PG) analogues, 16,16-dimethyl PG E2 and 16,16-dimethyl PG F2 alpha and of the cyclo-oxygenase inhibitor, flurbiprofen, on epithelial cell proliferation was assessed using a stathmokinetic technique. The epithelia examined were those of the jejunal crypts, the colonic crypts and that of dimethylhydrazine-induced adenocarcinomas of rat colon. The influence of the two prostaglandin analogues, and of flurbiprofen, on the growth of a human colorectal tumour propagated as xenografts in immune-deprived mice was also assessed. The PG E2 analogue transiently inhibited xenograft growth, but was without effect on the mitotic rate in the rat tissues. The PG F2 alpha analogue was also found to inhibit xenograft growth but, unlike the PG E2 analogue, it was found to be a strong inhibitor of cell proliferation in rat colonic tumours, and an accelerator of proliferation in jejunal-crypt cells. The only statistically significant effect of flurbiprofen was to accelerate cell division in the rat colonic tumours. PMID:7362778

Influence of prostaglandin analogues on epithelial cell proliferation and xenograft growth.

PubMed

Tutton, P J; Barkla, D H

1980-01-01

The influence of two prostaglandin (PG) analogues, 16,16-dimethyl PG E2 and 16,16-dimethyl PG F2 alpha and of the cyclo-oxygenase inhibitor, flurbiprofen, on epithelial cell proliferation was assessed using a stathmokinetic technique. The epithelia examined were those of the jejunal crypts, the colonic crypts and that of dimethylhydrazine-induced adenocarcinomas of rat colon. The influence of the two prostaglandin analogues, and of flurbiprofen, on the growth of a human colorectal tumour propagated as xenografts in immune-deprived mice was also assessed. The PG E2 analogue transiently inhibited xenograft growth, but was without effect on the mitotic rate in the rat tissues. The PG F2 alpha analogue was also found to inhibit xenograft growth but, unlike the PG E2 analogue, it was found to be a strong inhibitor of cell proliferation in rat colonic tumours, and an accelerator of proliferation in jejunal-crypt cells. The only statistically significant effect of flurbiprofen was to accelerate cell division in the rat colonic tumours.

Phosphoglycerate Mutase 1 Coordinates Glycolysis and Biosynthesis to Promote Tumor Growth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hitosugi, Taro; Zhou, Lu; Elf, Shannon

2012-11-12

It is unclear how cancer cells coordinate glycolysis and biosynthesis to support rapidly growing tumors. We found that the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1), commonly upregulated in human cancers due to loss of TP53, contributes to biosynthesis regulation partially by controlling intracellular levels of its substrate, 3-phosphoglycerate (3-PG), and product, 2-phosphoglycerate (2-PG). 3-PG binds to and inhibits 6-phosphogluconate dehydrogenase in the oxidative pentose phosphate pathway (PPP), while 2-PG activates 3-phosphoglycerate dehydrogenase to provide feedback control of 3-PG levels. Inhibition of PGAM1 by shRNA or a small molecule inhibitor PGMI-004A results in increased 3-PG and decreased 2-PG levels in cancermore » cells, leading to significantly decreased glycolysis, PPP flux and biosynthesis, as well as attenuated cell proliferation and tumor growth.« less

Amidase Activity of AmiC Controls Cell Separation and Stem Peptide Release and Is Enhanced by NlpD in Neisseria gonorrhoeae.

PubMed

Lenz, Jonathan D; Stohl, Elizabeth A; Robertson, Rosanna M; Hackett, Kathleen T; Fisher, Kathryn; Xiong, Kalia; Lee, Mijoon; Hesek, Dusan; Mobashery, Shahriar; Seifert, H Steven; Davies, Christopher; Dillard, Joseph P

2016-05-13

The human-restricted pathogen Neisseria gonorrhoeae encodes a single N-acetylmuramyl-l-alanine amidase involved in cell separation (AmiC), as compared with three largely redundant cell separation amidases found in Escherichia coli (AmiA, AmiB, and AmiC). Deletion of amiC from N. gonorrhoeae results in severely impaired cell separation and altered peptidoglycan (PG) fragment release, but little else is known about how AmiC functions in gonococci. Here, we demonstrated that gonococcal AmiC can act on macromolecular PG to liberate cross-linked and non-cross-linked peptides indicative of amidase activity, and we provided the first evidence that a cell separation amidase can utilize a small synthetic PG fragment as substrate (GlcNAc-MurNAc(pentapeptide)-GlcNAc-MurNAc(pentapeptide)). An investigation of two residues in the active site of AmiC revealed that Glu-229 is critical for both normal cell separation and the release of PG fragments by gonococci during growth. In contrast, Gln-316 has an autoinhibitory role, and its mutation to lysine resulted in an AmiC with increased enzymatic activity on macromolecular PG and on the synthetic PG derivative. Curiously, the same Q316K mutation that increased AmiC activity also resulted in cell separation and PG fragment release defects, indicating that activation state is not the only factor determining normal AmiC activity. In addition to displaying high basal activity on PG, gonococcal AmiC can utilize metal ions other than the zinc cofactor typically used by cell separation amidases, potentially protecting its ability to function in zinc-limiting environments. Thus gonococcal AmiC has distinct differences from related enzymes, and these studies revealed parameters for how AmiC functions in cell separation and PG fragment release. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Crystallographic and Molecular Dynamics Analysis of Loop Motions Unmasking the Peptidoglycan-Binding Site in Stator Protein MotB of Flagellar Motor

PubMed Central

Nahar, Musammat F.; Buckle, Ashley M.; Roujeinikova, Anna

2011-01-01

Background The C-terminal domain of MotB (MotB-C) shows high sequence similarity to outer membrane protein A and related peptidoglycan (PG)-binding proteins. It is believed to anchor the power-generating MotA/MotB stator unit of the bacterial flagellar motor to the peptidoglycan layer of the cell wall. We previously reported the first crystal structure of this domain and made a puzzling observation that all conserved residues that are thought to be essential for PG recognition are buried and inaccessible in the crystal structure. In this study, we tested a hypothesis that peptidoglycan binding is preceded by, or accompanied by, some structural reorganization that exposes the key conserved residues. Methodology/Principal Findings We determined the structure of a new crystalline form (Form B) of Helicobacter pylori MotB-C. Comparisons with the existing Form A revealed conformational variations in the petal-like loops around the carbohydrate binding site near one end of the β-sheet. These variations are thought to reflect natural flexibility at this site required for insertion into the peptidoglycan mesh. In order to understand the nature of this flexibility we have performed molecular dynamics simulations of the MotB-C dimer. The results are consistent with the crystallographic data and provide evidence that the three loops move in a concerted fashion, exposing conserved MotB residues that have previously been implicated in binding of the peptide moiety of peptidoglycan. Conclusion/Significance Our structural analysis provides a new insight into the mechanism by which MotB inserts into the peptidoglycan mesh, thus anchoring the power-generating complex to the cell wall. PMID:21533052

Imprinted propyl gallate electrochemical sensor based on graphene/single walled carbon nanotubes/sol-gel film.

PubMed

Xu, Guilin; Chi, Yu; Li, Lu; Liu, Shouhua; Kan, Xianwen

2015-06-15

A novel imprinted sol-gel electrochemical sensor for the determination of propyl gallate (PG) was developed based on a composite of graphene and single walled carbon nanotubes (GR-SWCNTs). It was fabricated by stepwise modifying GR-SWCNTs and molecularly imprinted polymers and stored in 0.10 mol L(-1) phosphate buffer solution pH 6.0, which endowed the sensor good sensitivity and selective recognition towards template molecules. The morphology and specific adsorption capacity of the sensor was characterized by scanning electron microscope and electrochemical methods, respectively. Under the optimized conditions, a linear range of the sensor to PG was 8.0 × 10(-8)-2.6 × 10(-3)mo lL(-1) with a limit of detection of 5.0 × 10(-8)mol L(-1) (S/N=3). The sensor exhibited specificity and selectivity towards template molecules as well as excellent reproducibility, regeneration and stability. Furthermore, the sensor could be applied to determine PG in edible oils, instant noodles and cookies with satisfactory results. Copyright © 2015 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsukiyama, Fuyo; Nakai, Yumi; Yoshida, Masataka

Catechins have recently been reported to increase the cellular content of the hypoxia-inducible factor (HIF)-1{alpha} within mammalian cells. These catechins have a gallate moiety as a common structure. We now report that n-propyl gallate (nPG) also increases the HIF-1{alpha} protein in the rat heart-derived H9c2 cells. The increase was dose-dependent and reached a maximum at 2-4 h after the addition of nPG to the cells. nPG did not change the HIF-1{alpha} mRNA level, showing that the increase is a posttranscriptional event. Although nPG did not inhibit the HIF prolyl hydroxylase, gallate, the hydrolysis product of nPG, inhibited the enzyme completelymore » at submillimolar concentrations. Model building studies on the human HIF prolyl hydroxylase 2 showed that the two phenolate oxygen atoms of gallate form a chelate with the active site Fe{sup 2+}, while the carboxyl group of gallate forms a strong ionic/hydrogen bonding interaction with Arg383, explaining why nPG, which has an esterified carboxyl group, is unable to inhibit the hydroxylase. Together with the observation that gallate was detected in the H9c2 cells treated with nPG, these results suggest that nPG incorporated into the cells is hydrolyzed and the released gallate inhibits the HIF prolyl hydroxylase, thereby reducing the HIF degradation rate and increasing the HIF-1{alpha} content.« less

Scavengers modifying the phototoxicity induced by ALA-mediated photodynamic therapy

NASA Astrophysics Data System (ADS)

Casas, Adriana; Perotti, Christian; Fukuda, Haydee; Batlle, Alcira

2001-04-01

The exogenously stimulated formation of intracellularly generated Protoporphyrin IX, a precursor of heme, is becoming one of the fastest developing areas in the field of Photodynamic Therapy (PDT). We have examined the degree of protection of several scavengers, aminoacids and compounds related to glutathione metabolism, to the photodamage induced by 5-aminolevulinic acid (ALA)-mediated PDT, employing the LM2 cell line, derived from a mammary murine adenocarcinoma. We have exposed the cells to different concentrations of the scavengers, 24 before PDT, during PDT, and 19 hr after treatment. We defined the protection grade (PG) as the ratio between cell survival after ALA-PDT treatment in the presence of the protector and cell survival after ALA-PDT treatment. We found that L-tryptophan (PG=8.3 at 2mM), N-acetyl-L-cysteine (PG= 7.9 at 30 mM), L-cysteine (PG=7.81 at 8mM), S-adenosyl-L-methionine (PG= 7.86 at 8mM), melatonin (PG=6.81 at 8mM) and glycine (PG=6.8 at 40 mM) are the best protectors to PDT damage, followed by L-methionine (PG=4.38 at 0.8 mM), mannitol (PG=2.32 at 2 mM) and reduced glutathione (PG=3.41 at 0.8 mM), whereas oxidized glutathione does not exert any protection. The implications of these results in the photodamage induced by ALA-PDT is discussed.

Induction of antibody response in the oral cavity of dogs following intraocular (eye drop) immunization with Porphyromonas gingivalis cell lysate incorporated in pH-sensitive fusogenic polymer-modified liposomes.

PubMed

Shimizu, Yosuke; Iwasaki, Tadashi; Tajima, Tomoko; Yuba, Eiji; Kono, Kenji; Watarai, Shinobu

2017-02-14

Induction of mucosal immune responses against Porphyromonas gingivalis within the oral cavity of dogs was studied by immunizing with pH-sensitive fusogenic polymer (MGluPG)-modified liposome-associated cell lysate. Dogs immunized with P. gingivalis cell lysate-containing MGluPG-modified liposomes by intraocular (eye drop) route displayed significant levels of P. gingivalis cell lysate-specific serum IgG and IgA as well as mucosal IgA antibodies in saliva secretion. Serum and salivary antibodies generated by intraocularly immunized with MGluPG-modified liposome-associated P. gingivalis cell lysate revealed a significant aggregation activity against P. gingivalis, whereas serum and saliva from dogs receiving MGluPG-modified liposomes unentrapping P. gingivalis cell lysate did not show the aggregation activity against P. gingivalis. Furthermore, P. gingivalis-specific antibodies in saliva of immunized dogs inhibited the adherence of P. gingivalis to cultured HeLa cells. More importantly, salivary antibodies induced by intraocular immunization with P. gingivalis cell lysate-containing MGluPG-modified liposomes significantly inhibited the coaggregation of P. gingivalis with Actinomyces naeslundii and the cell damage activity of P. gingivalis against FaDu cells, an oral epithelial cell. These results suggest that intraocularly administered P. gingivalis cell lysate-containing MGluPG-modified liposomes should be an effective mucosal vaccine against P. gingivalis infection in dogs and may be an important tool for the prevention of periodontitis.

Peptidoglycan Synthesis Machinery in Agrobacterium tumefaciens During Unipolar Growth and Cell Division

PubMed Central

Cameron, Todd A.; Anderson-Furgeson, James; Zupan, John R.; Zik, Justin J.

2014-01-01

ABSTRACT The synthesis of peptidoglycan (PG) in bacteria is a crucial process controlling cell shape and vitality. In contrast to bacteria such as Escherichia coli that grow by dispersed lateral insertion of PG, little is known of the processes that direct polar PG synthesis in other bacteria such as the Rhizobiales. To better understand polar growth in the Rhizobiales Agrobacterium tumefaciens, we first surveyed its genome to identify homologs of (~70) well-known PG synthesis components. Since most of the canonical cell elongation components are absent from A. tumefaciens, we made fluorescent protein fusions to other putative PG synthesis components to assay their subcellular localization patterns. The cell division scaffolds FtsZ and FtsA, PBP1a, and a Rhizobiales- and Rhodobacterales-specific l,d-transpeptidase (LDT) all associate with the elongating cell pole. All four proteins also localize to the septum during cell division. Examination of the dimensions of growing cells revealed that new cell compartments gradually increase in width as they grow in length. This increase in cell width is coincident with an expanded region of LDT-mediated PG synthesis activity, as measured directly through incorporation of exogenous d-amino acids. Thus, unipolar growth in the Rhizobiales is surprisingly dynamic and represents a significant departure from the canonical growth mechanism of E. coli and other well-studied bacilli. PMID:24865559

The Role of Ethylene and Cold Temperature in the Regulation of the Apple POLYGALACTURONASE1 Gene and Fruit Softening1[W][OA

PubMed Central

Tacken, Emma; Ireland, Hilary; Gunaseelan, Kularajathevan; Karunairetnam, Sakuntala; Wang, Daisy; Schultz, Keith; Bowen, Judith; Atkinson, Ross G.; Johnston, Jason W.; Putterill, Jo; Hellens, Roger P.; Schaffer, Robert J.

2010-01-01

Fruit softening in apple (Malus × domestica) is associated with an increase in the ripening hormone ethylene. Here, we show that in cv Royal Gala apples that have the ethylene biosynthetic gene ACC OXIDASE1 suppressed, a cold treatment preconditions the apples to soften independently of added ethylene. When a cold treatment is followed by an ethylene treatment, a more rapid softening occurs than in apples that have not had a cold treatment. Apple fruit softening has been associated with the increase in the expression of cell wall hydrolase genes. One such gene, POLYGALACTURONASE1 (PG1), increases in expression both with ethylene and following a cold treatment. Transcriptional regulation of PG1 through the ethylene pathway is likely to be through an ETHYLENE-INSENSITIVE3-like transcription factor, which increases in expression during apple fruit development and transactivates the PG1 promoter in transient assays in the presence of ethylene. A cold-related gene that resembles a COLD BINDING FACTOR (CBF) class of gene also transactivates the PG1 promoter. The transactivation by the CBF-like gene is greatly enhanced by the addition of exogenous ethylene. These observations give a possible molecular mechanism for the cold- and ethylene-regulated control of fruit softening and suggest that either these two pathways act independently and synergistically with each other or cold enhances the ethylene response such that background levels of ethylene in the ethylene-suppressed apples is sufficient to induce fruit softening in apples. PMID:20237022

Prolonged early G1 arrest by selective CDK4/CDK6 inhibition sensitizes myeloma cells to cytotoxic killing through cell cycle–coupled loss of IRF4

PubMed Central

Huang, Xiangao; Di Liberto, Maurizio; Jayabalan, David; Liang, Jun; Ely, Scott; Bretz, Jamieson; Shaffer, Arthur L.; Louie, Tracey; Chen, Isan; Randolph, Sophia; Hahn, William C.; Staudt, Louis M.; Niesvizky, Ruben; Moore, Malcolm A. S.

2012-01-01

Dysregulation of cyclin-dependent kinase 4 (CDK4) and CDK6 by gain of function or loss of inhibition is common in human cancer, including multiple myeloma, but success in targeting CDK with broad-spectrum inhibitors has been modest. By selective and reversible inhibition of CDK4/CDK6, we have developed a strategy to both inhibit proliferation and enhance cytotoxic killing of cancer cells. We show that induction of prolonged early-G1 arrest (pG1) by CDK4/CDK6 inhibition halts gene expression in early-G1 and prevents expression of genes programmed for other cell-cycle phases. Removal of the early-G1 block leads to S-phase synchronization (pG1-S) but fails to completely restore scheduled gene expression. Consequently, the IRF4 protein required to protect myeloma cells from apoptosis is markedly reduced in pG1 and further in pG1-S in response to cytotoxic agents, such as the proteasome inhibitor bortezomib. The coordinated loss of IRF4 and gain of Bim sensitize myeloma tumor cells to bortezomib-induced apoptosis in pG1 in the absence of Noxa and more profoundly in pG1-S in cooperation with Noxa in vitro. Induction of pG1 and pG1-S by reversible CDK4/CDK6 inhibition further augments tumor-specific bortezomib killing in myeloma xenografts. Reversible inhibition of CDK4/CDK6 in sequential combination therapy thus represents a novel mechanism-based cancer therapy. PMID:22718837

Prolonged early G(1) arrest by selective CDK4/CDK6 inhibition sensitizes myeloma cells to cytotoxic killing through cell cycle-coupled loss of IRF4.

PubMed

Huang, Xiangao; Di Liberto, Maurizio; Jayabalan, David; Liang, Jun; Ely, Scott; Bretz, Jamieson; Shaffer, Arthur L; Louie, Tracey; Chen, Isan; Randolph, Sophia; Hahn, William C; Staudt, Louis M; Niesvizky, Ruben; Moore, Malcolm A S; Chen-Kiang, Selina

2012-08-02

Dysregulation of cyclin-dependent kinase 4 (CDK4) and CDK6 by gain of function or loss of inhibition is common in human cancer, including multiple myeloma, but success in targeting CDK with broad-spectrum inhibitors has been modest. By selective and reversible inhibition of CDK4/CDK6, we have developed a strategy to both inhibit proliferation and enhance cytotoxic killing of cancer cells. We show that induction of prolonged early-G(1) arrest (pG1) by CDK4/CDK6 inhibition halts gene expression in early-G(1) and prevents expression of genes programmed for other cell-cycle phases. Removal of the early-G(1) block leads to S-phase synchronization (pG1-S) but fails to completely restore scheduled gene expression. Consequently, the IRF4 protein required to protect myeloma cells from apoptosis is markedly reduced in pG1 and further in pG1-S in response to cytotoxic agents, such as the proteasome inhibitor bortezomib. The coordinated loss of IRF4 and gain of Bim sensitize myeloma tumor cells to bortezomib-induced apoptosis in pG1 in the absence of Noxa and more profoundly in pG1-S in cooperation with Noxa in vitro. Induction of pG1 and pG1-S by reversible CDK4/CDK6 inhibition further augments tumor-specific bortezomib killing in myeloma xenografts. Reversible inhibition of CDK4/CDK6 in sequential combination therapy thus represents a novel mechanism-based cancer therapy.

Anticancer activity of silver nanoparticles from Panax ginseng fresh leaves in human cancer cells.

PubMed

Castro-Aceituno, Verónica; Ahn, Sungeun; Simu, Shakina Yesmin; Singh, Priyanka; Mathiyalagan, Ramya; Lee, Hyun A; Yang, Deok Chun

2016-12-01

The pharmaceutical role of silver nanoparticles has been increased over the last decades, especially those synthesized through herbal medicinal plants, due to their variety of pharmacological importance. Panax ginseng Meyer (P. ginseng) has been widely used as a therapeutic herbal medicine for a long time in cancer treatment. In this study, the cytotoxic and oxidative effect of a novel silver nanoparticles synthesized from P. ginseng fresh leaves (P.g AgNPs) were evaluated in different human cancer cell lines. In addition, the effect of P.g AgNPs on cell migration, apoptosis and the determination of the mechanism involve was determinate by the use of A549 lung cancer cell line. It was found that P.g AgNPs treatment inhibited cell viability and induced oxidative stress in A549, MCF7 and HepG2 cancer cell lines. Likewise, P.g AgNPs treatment inhibited the epidermal growth factor (EGF)-enhanced migration, as well as decreased the mRNA levels and phosphorylation of EGF receptors in A549 cells. Moreover, P.g AgNPs modified the morphology of the cell nucleus and increase apoptosis percentage; this effect was linked to the stimulation of p38 MAPK/p53 pathway. Taken together, our results showed that P.g AgNPs exhibited anti-cancer activity in A549 and the regulation of EGFR/p38 MAPK/p53 pathway might be the possible mechanism of its anti-activity. Further experiments are suggested to determinate the mechanism by which P.g AgNPs induce cytotoxicity and ROS generation in MCF-7 and HepG2 cells. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

The RET p.G533C mutation confers predisposition to multiple endocrine neoplasia type 2A in a Brazilian kindred and is able to induce a malignant phenotype in vitro and in vivo.

PubMed

Oliveira, Mariana N L; Hemerly, Jefferson P; Bastos, André U; Tamanaha, Rosana; Latini, Flavia R M; Camacho, Cléber P; Impellizzeri, Anelise; Maciel, Rui M B; Cerutti, Janete M

2011-09-01

We have previously described a p.G533C substitution in the rearranged during transfection (RET) oncogene in a large family with medullary thyroid carcinoma. Here, we explore the functional transforming potential of RET p.G533C mutation. Plasmids expressing RET mutants (p.G533C and p.C634Y) and RET wild type were stable transfected into a rat thyroid cell line (PCCL3). Biological and biochemical effects of RET p.G533C were investigated both in vitro and in vivo. Moreover, we report the first case of pheochromocytoma among the RET p.G533C-carriers in this Brazilian family and explore the RET mutational status in DNA isolated from pheochromocytoma. Ectopic expression of RET p.G533C and p.C634Y activates RET/MAPK/ERK pathway at similar levels and significantly increased cell proliferation, compared with RET wild type. We additionally show that p.G533C increased cell viability, anchorage-independent growth, and micronuclei formation while reducing apoptosis, hallmarks of the malignant phenotype. RET p.G533C down-regulates the expression of thyroid specific genes in PCCL3. Moreover, RET p.G533C-expressing cells were able to induce liver metastasis in nude mice. Finally, we described two novel RET variants (G548V and S556T) in the DNA isolated from pheochromocytoma while they were absent in the DNA isolated from blood. Our in vitro and in vivo analysis indicates that this mutation confers a malignant phenotype to PCCL3 cells. These findings, in association with the report of first case of pheochromocytoma in the Brazilian kindred, suggest that this noncysteine mutation may be more aggressive than was initially considered.

Comparison of usefulness of N-terminal pro-brain natriuretic peptide as an independent predictor of cardiac function among admission cardiac serum biomarkers in patients with anterior wall versus nonanterior wall ST-segment elevation myocardial infarction undergoing primary percutaneous coronary intervention.

PubMed

Haeck, Joost D E; Verouden, Niels J W; Kuijt, Wichert J; Koch, Karel T; Van Straalen, Jan P; Fischer, Johan; Groenink, Maarten; Bilodeau, Luc; Tijssen, Jan G P; Krucoff, Mitchell W; De Winter, Robbert J

2010-04-15

The purpose of the present study was to determine the prognostic value of N-terminal pro-brain natriuretic peptide (NT-pro-BNP), among other serum biomarkers, on cardiac magnetic resonance (CMR) imaging parameters of cardiac function and infarct size in patients with ST-segment elevation myocardial infarction undergoing primary percutaneous coronary intervention. We measured NT-pro-BNP, cardiac troponin T, creatinine kinase-MB fraction, high-sensitivity C-reactive protein, and creatinine on the patients' arrival at the catheterization laboratory in 206 patients with ST-segment elevation myocardial infarction. The NT-pro-BNP levels were divided into quartiles and correlated with left ventricular function and infarct size measured by CMR imaging at 4 to 6 months. Compared to the lower quartiles, patients with nonanterior wall myocardial infarction in the highest quartile of NT-pro-BNP (> or = 260 pg/ml) more often had a greater left ventricular end-systolic volume (68 vs 39 ml/m(2), p <0.001), a lower left ventricular ejection fraction (42% vs 54%, p <0.001), a larger infarct size (9 vs 4 g/m(2), p = 0.002), and a larger number of transmural segments (11% of segments vs 3% of segments, p <0.001). Multivariate analysis revealed that a NT-pro-BNP level of > or = 260 pg/ml was the strongest independent predictor of left ventricular ejection fraction in patients with nonanterior wall myocardial infarction compared to the other serum biomarkers (beta = -5.8; p = 0.019). In conclusion, in patients with nonanterior wall myocardial infarction undergoing primary percutaneous coronary intervention, an admission NT-pro-BNP level of > or = 260 pg/ml was a strong, independent predictor of left ventricular function assessed by CMR imaging at follow-up. Our findings suggest that NT-pro-BNP, a widely available biomarker, might be helpful in the early risk stratification of patients with nonanterior wall myocardial infarction. Copyright 2010 Elsevier Inc. All rights reserved.

Interaction of Mycoplasma hominis PG21 with Human Dendritic Cells: Interleukin-23-Inducing Mycoplasmal Lipoproteins and Inflammasome Activation of the Cell.

PubMed

Goret, J; Béven, L; Faustin, B; Contin-Bordes, C; Le Roy, C; Claverol, S; Renaudin, H; Bébéar, C; Pereyre, S

2017-08-01

Mycoplasma hominis lacks a cell wall, and lipoproteins anchored to the extracellular side of the plasma membrane are in direct contact with the host components. A Triton X-114 extract of M. hominis enriched with lipoproteins was shown to stimulate the production of interleukin-23 (IL-23) by human dendritic cells (hDCs). The inflammasome activation of the host cell has never been reported upon M. hominis infection. We studied here the interaction between M. hominis PG21 and hDCs by analyzing both the inflammation-inducing mycoplasmal lipoproteins and the inflammasome activation of the host cell. IL-23-inducing lipoproteins were determined using a sequential extraction strategy with two nondenaturing detergents, Sarkosyl and Triton X-114, followed by SDS-PAGE separation and mass spectrometry identification. The activation of the hDC inflammasome was assessed using PCR array and enzyme-linked immunosorbent assay (ELISA). We defined a list of 24 lipoproteins that could induce the secretion of IL-23 by hDCs, 5 with a molecular mass between 20 and 35 kDa and 19 with a molecular mass between 40 and 100 kDa. Among them, lipoprotein MHO_4720 was identified as potentially bioactive, and a synthetic lipopeptide corresponding to the N-terminal part of the lipoprotein was subsequently shown to induce IL-23 release by hDCs. Regarding the hDC innate immune response, inflammasome activation with caspase-dependent production of IL-1β was observed. After 24 h of coincubation of hDCs with M. homini s, downregulation of the NLRP3-encoding gene and of the adaptor PYCARD-encoding gene was noticed. Overall, this study provides insight into both protagonists of the interaction of M. hominis and hDCs. IMPORTANCE Mycoplasma hominis is a human urogenital pathogen involved in gynecologic and opportunistic infections. M. hominis lacks a cell wall, and its membrane contains many lipoproteins that are anchored to the extracellular side of the plasma membrane. In the present study, we focused on the interaction between M. hominis and human dendritic cells and examined both sides of the interaction, the mycoplasmal lipoproteins involved in the activation of the host cell and the immune response of the cell. On the mycoplasmal side, we showed for the first time that M. hominis lipoproteins with high molecular mass were potentially bioactive. On the cell side, we reported an activation of the inflammasome, which is involved in the innate immune response. Copyright © 2017 American Society for Microbiology.

PG545 enhances anti-cancer activity of chemotherapy in ovarian models and increases surrogate biomarkers such as VEGF in preclinical and clinical plasma samples.

PubMed

Winterhoff, Boris; Freyer, Luisa; Hammond, Edward; Giri, Shailendra; Mondal, Susmita; Roy, Debarshi; Teoman, Attila; Mullany, Sally A; Hoffmann, Robert; von Bismarck, Antonia; Chien, Jeremy; Block, Matthew S; Millward, Michael; Bampton, Darryn; Dredge, Keith; Shridhar, Viji

2015-05-01

Despite the utility of antiangiogenic drugs in ovarian cancer, efficacy remains limited due to resistance linked to alternate angiogenic pathways and metastasis. Therefore, we investigated PG545, an anti-angiogenic and anti-metastatic agent which is currently in Phase I clinical trials, using preclinical models of ovarian cancer. PG545's anti-cancer activity was investigated in vitro and in vivo as a single agent, and in combination with paclitaxel, cisplatin or carboplatin using various ovarian cancer cell lines and tumour models. PG545, alone, or in combination with chemotherapeutics, inhibited proliferation of ovarian cancer cells, demonstrating synergy with paclitaxel in A2780 cells. PG545 inhibited growth factor-mediated cell migration and reduced HB-EGF-induced phosphorylation of ERK, AKT and EGFR in vitro and significantly reduced tumour burden which was enhanced when combined with paclitaxel in an A2780 model or carboplatin in a SKOV-3 model. Moreover, in the immunocompetent ID8 model, PG545 also significantly reduced ascites in vivo. In the A2780 maintenance model, PG545 initiated with, and following paclitaxel and cisplatin treatment, significantly improved overall survival. PG545 increased plasma VEGF levels (and other targets) in preclinical models and in a small cohort of advanced cancer patients which might represent a potential biomarker of response. Our results support clinical testing of PG545, particularly in combination with paclitaxel, as a novel therapeutic strategy for ovarian cancer. Copyright © 2015 Elsevier Ltd. All rights reserved.

Hydrogen storage capacity on Ti-decorated porous graphene: First-principles investigation

NASA Astrophysics Data System (ADS)

Yuan, Lihua; Kang, Long; Chen, Yuhong; Wang, Daobin; Gong, Jijun; Wang, Chunni; Zhang, Meiling; Wu, Xiaojuan

2018-03-01

Hydrogen storage capacity on Titanium (Ti) decorated porous graphene (PG) has been investigated using density functional theory simulations with generalized gradient approximation method. The possible adsorption sites of Ti atom on PG and electronic properties of Ti-PG system are also discussed.The results show a Ti atom prefers to strongly adsorb on the center site above the C hexagon with the binding energy of 3.65 eV, and the polarization and the hybridization mechanisms both contribute to the Ti atom adsorption on PG. To avoid a tendency of clustering among Ti atoms, the single side of the PG unit cell should only contain one Ti atom. For the single side of PG, four H2 molecules can be adsorbed around Ti atom, and the adsorption mechanism of H2 molecules come from not only the polarization mechanism between Ti and H atoms but also the orbital hybridization among Ti atom, H2 molecules and C atoms. For the case of double sides of PG, eight H2 molecules can be adsorbed on Ti-decorated PG unit cell with the average adsorption energy of -0.457 eV, and the gravimetric hydrogen storage capacity is 6.11 wt.%. Furthermore, ab inito molecular-dynaics simulation result shows that six H2 molecules can be adsorbed on double sides of unit cell of Ti-PG system and the configuration of Ti-PG is very stable at 300 K and without external pressure, which indicates Ti-decorated PG could be considered as a potential hydrogen storage medium at ambient conditions.

A systems analysis of the chemosensitivity of breast cancer cells to the polyamine analogue PG-11047

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuo, Wen-Lin; Das, Debopriya; Ziyad, Safiyyah

2009-11-14

Polyamines regulate important cellular functions and polyamine dysregulation frequently occurs in cancer. The objective of this study was to use a systems approach to study the relative effects of PG-11047, a polyamine analogue, across breast cancer cells derived from different patients and to identify genetic markers associated with differential cytotoxicity. A panel of 48 breast cell lines that mirror many transcriptional and genomic features present in primary human breast tumours were used to study the antiproliferative activity of PG-11047. Sensitive cell lines were further examined for cell cycle distribution and apoptotic response. Cell line responses, quantified by the GI50 (dosemore » required for 50% relative growth inhibition) were correlated with the omic profiles of the cell lines to identify markers that predict response and cellular functions associated with drug sensitivity. The concentrations of PG-11047 needed to inhibit growth of members of the panel of breast cell lines varied over a wide range, with basal-like cell lines being inhibited at lower concentrations than the luminal cell lines. Sensitive cell lines showed a significant decrease in S phase fraction at doses that produced little apoptosis. Correlation of the GI50 values with the omic profiles of the cell lines identified genomic, transcriptional and proteomic variables associated with response. A 13-gene transcriptional marker set was developed as a predictor of response to PG-11047 that warrants clinical evaluation. Analyses of the pathways, networks and genes associated with response to PG-11047 suggest that response may be influenced by interferon signaling and differential inhibition of aspects of motility and epithelial to mesenchymal transition.« less

New insight into artifactual phenomena during in vitro toxicity assessment of engineered nanoparticles: study of TNF-α adsorption on alumina oxide nanoparticle.

PubMed

Pailleux, Mélanie; Boudard, Delphine; Pourchez, Jérémie; Forest, Valérie; Grosseau, Philippe; Cottier, Michèle

2013-04-01

Biomolecules can be adsorbed on nanoparticles (NPs) and degraded during in vitro toxicity assays. These artifactual phenomena could lead to misinterpretation of biological activity, such as false-negative results. To avoid possible underestimation of cytokine release after contact between NP and cells, we propose a methodology to account for these artifactual phenomena and lead to accurate measurements. We focused on the pro-inflammatory cytokine tumor necrosis factor TNF-α. We studied well-characterized boehmite engineered NP [aluminum oxide hydroxide, AlO(OH)]. The rate of TNF-α degradation and its adsorption (on boehmite and on the walls of wells) were determined in cell-free conditions by adding a known TNF-α concentration (1500 pg/ml) under various experimental conditions. After a 24-h incubation, we quantified that 7 wt.% of the initial TNF-α was degraded over time, 6 wt.% adsorbed on the walls of 96-well plates, and 13 wt.% adsorbed on the boehmite surface. Finally, boehmite NP were incubated with murine macrophages (RAW 264.7 cell line). The release of TNF-α was assessed for boehmite NP and the experimental data were corrected considering the artifactual phenomena, which accounted for about 20-30% of the total. Copyright © 2013 Elsevier Ltd. All rights reserved.

White blood cell count may identify abnormal cardiometabolic phenotype and preclinical organ damage in overweight/obese children.

PubMed

Di Bonito, P; Pacifico, L; Chiesa, C; Invitti, C; Miraglia Del Giudice, E; Baroni, M G; Moio, N; Pellegrin, M C; Tomat, M; Licenziati, M R; Manco, M; Maffeis, C; Valerio, G

2016-06-01

Subclinical inflammation is a central component of cardiometabolic disease risk in obese subjects. The aim of the study was to evaluate whether the white blood cell count (WBCc) may help to identify an abnormal cardiometabolic phenotype in overweight (Ow) or obese (Ob) children. A cross-sectional sample of 2835 Ow/Ob children and adolescents (age 6-18 years) was recruited from 10 Italian centers for the care of obesity. Anthropometric and biochemical variables were assessed in the overall sample. Waist to height ratio (WhtR), alanine aminotransferase (ALT), lipids, 2 h post-load plasma glucose (2hPG), left ventricular (LV) geometry and carotid intima-media thickness (cIMT) were assessed in 2128, 2300, 1834, 535 and 315 children, respectively. Insulin resistance and whole body insulin sensitivity index (WBISI) were analyzed using homeostatic model assessment (HOMA-IR) and Matsuda's test. Groups divided in quartiles of WBCc significantly differed for body mass index, WhtR, 2hPG, HOMA-IR, WBISI, lipids, ALT, cIMT, LV mass and relative wall thickness. Children with high WBCc (≥8700 cell/mm(3)) showed a 1.3-2.5 fold increased probability of having high normal 2hPG, high ALT, high cIMT, or LV remodeling/concentric LV hypertrophy, after adjustment for age, gender, pubertal status, BMI and centers. This study shows that WBCc is associated with early derangements of glucose metabolism and preclinical signs of liver, vascular and cardiac damage. The WBCc may be an effective and low-cost tool for identifying Ow and Ob children at the greatest risk of potential complications. Copyright © 2016 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.

Effect of oridonin-mediated hallmark changes on inflammatory pathways in human pancreatic cancer (BxPC-3) cells.

PubMed

Chen, Ru-Yi; Xu, Bin; Chen, Su-Feng; Chen, Si-Si; Zhang, Ting; Ren, Jun; Xu, Jian

2014-10-28

To investigate the effect of oridonin on nuclear transcription factors and to study the relationship between biological behavior and inflammatory factors in human pancreatic cancer (BxPC-3) cells. BxPC-3 cells were treated with various concentrations of oridonin, and viability curves were generated to test for inhibitory effects of the drug on cells. The expression of cytokines such as interleukin-1β (IL-1β), IL-6, or IL-33 was detected in BxPC-3 cell supernatants using an enzyme-linked immunosorbent assay (ELISA), and the protein expression of nuclear transcription factors including nuclear factor κB, activating protein-1, signal transducer and activator of transcription 3, bone morphogenetic protein 2, transforming growth factor β1 and sma and mad homologues in BxPC-3 cells was detected using Western blot. Carcinoma hallmark-related proteins such as survivin, vascular endothelial growth factor, and matrix metallopeptidase 2 were also detected using immunoblotting, and intra-nuclear IL-33 expression was detected using immunofluorescent staining. Treatment with oridonin reduced the viability of BxPC-3 cells in a dose dependent manner. The cells exhibited reduced growth following treatment with 8 μg/mL oridonin (13.05% ± 3.21%, P < 0.01), and the highest inhibitory ratio was 90.64% ± 0.70%, which was achieved with oridonin at a dose of 32 μg/mL. The IC50 value of oridonin in BxPC-3 cells was 19.32 μg/mL. ELISA analysis revealed that oridonin down-regulated the inflammatory factors IL-1β, IL-6, and IL-33 in a dose-dependent manner. IL-1β expression was significantly reduced in the 16 and 32 μg/mL treatment groups compared to the control group (12.97 ± 0.45 pg/mL, 11.17 ± 0.63 pg/mL vs 14.40 ± 0.38 pg/mL, P < 0.01). Similar trends were observed for IL-6 expression, which was significantly reduced in the 16 and 32 μg/mL treatment groups compared to the control group (4.05 ± 0.14 pg/mL vs 4.45 ± 0.43 pg/mL, P < 0.05; 3.95 ± 0.13 pg/mL vs 4.45 ± 0.43 pg/mL, P < 0.01). IL-33 expression was significantly reduced in the 8, 16, and 32 μg/mL treatment groups compared to the control group (911.05 ± 14.18 pg/mL vs 945.25 ± 12.09 pg/mL, P < 0.05; 802.70 ± 11.88 pg/mL, 768.54 ± 10.98 pg/mL vs 945.25 ± 12.09 pg/mL, P < 0.01). Western blot and immunofluorescent staining analyses suggested that oridonin changed the hallmarks and regulated the expression of various nuclear transcription factors. The results obtained suggest that oridonin alters the hallmarks of pancreatic cancer cells through the regulation of nuclear transcription factors.

Dynamic FtsA and FtsZ localization and outer membrane alterations during polar growth and cell division in Agrobacterium tumefaciens

PubMed Central

Zupan, John R.; Cameron, Todd A.; Anderson-Furgeson, James; Zambryski, Patricia C.

2013-01-01

Growth and cell division in rod-shaped bacteria have been primarily studied in species that grow predominantly by peptidoglycan (PG) synthesis along the length of the cell. Rhizobiales species, however, predominantly grow by PG synthesis at a single pole. Here we characterize the dynamic localization of several Agrobacterium tumefaciens components during the cell cycle. First, the lipophilic dye FM 4-64 predominantly stains the outer membranes of old poles versus growing poles. In cells about to divide, however, both poles are equally labeled with FM 4-64, but the constriction site is not. Second, the cell-division protein FtsA alternates from unipolar foci in the shortest cells to unipolar and midcell localization in cells of intermediate length, to strictly midcell localization in the longest cells undergoing septation. Third, the cell division protein FtsZ localizes in a cell-cycle pattern similar to, but more complex than, FtsA. Finally, because PG synthesis is spatially and temporally regulated during the cell cycle, we treated cells with sublethal concentrations of carbenicillin (Cb) to assess the role of penicillin-binding proteins in growth and cell division. Cb-treated cells formed midcell circumferential bulges, suggesting that interrupted PG synthesis destabilizes the septum. Midcell bulges contained bands or foci of FtsA-GFP and FtsZ-GFP and no FM 4-64 label, as in untreated cells. There were no abnormal morphologies at the growth poles in Cb-treated cells, suggesting unipolar growth uses Cb-insensitive PG synthesis enzymes. PMID:23674672

PG1058 Is a Novel Multidomain Protein Component of the Bacterial Type IX Secretion System

PubMed Central

Veith, Paul D.; Butler, Catherine A.; Nor Muhammad, Nor A.; Chen, Yu-Yen; Slakeski, Nada; Peng, Benjamin; Zhang, Lianyi; Dashper, Stuart G.; Cross, Keith J.; Cleal, Steven M.; Moore, Caroline; Reynolds, Eric C.

2016-01-01

Porphyromonas gingivalis utilises the Bacteroidetes-specific type IX secretion system (T9SS) to export proteins across the outer membrane (OM), including virulence factors such as the gingipains. The secreted proteins have a conserved carboxy-terminal domain essential for type IX secretion that is cleaved upon export. In P. gingivalis the T9SS substrates undergo glycosylation with anionic lipopolysaccharide (A-LPS) and are attached to the OM. In this study, comparative analyses of 24 Bacteroidetes genomes identified ten putative novel components of the T9SS in P. gingivalis, one of which was PG1058. Computer modelling of the PG1058 structure predicted a novel N- to C-terminal architecture comprising a tetratricopeptide repeat (TPR) domain, a β-propeller domain, a carboxypeptidase regulatory domain-like fold (CRD) and an OmpA_C-like putative peptidoglycan binding domain. Inactivation of pg1058 in P. gingivalis resulted in loss of both colonial pigmentation and surface-associated proteolytic activity; a phenotype common to T9SS mutants. Immunoblot and LC-MS/MS analyses of subcellular fractions revealed T9SS substrates accumulated within the pg1058 mutant periplasm whilst whole-cell ELISA showed the Kgp gingipain was absent from the cell surface, confirming perturbed T9SS function. Immunoblot, TEM and whole-cell ELISA analyses indicated A-LPS was produced and present on the pg1058 mutant cell surface although it was not linked to T9SS substrate proteins. This indicated that PG1058 is crucial for export of T9SS substrates but not for the translocation of A-LPS. PG1058 is a predicted lipoprotein and was localised to the periplasmic side of the OM using whole-cell ELISA, immunoblot and LC-MS/MS analyses of subcellular fractions. The structural prediction and localisation of PG1058 suggests that it may have a role as an essential scaffold linking the periplasmic and OM components of the T9SS. PMID:27711252

Novel anticancer activity of phloroglucinol against breast cancer stem-like cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Rae-Kwon; Uddin, Nizam; Hyun, Jin-Won

Poor prognosis of breast cancer patients is closely associated with metastasis and relapse. There is substantial evidence supporting that cancer stem-like cells (CSCs) are primarily responsible for relapse in breast cancer after anticancer treatment. However, there is a lack of suitable drugs that target breast cancer stem-like cells (BCSCs). Here, we report that phloroglucinol (PG), a natural phlorotannin component of brown algae, suppresses sphere formation, anchorage-independent colony formation and in vivo tumorigenicity. In line with these observations, treatment with PG also decreased CD44{sup +} cancer cell population as well as expression of CSC regulators such as Sox2, CD44, Oct4, Notch2more » and β-catenin. Also, treatment with PG sensitized breast cancer cells to anticancer drugs such as cisplatin, etoposide, and taxol as well as to ionizing radiation. Importantly, PG inhibited KRAS and its downstream PI3K/AKT and RAF-1/ERK signaling pathways that regulate the maintenance of CSCs. Taken together, our findings implicate PG as a good candidate to target BCSCs and to prevent the disease relapse. - Highlights: • Phloroglucinol suppresses in vivo tumor formation. • Phloroglucinol sensitizes breast cancer cells to anticancer agents. • Phloroglucinol inhibits breast cancer stem-like cells. • Phloroglucinol inhibits PI3K/AKT and KRAS/RAF/ERK signaling pathways.« less

Toll-Like Receptor 4 Expression in the Epithelium of Inflammatory Periapical Lesions.

PubMed Central

Leonardi, R.; Perrotta, R.E.; Musumeci, G.; Crimi, S.; dos Santos, J.N.; Rusu, M.C.; Bufo, P.; Barbato, E.; Pannone, G.

2015-01-01

Toll-like receptors (TLR) are essential for the innate immune response against invading pathogens and have been described in immunocompetent cells of areas affected by periapical disease. Besides initiating the inflammatory response, they also directly regulate epithelial cell proliferation and survival in a variety of settings. This study evaluates the in situ expression of TLR4 in periapical granulomas (PG) and radicular cysts, focusing on the epithelial compartment. Twenty-one periapical cysts (PC) and 10 PG were analyzed; 7 dentigerous non-inflamed follicular cyst (DC) served as control. TLR4 expression was assessed by immunohistochemistry. TLR4 immunoreaction products were detected in the epithelium of all specimens, with a higher percentage of immunostained cells in PG. Although TLR4 overexpression was detected in both PG and PC, there were differences that seemed to be related to the nature of the lesion, since in PG all epithelial cells of strands, islands and trabeculae were strongly immunoreactive for TLR4, whereas in PC only some areas of the basal and suprabasal epithelial layers were immunostained. This staining pattern is consistent with the action of TLR4: in PG it could promote formation of epithelial cell rests of Malassez and in epithelial strands and islands the enhancement of cell survival, proliferation and migration, whereas in PC TLR4 could protect the lining epithelium from extensive apoptosis. These findings go some way towards answering the intriguing question of why many epithelial strands or islands in PG and the lining epithelium of apical cysts regress after non-surgical endodontic therapy, and suggest that TLR4 plays a key role in the pathobiology of the inflammatory process related to periapical disease. PMID:26708181

Toll-like receptor 4 expression in the epithelium of inflammatory periapical lesions. An immunohistochemical study.

PubMed

Leonardi, R; Perrotta, R E; Loreto, C; Musumeci, G; Crimi, S; Dos Santos, J N; Rusu, M C; Bufo, P; Barbato, E; Pannone, G

2015-10-26

Toll-like receptors (TLR) are essential for the innate immune response against invading pathogens and have been described in immunocompetent cells of areas affected by periapical disease. Besides initiating the inflammatory response, they also directly regulate epithelial cell proliferation and survival in a variety of settings. This study evaluates the in situ expression of TLR4 in periapical granulomas (PG) and radicular cysts, focusing on the epithelial compartment. Twenty-one periapical cysts (PC) and 10 PG were analyzed; 7 dentigerous non-inflamed follicular cyst (DC) served as control. TLR4 expression was assessed by immunohistochemistry. TLR4 immunoreaction products were detected in the epithelium of all specimens, with a higher percentage of immunostained cells in PG. Although TLR4 overexpression was detected in both PG and PC, there were differences that seemed to be related to the nature of the lesion, since in PG all epithelial cells of strands, islands and trabeculae were strongly immunoreactive for TLR4, whereas in PC only some areas of the basal and suprabasal epithelial layers were immunostained. This staining pattern is consistent with the action of TLR4: in PG it could promote formation of epithelial cell rests of Malassez and in epithelial strands and islands the enhancement of cell survival, proliferation and migration, whereas in PC TLR4 could protect the lining epithelium from extensive apoptosis. These findings go some way towards answering the intriguing question of why many epithelial strands or islands in PG and the lining epithelium of apical cysts regress after non-surgical endodontic therapy, and suggest that TLR4 plays a key role in the pathobiology of the inflammatory process related to periapical disease.

A new fluorescent dye accumulation assay for parallel measurements of the ABCG2, ABCB1 and ABCC1 multidrug transporter functions.

PubMed

Szabó, Edit; Türk, Dóra; Telbisz, Ágnes; Kucsma, Nóra; Horváth, Tamás; Szakács, Gergely; Homolya, László; Sarkadi, Balázs; Várady, György

2018-01-01

ABC multidrug transporters are key players in cancer multidrug resistance and in general xenobiotic elimination, thus their functional assays provide important tools for research and diagnostic applications. In this study we have examined the potential interactions of three key human ABC multidrug transporters with PhenGreen diacetate (PGD), a cell permeable fluorescent metal ion indicator. The non-fluorescent, hydrophobic PGD rapidly enters the cells and, after cleavage by cellular esterases, in the absence of quenching metal ions, PhenGreen (PG) becomes highly fluorescent. We found that in cells expressing functional ABCG2, ABCB1, or ABCC1 transporters, cellular PG fluorescence is strongly reduced. This fluorescence signal in the presence of specific transporter inhibitors is increased to the fluorescence levels in the control cells. Thus the PG accumulation assay is a new, unique tool for the parallel determination of the function of the ABCG2, ABCB1, and ABCC1 multidrug transporters. Since PG has very low cellular toxicity, the PG accumulation assay also allows the selection, separation and culturing of selected cell populations expressing either of these transporters.

Polarization of T-helper lymphocytes toward the Th2 phenotype in uremic patients.

PubMed

Libetta, C; Rampino, T; Dal Canton, A

2001-08-01

T-helper (Th) lymphocytes consist of Th1 and Th2 subsets. Th1 cells are effectors of cell-mediated immunity and secrete interferon-gamma (IFN-gamma), which recruits new Th1 cells in cooperation with interleukin-12 (IL-12; produced by monocytes) and inhibits Th2 differentiation. Th2 cells produce IL-4 and IL-10, which inhibit IFN-gamma secretion and cell immunity. We investigated whether the impaired immune response in uremia is associated with an altered balance of Th1/Th2. Peripheral-blood mononuclear cells (PBMCs) were collected from patients with chronic renal failure (CRF) on conservative treatment (CRF patients), patients with end-stage renal disease (ESRD) on regular hemodialysis therapy (ESRD-HD patients), and healthy controls (CON). CD4(+) cells were isolated from PBMCs by negative selection using a magnetic labeling system. PBMCs and purified CD4(+) cells were cultured in Iscove's medium and Iscove's medium plus mitogens (phytohemagglutinin and lipopolysaccharide). IFN-gamma, IL-12, IL-4, and IL-10 were measured in supernatant. The constitutive release of IL-4 and IL-10 by PBMCs and CD4(+) cells of CRF and ESRD-HD patients was increased by five to eight times in comparison with CON (P < 0.001). Constitutive IFN-gamma release by PBMCs of ESRD-HD patients was undetectable, although they secreted an increased amount of IL-12. Mitogen-stimulated release of IFN-gamma by PBMCs and CD4(+) cells of CRF and ESRD-HD patients was blunted (average PBMCs: CON, 115.8 pg/2 x10(6) cells; CRF, 81.8 pg/2 x10(6) cells; ESRD-HD, 9.3 pg/2 x10(6) cells; CD4(+) cells: CON, 358.0 pg/5 x 10(5) cells; CRF, 165.4 pg/5 x 10(5) cells; ESRD-HD, 43.5 pg/5 x 10(5) cells). The ability of PBMCs of ESRD-HD patients to secrete IFN-gamma was recovered after IL-4 and IL-10 neutralization. Uremia is associated with a prevalence of Th1 over Th2 cells and a configuration of cytokine network that depresses cell-mediated immunity.

Multifuntional Nanotherapeutics for the Combinatorial Drug and Gene Therapy in the Treatment of Glioblastoma Multiforme

NASA Astrophysics Data System (ADS)

Hourigan, Breanne

Glioblastoma multiforme (GBM), a grade IV glioma, is the most common primary brain tumor, affecting about 3 out of 100,000 persons per year in the United States. GBM accounts for about 80% of primary malignant brain tumors, and is also the most aggressive of malignant brain tumors. With exhaustive treatment, survival only averages between 12 and 15 months, with a 2-year survival rate less than 25%. New therapeutic strategies are necessary to improve the outcomes of this disease. Chemotherapy with temozolomide (TMZ), a DNA alkylating agent, is used as a first-line of treatment for GBM. However, GBM tumors develop resistance to TMZ over time due to increased expression of O6-methylguanine-DNA methyltransferase (MGMT), a gene responsible for DNA repair. We previously developed cationic, amphiphilic copolymer poly(lactide-co-glycolide)-g-polyethylenimine (PgP) and demonstrated its utility for nucleic acid delivery. Here, we examine the ability of PgP polyplexes to overcome TMZ resistance and improve therapeutic efficacy through combination drug and gene therapy for GBM treatment. In this study, we evaluated the ability of PgP to deliver siRNA targeting to MGMT (siMGMT), a gene responsible for drug resistance in GBM. Our results demonstrated that PgP effectively forms stable complex with siRNA and protects siRNAs from heparin competition assay, serum- and ribonuclease-mediated degradation, confirming the potential of the polyplex for in vivo delivery. Results from MTT assays showed that PgP/siRNA polyplexes exhibited minimal cytotoxicity compared to untreated cells when incubated with T98G human GBM cells. We also demonstrated that PgP/siMGMT polyplexes mediate knockdown of MGMT protein as well as a significant ˜56% and ˜68% knockdown of MGMT mRNA in T98G GBM cells compared to cells treated with PgP complexed with non-targeting siRNA (siNT) at a 60:1 and 80:1 nitrogen:phosphate (N:P) ratio, respectively. Further, co-incubation of PgP/siMGMT polyplexes with TMZ enhanced therapeutic efficacy in T98G GBM cells compared to treatment with the polyplex or TMZ alone. After generation of athymic mouse GBM model, PgP/siMGMT polyplexes were locally injected into the tumor. Relative to untreated injury only, PgP/siMGMT polyplexes significantly reduced MGMT mRNA and protein expression at 3 days post-injection. These studies demonstrate that PgP is an efficient non-viral delivery carrier for therapeutic siMGMT to the tumor cells and may be a promising platform for the combinatorial siRNA/drug therapy for GBM treatment. In the future, we will study the therapeutic efficacy of combination of PgP/siMGMT and TMZ in athymic mouse GBM model.

Cardiovascular progenitor-derived extracellular vesicles recapitulate the beneficial effects of their parent cells in the treatment of chronic heart failure.

PubMed

Kervadec, Anaïs; Bellamy, Valérie; El Harane, Nadia; Arakélian, Lousineh; Vanneaux, Valérie; Cacciapuoti, Isabelle; Nemetalla, Hany; Périer, Marie-Cécile; Toeg, Hadi D; Richart, Adèle; Lemitre, Mathilde; Yin, Min; Loyer, Xavier; Larghero, Jérôme; Hagège, Albert; Ruel, Marc; Boulanger, Chantal M; Silvestre, Jean-Sébastien; Menasché, Philippe; Renault, Nisa K E

2016-06-01

Cell-based therapies are being explored as a therapeutic option for patients with chronic heart failure following myocardial infarction. Extracellular vesicles (EV), including exosomes and microparticles, secreted by transplanted cells may orchestrate their paracrine therapeutic effects. We assessed whether post-infarction administration of EV released by human embryonic stem cell-derived cardiovascular progenitors (hESC-Pg) can provide equivalent benefits to administered hESC-Pg and whether hESC-Pg and EV treatments activate similar endogenous pathways. Mice underwent surgical occlusion of their left coronary arteries. After 2-3 weeks, 95 mice included in the study were treated with hESC-Pg, EV, or Minimal Essential Medium Alpha Medium (alpha-MEM; vehicle control) delivered by percutaneous injections under echocardiographic guidance into the peri-infarct myocardium. functional and histologic end-points were blindly assessed 6 weeks later, and hearts were processed for gene profiling. Genes differentially expressed between control hearts and hESC-Pg-treated and EV-treated hearts were clustered into functionally relevant pathways. At 6 weeks after hESC-Pg administration, treated mice had significantly reduced left ventricular end-systolic (-4.20 ± 0.96 µl or -7.5%, p = 0.0007) and end-diastolic (-4.48 ± 1.47 µl or -4.4%, p = 0.009) volumes compared with baseline values despite the absence of any transplanted hESC-Pg or human embryonic stem cell-derived cardiomyocytes in the treated mouse hearts. Equal benefits were seen with the injection of hESC-Pg-derived EV, whereas animals injected with alpha-MEM (vehicle control) did not improve significantly. Histologic examination suggested a slight reduction in infarct size in hESC-Pg-treated animals and EV-treated animals compared with alpha-MEM-treated control animals. In the hESC-Pg-treated and EV-treated groups, heart gene profiling identified 927 genes that were similarly upregulated compared with the control group. Among the 49 enriched pathways associated with these up-regulated genes that could be related to cardiac function or regeneration, 78% were predicted to improve cardiac function through increased cell survival and/or proliferation or DNA repair as well as pathways related to decreased fibrosis and heart failure. In this post-infarct heart failure model, either hESC-Pg or their secreted EV enhance recovery of cardiac function and similarly affect cardiac gene expression patterns that could be related to this recovery. Although the mechanisms by which EV improve cardiac function remain to be determined, these results support the idea that a paracrine mechanism is sufficient to effect functional recovery in cell-based therapies for post-infarction-related chronic heart failure. Copyright © 2016 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.

Reassessing ecdysteroidogenic cells from the cell membrane receptors' perspective.

PubMed

Alexandratos, Alexandros; Moulos, Panagiotis; Nellas, Ioannis; Mavridis, Konstantinos; Dedos, Skarlatos G

2016-02-05

Ecdysteroids secreted by the prothoracic gland (PG) cells of insects control the developmental timing of their immature life stages. These cells have been historically considered as carrying out a single function in insects, namely the biochemical conversion of cholesterol to ecdysteroids and their secretion. A growing body of evidence shows that PG cells receive multiple cues during insect development so we tested the hypothesis that they carry out more than just one function in insects. We characterised the molecular nature and developmental profiles of cell membrane receptors in PG cells of Bombyx mori during the final larval stage and determined what receptors decode nutritional, developmental and physiological signals. Through iterative approaches we identified a complex repertoire of cell membrane receptors that are expressed in intricate patterns and activate previously unidentified signal transduction cascades in PG cells. The expression patterns of some of these receptors explain precisely the mechanisms that are known to control ecdysteroidogenesis. However, the presence of receptors for the notch, hedgehog and wingless signalling pathways and the expression of innate immunity-related receptors such as phagocytosis receptors, receptors for microbial ligands and Toll-like receptors call for a re-evaluation of the role these cells play in insects.

Unusual cardiac paraganglioma mimicking an atypical carcinoid tumor of the lung

PubMed Central

Evans, Mark; Wang, Beverly; Delrosario, J. Lawrence; Cheng, Timmy; Milliken, Jeffrey

2018-01-01

We present a case of unusual cardiac paraganglioma (PG) initially misdiagnosed as atypical carcinoid tumor of the lung and discuss key clinical and pathologic characteristics that guide surgical management of these rare chromaffin cell tumors. A 64-year-old female with persistent cough and back pain was found to have a 4 cm × 3 cm mass abutting multiple cardiopulmonary structures. A biopsy was performed at an outside institution and pathology reported “atypical neuroendocrine carcinoma, consistent with carcinoid”. The patient was transferred to our institution and pericardial resection with right pneumonectomy was performed to excise the tumor. Histology of the mass was that of PG with multiple ethanol embolizations. Immunohistochemical examination revealed that type I (chief) cells were positive for neuroendocrine markers (chromogranin A and synaptophysin), while type II (sustentacular) cells were positive for S100. There was no evidence of atypical carcinoid tumor in the lung. PG is an entity of chromaffin cell tumors that often affects the adrenal glands and carotid body. PG rarely occurs in the thoracic region, accounting for just 1–2% of all PG. Proper diagnosis of cardiac PG is challenging owing to its rare prevalence, subtle symptoms of presentation, and the neuroendocrine histopathological features it shares with atypical carcinoids. These tumors are typically benign and are best treated by surgical resection. Our report examines the approach to appropriate diagnosis of cardiac PG vs. atypical carcinoid, preoperative management, and surgical treatment by describing successful resection through thoracotomy without the use of cardiopulmonary bypass. PMID:29600100

Bacterial SPOR domains are recruited to septal peptidoglycan by binding to glycan strands that lack stem peptides

PubMed Central

Yahashiri, Atsushi; Jorgenson, Matthew A.; Weiss, David S.

2015-01-01

Bacterial SPOR domains bind peptidoglycan (PG) and are thought to target proteins to the cell division site by binding to “denuded” glycan strands that lack stem peptides, but uncertainties remain, in part because septal-specific binding has yet to be studied in a purified system. Here we show that fusions of GFP to SPOR domains from the Escherichia coli cell-division proteins DamX, DedD, FtsN, and RlpA all localize to septal regions of purified PG sacculi obtained from E. coli and Bacillus subtilis. Treatment of sacculi with an amidase that removes stem peptides enhanced SPOR domain binding, whereas treatment with a lytic transglycosylase that removes denuded glycans reduced SPOR domain binding. These findings demonstrate unequivocally that SPOR domains localize by binding to septal PG, that the physiologically relevant binding site is indeed a denuded glycan, and that denuded glycans are enriched in septal PG rather than distributed uniformly around the sacculus. Accumulation of denuded glycans in the septal PG of both E. coli and B. subtilis, organisms separated by 1 billion years of evolution, suggests that sequential removal of stem peptides followed by degradation of the glycan backbone is an ancient feature of PG turnover during bacterial cell division. Linking SPOR domain localization to the abundance of a structure (denuded glycans) present only transiently during biogenesis of septal PG provides a mechanism for coordinating the function of SPOR domain proteins with the progress of cell division. PMID:26305949

The Ratio of Estimated Average Glucose to Fasting Plasma Glucose Level Is Superior to Glycated Albumin, Hemoglobin A1c, Fructosamine, and GA/A1c Ratio for Assessing β-Cell Function in Childhood Diabetes

PubMed Central

Lee, Ji Eun; Lee, Ji Woo; Fujii, Tatsuyoshi; Fujii, Noriyoshi; Choi, Jong Weon

2014-01-01

Objective. This study investigated the use of the estimated average glucose to fasting plasma glucose ratio (eAG/fPG ratio) to screen for β-cell function in pediatric diabetes. Methods. Glycated hemoglobin (HbA1c), glycated albumin (GA), fructosamine, insulin, and C-peptide levels were measured. The ratio of GA to HbA1c (GA/A1c ratio) was calculated, and the homeostasis model assessment of β-cell function (HOMA-β) was determined. Results. Median values of C-peptide, insulin, and HOMA-β levels were significantly higher in patients with an increased eAG/fPG ratio than in those with a decreased eAG/fPG ratio. C-peptide and HOMA-β levels were more closely correlated with the eAG/fPG ratio than with GA, HbA1c, the GA/A1c ratio, and fructosamine. In contrast, body mass index was significantly associated with GA, GA/A1c ratio, and fructosamine, but not with the eAG/fPG ratio and HbA1c levels. To test the diagnostic accuracies of the eAG/fPG ratio for identifying HOMA-β > 30.0% in patients with type 2 diabetes, the area under the ROC curve of the eAG/fPG ratio was significantly larger than that of the GA/A1c ratio [0.877 (95% CI, 0.780–0.942) versus 0.775 (95% CI, 0.664–0.865), P = 0.039]. Conclusions. A measurement of the eAG/fPG ratio may provide helpful information for assessing β-cell function in pediatric patients with diabetes. PMID:25013775

Effect of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on the expression of EphA2 in osteoblasts and osteoclasts.

PubMed

Gao, Aichao; Wang, Xichao; Yu, Haiyan; Li, Na; Hou, Yubo; Yu, Weixian

2016-02-01

Porphyromonas gingivalis (Pg) as the major pathogenic bacterium of chronic periodontitis can cause alveolar bone resorption. Lipopolysaccharide (LPS) is its main virulence factor. The Eph family plays an important role in maintaining bone homeostasis. In this study, the effects of P. gingivalis lipopolysaccharide (Pg-LPS) on the expression of EphA2 in osteoblasts and osteoclasts were investigated. MC3T3-E1 cells and RAW264.7 cells were separately cultured in osteoblast-conditioned medium and osteoclast-conditioned medium to induce their differentiation into osteoblasts and osteoclasts, respectively. MC3T3-E1 cells were treated with 1 μg/mL of Pg-LPS 3, 7, and 14 d later, while RAW264.7 cells were treated with 10 μg/mL of Pg-LPS 1, 3, and 5 d later. The results have shown that Pg-LPS increased the expression of EphA2 both in osteoblasts and osteoclasts, decreased the expression of osteogenic-related genes (ALP, Sp7), and increased the expression of osteoclast-related genes (MMP9, c-fos, ACP5, CtsK, and NFATc1). Tartrate-resistant acid phosphatase (TRAP) staining illustrated that Pg-LPS promoted osteoclast differentiation and decreased the activity of alkaline phosphatase. Therefore, analysis indicates that, when treated with Pg-LPS, the expression of EphA2 is upregulated while the activity of osteoblasts and osteoclasts was reduced and increased, respectively. Our data suggest that EphA2 is closely related to the formation of osteoblasts and resorption of osteoclast and is likely to play an role in bone resorption induced in chronic periodontitis. These findings may provide information on new targets for prevention and treatment of chronic periodontitis.

Three-dimensional endothelial cell morphogenesis under controlled ion release from copper-doped phosphate glass.

PubMed

Stähli, Christoph; James-Bhasin, Mark; Nazhat, Showan N

2015-02-28

Copper ions represent a promising angiogenic agent but are associated with cytotoxicity at elevated concentrations. Phosphate-based glasses (PGs) exhibit adjustable dissolution properties and allow for controlled ion release. This study examined the formation of capillary-like networks by SVEC4-10 endothelial cells (ECs) seeded in a three-dimensional (3D) type I collagen hydrogel matrix mixed with PG particles of the formulation 50P2O5-30CaO-(20-x)Na2O-xCuO (x=0 and 10 mol%). Copper and total phosphorus release decreased over time and was more sustained in the case of 10% CuO PG. Moreover, increasing the concentration of 10% CuO PG in collagen substantially delayed dissolution along with preferential release of copper. A 3D morphometric characterization method based on confocal laser scanning microscopy image stacks was developed in order to quantify EC network length, connectivity and branching. Network length was initially reduced in a concentration-dependent fashion by 10% CuO PG and, to a lesser extent, by 0% CuO PG, but reached values identical to the non-PG control by day 5 in culture. This reduction was attributed to a PG-mediated decrease in cell metabolic activity while cell proliferation as well as network connectivity and branching were independent of PG content. Gene expression of matrix metalloproteinases (MMP)-1 and -2 was up-regulated by PGs, indicating that MMPs did not play a critical role in network growth. The relationship between ion release and EC morphogenesis in 3D provided in this study is expected to contribute to an ultimately successful pro-angiogenic application of CuO-doped PGs. Copyright © 2015 Elsevier B.V. All rights reserved.

The coefficient of restitution of pressurized balls: a mechanistic model

NASA Astrophysics Data System (ADS)

Georgallas, Alex; Landry, Gaëtan

2016-01-01

Pressurized, inflated balls used in professional sports are regulated so that their behaviour upon impact can be anticipated and allow the game to have its distinctive character. However, the dynamics governing the impacts of such balls, even on stationary hard surfaces, can be extremely complex. The energy transformations, which arise from the compression of the gas within the ball and from the shear forces associated with the deformation of the wall, are examined in this paper. We develop a simple mechanistic model of the dependence of the coefficient of restitution, e, upon both the gauge pressure, P_G, of the gas and the shear modulus, G, of the wall. The model is validated using the results from a simple series of experiments using three different sports balls. The fits to the data are extremely good for P_G > 25 kPa and consistent values are obtained for the value of G for the wall material. As far as the authors can tell, this simple, mechanistic model of the pressure dependence of the coefficient of restitution is the first in the literature. *%K Coefficient of Restitution, Dynamics, Inflated Balls, Pressure, Impact Model

Characterization of N-Succinylation of L-Lysylphosphatidylglycerol in Bacillus subtilis Using Tandem Mass Spectrometry

NASA Astrophysics Data System (ADS)

Atila, Metin; Katselis, George; Chumala, Paulos; Luo, Yu

2016-10-01

Phospholipids generally dominate in bacterial lipids. The negatively charged nature of phospholipids renders bacteria susceptible to cationic antibiotic peptides. In comparison with Gram-negative bacteria, Gram-positive bacteria in general have much less zwitterionic phosphatidylethanolamine. However, they are known for producing aminoacylated phosphatidylglycerol (PG), especially positively charged l-lysyl-PG, which is catalyzed by lysyl-PG synthase MprF, which appears to have a broad range of specificity for l-aminoacyl transfer RNAs. In addition, many Gram-positive bacteria also have a dlt-gene-coded d-alanylation pathway for lipoteichoic acids and wall teichoic acids covalently attached to a glycolipid or peptidoglycan. d-Alanylation also masks the dominant negative charge of the phosphate-rich polymers of teichoic acids. Using mass spectrometry, we have recently observed that precursor scans in negative mode for deprotonated amino acid fragments were most sensitive for ester-linked amino acids. Such a scan for precursors generating an m/ z 145 lysyl anion revealed lysyl-PG as well as an additional species 100 m/ z units greater than lysyl-PG. This unexpected species corresponded precisely to the expected mass of N-succinylated lysyl-PG. Tandem mass spectrometry revealed a precise match to the fragmentation pattern of this putative new species. PG, lysyl-PG, and N-succinyl-lysyl-PG may form a complete loop of charge reversal from -1 to +1 and then back to -1. Analogous charge reversal by N-succinylation of lysine residues in the bacterial as well as eukaryotic proteomes has been recently discovered as a major posttranslational modification. Such modification in bacterial lipids is possibly catalyzed by an enzyme homologous to the enzymes that modify lysine residues in proteins.

Cytotoxicity of Pomegranate Polyphenolics in Breast Cancer Cells in Vitro and Vivo - Potential Role of miRNA-27a and miRNA-155 in Cell Survival and Inflammation

PubMed Central

Banerjee, Nivedita; Talcott, Stephen; Safe, Stephen; Mertens –Talcott, Susanne U

2012-01-01

Several studies have demonstrated that polyphenolics from pomegranate (Punica granatum L.) are potent inhibitors of cancer cell proliferation and induce apoptosis, cell cycle arrest, and also decrease inflammation in vitro and vivo. There is growing evidence that botanicals exert their cytotoxic and anti-inflammatory activities, at least in part, by decreasing specificity protein (Sp) transcription factors. These are overexpressed in breast-tumors and regulate genes important for cancer cell survival and inflammation such as the p65 unit of NF-κB. Moreover, previous studies have shown that Pg extracts decrease inflammation in lung cancer cell lines by inhibiting phosphatidylinositol 3,4,5-trisphosphate (PI3K)-dependent phosphorylation of AKT in vitro and inhibiting the activation of NF-kB in vivo. The objective of this study was to investigate the roles of miR-27a-ZBTB10-Sp and miR-155-SHIP-1-PI3K on the anti-inflammatory and cytotoxic activity of pomegranate extract. Pg extract (2.5–50 µg/ml) inhibited growth of BT-474 and MDA-MB-231 cells but not the non-cancer MCF-10F and MCF-12F cells. Pg extract significantly decreased Sp1, Sp3, and Sp4 as well as miR-27a in BT474 and MDA-MB-231 cells and increased expression of the transcriptional repressor ZBTB10. A significant decrease in Sp proteins and Sp-regulated genes was also observed. Pg extract also induced SHIP-1 expression and this was accompanied by downregulation of miRNA-155 and inhibition of PI3K-dependent phosphorylation of AKT. Similar results were observed in tumors from nude mice bearing BT474 cells as xenografts and treated with Pg extract. The effects of antagomirs and knockdown of SHIP-1 by RNA interference confirmed that the anti-inflammatory and cytotoxic effects of Pg extract were partly due to the disruption of both miR-27a-ZBTB10 and miR-155-SHIP-1. In summary the anticancer activities of Pg extract in breast cancer cells were due in part to targeting microRNAs155 and 27a. Both pathways play an important role in the proliferative/inflammatory phenotype exhibited by these cell lines PMID:22941571

Highly exposed Pt nanoparticles supported on porous graphene for electrochemical detection of hydrogen peroxide in living cells.

PubMed

Liu, Jian; Bo, Xiangjie; Zhao, Zheng; Guo, Liping

2015-12-15

In this study, we developed a novel biosensor based on highly exposed Pt nanoparticles (Pt NPs) decorated porous graphene (PG) for the reliable detection of extracellular hydrogen peroxide (H2O2) released from living cells. The commercially available low-cost hydrophilic CaCO3 spheres were used as template for preparing PG. The porous structure provided larger surface area and more active sites. Due to the porous structure of PG, the Pt NPs supported on PG were not secluded by aggregated graphene layers and were highly exposed to target molecules. Ultrafine Pt NPs were well dispersed and loaded on PG by a method of microwave assistance. Electrochemical performances of the Pt/PG nanocomposites modified glassy carbon electrode (GCE) were investigated. The electrocatalytic reduction of H2O2 showed a wide linear range from 1 to 1477 μM, with a high sensitivity of 341.14 μA mM(-1) cm(-2) and a limit of detection (LOD) as low as 0.50 μM. Moreover, the Pt/PG/GCE exhibited excellent anti-interference property, reproducibility and long-term storage stability. Because of these remarkable analytical advantages, the constructed sensor was used to determine H2O2 released from living cells with satisfactory results. The superior catalytic activity makes Pt/PG nanocomposites a promising candidate for electrochemical sensors and biosensors design. Copyright © 2015 Elsevier B.V. All rights reserved.

Distribution and Metabolism of Lipocurc™ (Liposomal Curcumin) in Dog and Human Blood Cells: Species Selectivity and Pharmacokinetic Relevance.

PubMed

Bolger, Gordon T; Licollari, Albert; Tan, Aimin; Greil, Richard; Vcelar, Brigitta; Majeed, Muhammad; Helson, Lawrence

2017-07-01

The aim of this study was to investigate the distribution of curcumin (in the form of Lipocurc™) and its major metabolite tetrahydrocurcumin (THC) in Beagle dog and human red blood cells, peripheral blood mononuclear cells (PBMC) and hepatocytes. Lipocurc™ was used as the source of curcumin for the cell distribution assays. In vitro findings with red blood cells were also compared to in vivo pharmacokinetic data available from preclinical studies in dogs and phase I clinical studies in humans. High levels of curcumin were measured in PBMCs (625.5 ng/g w.w. cell pellet or 7,297 pg/10 6 cells in dog and 353.7 ng/g w.w. cell pellet or 6,809 pg/10 6 cells in human) and in hepatocytes (414.5 ng/g w.w. cell pellet or 14,005 pg/10 6 cells in dog and 813.5 ng/g w.w. cell pellet or 13,780 pg/10 6 cells in human). Lower curcumin levels were measured in red blood cells (dog: 78.4 ng/g w.w. cell pellet or 7.2 pg/10 6 cells, human: 201.5 ng/g w.w. cell pellet or 18.6 pg/10 6 cells). A decrease in the medium concentration of curcumin was observed in red blood cells and hepatocytes, but not in PBMCs. Red blood cell levels of THC were ~5-fold higher in dog compared to human and similar between dog and human for hepatocytes and PBMCs. The ratio of THC to curcumin found in the red blood cell medium following incubation was 6.3 for dog compared to 0.006 for human, while for PBMCs and hepatocytes the ratio of THC to curcumin in the medium did not display such marked species differences. There was an excellent correlation between the in vitro disposition of curcumin and THC following incubation with red blood cells and in vivo plasma levels of curcumin and THC in dog and human following intravenous infusion. The disposition of curcumin in blood cells is, therefore, species-dependent and of pharmacokinetic relevance. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Accumulation of pro-cancer cytokines in the plasma fraction of stored packed red cells.

PubMed

Benson, Douglas D; Beck, Adam W; Burdine, Marie S; Brekken, Rolf; Silliman, Christopher C; Barnett, Carlton C

2012-03-01

Perioperative blood transfusion has been linked to decreased survival in pancreatic cancer; however, the exact causal mechanism has not been elucidated. Allogeneic transfusions are known to expose patients to foreign cells and lipid mediators. We hypothesize that stored packed red cells (pRBCs) contain pro-cancer cytokines that augment tumor progression. We analyzed the plasma fraction of stored pRBCs for pro-cancer cytokines and evaluated the affect of both storage time and leukocyte reduction on these mediators. Chemiarray™ analysis for pro-cancer cytokines was performed on the acellular plasma fraction of stored leukocyte-reduced (LR) and non-leukocyte-reduced (NLR) pRBCs at day 1 (D.1-fresh) and day 42 (D.42-outdate) of storage. Elevated expression of monocyte chemotactic protein-1 (MCP-1), regulated on activation, normal T cell expressed and secreted (RANTES), angiogenin, tumor necrosis factor-alpha (TNF-α), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) was found. Specific enzyme-linked immunosorbent assay was performed for each of these factors in LR and NLR blood at D.1, day 28, and D.42. Data were analyzed by ANOVA. A p value ≤ 0.05 was considered significant; N ≥ 4 per group. Migration assays were performed using inhibitors of EGF (gefitinib) and PDGF (imatinib) on murine pancreatic adenocarcinoma cells (Pan02) exposed to D.1 and D.42 LR and NLR plasma. Proliferation assays were performed on Pan02 cells to test the inhibition of PDGF. MCP-1 levels increased with storage time in LR blood, 86.3 ± 6.3 pg/ml at D.1 vs. 121.2 ± 6.1 pg/ml at D.42 (p = 0.007), and NLR blood, 78.2 ± 7.3 pg/ml at D.1 vs. 647.8 ± 220.7 pg/ml at D.42 (p = 0.02). RANTES levels are lower in LR compared to NLR stored blood, 3.0 ± 1.9 vs. 15.8 ± 0.7 pg/ml at D.42 (p < 0.001), but similar in D.1 blood, 13.8 ± 1.8 pg/ml in LR vs. 12.0 ± 1.6 pg/ml in NLR. Angiogenin levels were different between LR and NLR blood, 0 pg/ml (undetectable) vs. 44.2 ± 3.7 pg/ml (p < 0.001). Storage time did not affect concentration. TNF-α levels were not different between LR and NLR blood, and there was no storage time effect on concentration. EGF and PDGF levels increased with storage time in NLR blood only, 216.4 ± 3.8 pg/ml at D.1 vs. 1,436.4 ± 238.6 pg/ml at D.42 for EGF (p = 0.001), and 61.6 ± 6.0 pg/ml at D.1 vs. 76.5 ± 1.7 pg/ml at D.42 (p = 0.003) for PDGF. Inhibition of EGF reduced migration in Pan02 cells treated with D.42 NLR blood, 245.9 ± 11.2 vs. 164.6 ± 10.6 cells/hpf (p < 0.001). Inhibition of PDGF had no effect on Pan02 migration and reduced cell proliferation in cells treated with D.42 NLR, 181.1 ± 1.5% over control vs. 157.5 ± 2.1% (p < 0.001). Pro-cancer cytokines that can augment tumor progression were identified in pRBCs. Some of these factors are present in fresh blood. The soluble factors identified herein may represent possible therapeutic targets to offset negative effects of transfusion. These data stress the need for efforts in cancer patients to reduce transfusion requirements if needed.

Association of hemoglobin A1c and glycated albumin with carotid atherosclerosis in community-dwelling Japanese subjects: the Hisayama Study.

PubMed

Mukai, Naoko; Ninomiya, Toshiharu; Hata, Jun; Hirakawa, Yoichiro; Ikeda, Fumie; Fukuhara, Masayo; Hotta, Taeko; Koga, Masafumi; Nakamura, Udai; Kang, Dongchon; Kitazono, Takanari; Kiyohara, Yutaka

2015-06-24

It is not clear which glucose measure is more useful in the assessment of atherosclerosis. We investigated the associations of hemoglobin A1c (HbA1c), glycated albumin (GA), 1,5-anhydroglucitol (1,5-AG), fasting plasma glucose (FPG), and 2-hour postload glucose (PG) with carotid intima-media thickness (IMT) in community-dwelling Japanese subjects. A total of 2702 subjects aged 40-79 years underwent a 75-g oral glucose tolerance test and measurements of HbA1c, GA, 1,5-AG, and carotid IMT by ultrasonography in 2007-2008. Carotid wall thickening was defined as a maximum IMT of >1.0 mm. The crude and multivariable-adjusted linear and logistic regression models were used to analyze cross-sectional associations between levels of glycemic measures and carotid IMT. The crude average of the maximum IMT increased significantly with rising quartiles of HbA1c, GA, FPG, and 2-hour PG levels in subjects with and without glucose intolerance (GI), while no clear association was observed for 1,5-AG. After adjustment for other confounding factors, positive trends for HbA1c, GA, and FPG (all p for trend < 0.05), but not 2-hour PG (p = 0.07) remained robust in subjects with GI, but no such associations were found in those without GI. When estimating multivariable-adjusted β values for the associations of 1 SD change in glycemic measures with the maximum IMT in subjects with GI, the magnitude of the influence of HbA1c (β = 0.021), GA (β = 0.024), and FPG (β = 0.024) was larger than that of 2-hour PG (β = 0.014) and 1,5-AG (β = 0.003). The multivariable-adjusted odds ratios for the presence of carotid wall thickening increased significantly with elevating HbA1c, GA, and FPG levels only in subjects with GI (all p for trend < 0.001). Among subjects with GI, the area under the receiver operating characteristic curve significantly increased by adding HbA1c (p = 0.04) or GA (p = 0.04), but not 1,5-AG, FPG, or 2-hour PG, to the model including other cardiovascular risk factors. In community-dwelling Japanese subjects with GI, elevated HbA1c, GA, and FPG levels were significantly associated with increased carotid IMT, and HbA1c and GA provided superior discrimination for carotid wall thickening compared to 1,5-AG, FPG, and 2-hour PG, suggesting that HbA1c and GA are useful for assessing carotid atherosclerosis.

Antioxidative pyranonigrins in rice mold starters and their suppressive effect on the expression of blood adhesion molecules.

PubMed

Miyake, Yoshiaki; Mochizuki, Mika; Ito, Chihiro; Itoigawa, Masataka; Osawa, Toshihiko

2008-06-01

Antioxidants having a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacity in rice mold starters, which are used for the preparation of various Japanese fermented foods, and their effectiveness against the expression of blood adhesion molecules were examined. An antioxidant was isolated from the rice mold starters used for shochu and identified as pyranonigrin-S (PG-S) by (1)H-NMR, (13)C-NMR, and FAB-MS analyses. It was a derivative of pyranonigrin-A (PG-A), which has been isolated as an antioxidant from the rice mold starters. Pyranonigrins PG-A and PG-S were found to exist in spores on rice mold starters which had been prepared by Aspergillus awamori, A. kawachii, and A. saitoi. PG-S exhibited a higher level of DPPH radical scavenging activity than PG-A. PG-A was found to have a significant suppressive effect on the expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) induced by tumor necrosis factor-alpha (TNF-alpha) (P<0.05).

IB-11PSEUDO-PROGRESSION (PsdPg) IS A HARBINGER OF A MORE EFFECTIVE ANTI-TUMOR RESPONSE

PubMed Central

Sturla, Lisa; Donahue, John; Machan, Jason; Delamonte, Suzanne; Jeyapalan, Suriya

2014-01-01

BACKGROUND: PsdPg is the increased contrast enhancement, high choline/creatine ratio and increased perfusion observed in the residual tumor bed of high-grade glioma patients after completion of temozolomide/radiation. It resolves within 3-6 months and incidence ranges from 10 - 31%. Though correlated with longer patient survival, its pathological basis is unclear. We used a cytokine/chemokine focused approach to compare the tumor microenvironment in pre- and post-treatment tumor tissue from patients with PsdPg to patients with true progression (TP). METHODS: We obtained pre-treatment formalin fixed paraffin embedded (FFPE) tissue from 35 GBM patients and post-treatment FFPE tissue from five patients with PsdPg and TP. A quantitative PCR array and custom Quantigene 2.0 multiplex was used to quantify gene expression corresponding to major cytokines/chemokines. An 18-gene signature was used to determine the macrophage polarization score (cumulative M2-associated cytokine expression - cumulative M1-associated cytokine expression). Immunohistochemistry (IHC) was used to confirm significantly different targets at the protein level. RESULTS: IHC revealed 7-fold higher B-cell infiltration in TP patients as compared to patients with PsdPg (p = 0.003). Macrophage and T-cell infiltration were not significantly different between the two groups. Nevertheless, the cytokines associated with macrophage polarization indicated pro-tumorigenic (M2) polarization in TP patients while PsdPg patients exhibited classical anti-tumorigenic (M1) polarization. TP patients had a 10-fold higher M2 score (p = 0.03) compared to PsdPg patients. The M1 score of tissue from PsdPg patients post-treatment was 25-fold higher than their pre-treatment tissue (p = 0.01). Analysis of a 7-gene signature associated with natural killer (NK) cell recruitment and activation showed a 8-fold higher expression in pre-treatment tissue from PsdPg patients compared to TP patients (p = 0.009) suggesting that NK cells, which are mediators of anti-tumor immunity, play an important role in pseudo-progression. CONCLUSIONS: These data suggest a more effective anti-tumor immune response in PsdPg patients, which may explain their longer overall survival.

Conditioning of native substrates by chondroitin sulfate proteoglycans during cardiac mesenchymal cell migration

PubMed Central

1986-01-01

It is generally proposed that embryonic mesenchymal cells use sulfated macromolecules during in situ migration. Attempts to resolve the molecular mechanisms for this hypothesis using planar substrates have been met with limited success. In the present study, we provide evidence that the functional significance of certain sulfated macromolecules during mesenchyme migration required the presence of the endogenous migratory template; i.e., native collagen fibrils. Using three-dimensional collagen gel lattices and whole embryo culture procedures to produce metabolically labeled sulfated macromolecules in embryonic chick cardiac tissue, we show that these molecules were primarily proteoglycan (PG) in nature and that their distribution was class specific; i.e., heparan sulfate PG, the minor labeled component (15%), remained pericellular while chondroitin sulfate (CS) PG, the predominately labeled PG (85%), was associated with collagen fibrils as "trails" of 50-60-nm particles when viewed by scanning electron microscopy. Progressive "conditioning" of collagen with CS-PG inhibited the capacity of the template to support subsequent cell migration. Lastly, metabolically labeled, PG-derived CS chains were compared with respect to degree of sulfation in either the C-6 or C-4 position by chromatographic separation of chondroitinase AC digestion products. Results from temporal and regional comparisons of in situ-labeled PGs indicated a positive correlation between the presence of mesenchyme and an enrichment of disaccharide-4S relative to that from regions lacking mesenchyme (i.e., principally myocardial tissue). The suggestion of a mesenchyme-specific CS-PG was substantiated by similarly examining the PGs synthesized solely by cardiac mesenchymal cells migrating within hydrated collagen lattice in culture. These data were incorporated into a model of "substratum conditioning" which provides a molecular mechanism by which secretion of mesenchyme-specific CS-PGs not only provides for directed and sustained cell movement, but ultimately inhibits migration of the cell population as a whole. PMID:3782305

Silver Nanoparticles Induce HePG-2 Cells Apoptosis Through ROS-Mediated Signaling Pathways

NASA Astrophysics Data System (ADS)

Zhu, Bing; Li, Yinghua; Lin, Zhengfang; Zhao, Mingqi; Xu, Tiantian; Wang, Changbing; Deng, Ning

2016-04-01

Recently, silver nanoparticles (AgNPs) have been shown to provide a novel approach to overcome tumors, especially those of hepatocarcinoma. However, the anticancer mechanism of silver nanoparticles is unclear. Thus, the purpose of this study was to estimate the effect of AgNPs on proliferation and activation of ROS-mediated signaling pathway on human hepatocellular carcinoma HePG-2 cells. A simple chemical method for preparing AgNPs with superior anticancer activity has been showed in this study. AgNPs were detected by transmission electronic microscopy (TEM) and energy dispersive X-ray (EDX). The size distribution and zeta potential of silver nanoparticles were detected by Zetasizer Nano. The average size of AgNPs (2 nm) observably increased the cellular uptake by endocytosis. AgNPs markedly inhibited the proliferation of HePG-2 cells through induction of apoptosis with caspase-3 activation and PARP cleavage. AgNPs with dose-dependent manner significantly increased the apoptotic cell population (sub-G1). Furthermore, AgNP-induced apoptosis was found dependent on the overproduction of reactive oxygen species (ROS) and affecting of MAPKs and AKT signaling and DNA damage-mediated p53 phosphorylation to advance HePG-2 cells apoptosis. Therefore, our results show that the mechanism of ROS-mediated signaling pathways may provide useful information in AgNP-induced HePG-2 cell apoptosis.

Trinitrotoluene Induces Endoplasmic Reticulum Stress and Apoptosis in HePG2 Cells.

PubMed

Song, Li; Wang, Yue; Wang, Jun; Yang, Fan; Li, Xiaojun; Wu, Yonghui

2015-11-09

This study aims to describe trinitrotoluene (TNT)-induced endoplasmic reticulum stress (ERS) and apoptosis in HePG2 cells. HePG2 cells were cultured in vitro with 0, 6, 12, or 24 μg/ml TNT solution for 12, 24, and 48 h. Western blotting was performed to detect intracellular ERS-related proteins, including glucose-regulated protein (GRP) 78, GRP94, Caspase 4, p-Jun N-terminal kinase (JNK), and C/EBP homologous protein (CHOP). Real-time PCR was used to measure mRNA expression from the respective genes. The expressions of ERS-related proteins GRP78 and GRP94 as well as mRNA and protein expression of ERS signaling apoptotic CHOP in the TNT treatment group were significantly increased. In addition, the mRNA and protein expression levels of ERS-induced apoptotic protein Caspase-4 were significantly increased. Flow cytometry revealed that after TNT treatment, the apoptosis rate also significantly increased. TNT could increase the expression levels of GRP78, GRP94, Caspase-4, and CHOP in HePG2 cells; this increase in protein expression might be involved in HePG2 apoptosis through the induction of the ERS pathway.

Trinitrotoluene Induces Endoplasmic Reticulum Stress and Apoptosis in HePG2 Cells

PubMed Central

Song, Li; Wang, Yue; Wang, Jun; Yang, Fan; Li, Xiaojun; Wu, Yonghui

2015-01-01

Background This study aims to describe trinitrotoluene (TNT)-induced endoplasmic reticulum stress (ERS) and apoptosis in HePG2 cells. Material/Methods HePG2 cells were cultured in vitro with 0, 6, 12, or 24 μg/ml TNT solution for 12, 24, and 48 h. Western blotting was performed to detect intracellular ERS-related proteins, including glucose-regulated protein (GRP) 78, GRP94, Caspase 4, p-Jun N-terminal kinase (JNK), and C/EBP homologous protein (CHOP). Real-time PCR was used to measure mRNA expression from the respective genes. Results The expressions of ERS-related proteins GRP78 and GRP94 as well as mRNA and protein expression of ERS signaling apoptotic CHOP in the TNT treatment group were significantly increased. In addition, the mRNA and protein expression levels of ERS-induced apoptotic protein Caspase-4 were significantly increased. Flow cytometry revealed that after TNT treatment, the apoptosis rate also significantly increased. Conclusions TNT could increase the expression levels of GRP78, GRP94, Caspase-4, and CHOP in HePG2 cells; this increase in protein expression might be involved in HePG2 apoptosis through the induction of the ERS pathway. PMID:26551326

Vascular endothelial growth factor expression and inhibition in uveal melanoma cell lines

PubMed Central

Logan, Patrick; Burnier, Julia; Burnier, Miguel N.

2013-01-01

Background: Uveal melanoma (UM) is a disease that affects approximately five people per million in the United States. This disease metastasises predominantly to the liver, and treatment options following the clinical detection of these sequelae are limited. Vascular endothelial growth factor-A (VEGF-A) is the primary activator of tumour angiogenesis and functions by binding to VEGF-Receptor 2 (VEGF-R2) and is often required for tumour growth beyond 2–3 mm. The purpose of this study was to investigate the expression of VEGF-A and the primary VEGF-R2 in three UM cell lines. Furthermore, we investigated the effects of VEGF-A inhibition on receptor activation and production of other cytokines. Finally, the effects of VEGF-A inhibition on the proliferation, migration, and invasion in the cell lines were ascertained. Materials: Three UM cell lines (92.1, OCM-1, and UW-1) were incubated with and without the addition of 100 μg/mL of bevacizumab. VEGF-A expression under both conditions was determined by sandwich enzyme-linked immunosorbent assay (ELISA), and phosphorylated VEGF-R2 expression was determined using western blot. The effects of VEGF-A inhibition on 20 cytokines (IL-1a, IL-2, IL-5, IL-8, IL-12p70, GM-CSF, IFNy, CCL3, MMP-9, TNF-a, IL-1b, IL-4, IL-6, IL-10, IL-13, GRO, MCP-1, MIP-1b, and RANTES) were determined using a multiplex sandwich ELISA. Proliferation rates before and after treatment were evaluated via sulforhodamine B assay, and migration and invasion assays implementing the Boyden chamber technique, the latter with artificial extracellular matrix, were used to assess their respective abilities. The Student’s t-test was used to compare changes in cytokine expression following VEGF-A inhibition. Analysis of variance was used to compare changes in the functional abilities of three uveal melanoma cell lines following VEGF-A inhibition. A P-value < 0.05 was considered statistically significant. Results: All three cell lines produced copious amounts of VEGF-A in culture (92.1, 11785.5 ± 231.8 pg/μL; OCM-1, 4608.0 ± 324.0 pg/μL; UW-1, 8309.3 ± 634.5 pg/μL), which was reduced to undetectable levels following the administration of bevacizumab (P< 0.05). Similarly, detectable phosphorylated VEGF-R2 was present in all cells, which was reduced significantly in all cell lines following bevacizumab treatment (107525.2 ± 8602.0 versus 1024.5 ± 98.2, 46587.3 ± 4192.9 versus 12821.1 ± 1666.7, and 60394.3 ± 4026.4 versus 6908.2 ± 607.2; 92.1, OCM-1, and UW-1, respectively; P< 0.05). Of the cytokines investigated, only MMP-9 and CCL3 were ubiquitously altered across all three cell lines following bevacizumab treatment; they were upregulated (CCL3: 1072.50 ± 18.77 pg/mL versus 1281.00 ± 72.34 pg/mL; 22.5 ± 7.85 pg/mL versus 62.00 ± 9.16 pg/mL; 20.33 ± 6.35 pg/mL versus 35.00 ± 6.22 pg/mL; control versus bevacizumab; MMP-9: 25.50 ± 5.47 pg/mL versus 88.25 ± 13.38 pg/mL; 19.75 ± 4.14 pg/mL versus 45.25 ± 8.36 pg/mL; 3.25 ± 1.09 pg/mL versus 19.25 ± 3.77 pg/mL; control versus bevacizumab; 92.1, OCM-1, and UW-1, respectively; P< 0.05). Bevacizumab significantly reduced the proliferation of one cell line (92.1: 0.405 ± 0.012 versus 0.509 ± 0.033; bevacizumab versus control; values OD; P< 0.05), the migration of two cell lines (92.1: 0.071 ± 0.003 versus 0.115 ± 0.003; OCM-1: 0.049 ± 0.005 versus 0.117 ± 0.014; bevacizumab versus control; values OD; P< 0.05), and did not significantly affect invasion. Conclusion: Despite the significant reduction in phosphorylated VEGF-R2 levels, bevacizumab did not have a dramatic impact on the functional abilities of the three UM cell lines studied. Our results indicate that compensatory mechanisms, such as the upregulation of MMP-9 and CCL-3, following bevacizumab administration may mitigate its effects on these abilities. PMID:23914254

Comprehensive QTL mapping survey dissects the complex fruit texture physiology in apple (Malus x domestica Borkh.).

PubMed

Longhi, Sara; Moretto, Marco; Viola, Roberto; Velasco, Riccardo; Costa, Fabrizio

2012-02-01

Fruit ripening is a complex physiological process in plants whereby cell wall programmed changes occur mainly to promote seed dispersal. Cell wall modification also directly regulates the textural properties, a fundamental aspect of fruit quality. In this study, two full-sib populations of apple, with 'Fuji' as the common maternal parent, crossed with 'Delearly' and 'Pink Lady', were used to understand the control of fruit texture by QTL mapping and in silico gene mining. Texture was dissected with a novel high resolution phenomics strategy, simultaneously profiling both mechanical and acoustic fruit texture components. In 'Fuji × Delearly' nine linkage groups were associated with QTLs accounting from 15.6% to 49% of the total variance, and a highly significant QTL cluster for both textural components was mapped on chromosome 10 and co-located with Md-PG1, a polygalacturonase gene that, in apple, is known to be involved in cell wall metabolism processes. In addition, other candidate genes related to Md-NOR and Md-RIN transcription factors, Md-Pel (pectate lyase), and Md-ACS1 were mapped within statistical intervals. In 'Fuji × Pink Lady', a smaller set of linkage groups associated with the QTLs identified for fruit texture (15.9-34.6% variance) was observed. The analysis of the phenotypic variance over a two-dimensional PCA plot highlighted a transgressive segregation for this progeny, revealing two QTL sets distinctively related to both mechanical and acoustic texture components. The mining of the apple genome allowed the discovery of the gene inventory underlying each QTL, and functional profile assessment unravelled specific gene expression patterns of these candidate genes.

Hepatitis B Virus Core Protein Dephosphorylation Occurs during Pregenomic RNA Encapsidation.

PubMed

Zhao, Qiong; Hu, Zhanying; Cheng, Junjun; Wu, Shuo; Luo, Yue; Chang, Jinhong; Hu, Jianming; Guo, Ju-Tao

2018-07-01

Hepatitis B virus (HBV) core protein consists of an N-terminal assembly domain and a C-terminal domain (CTD) with seven conserved serines or threonines that are dynamically phosphorylated/dephosphorylated during the viral replication cycle. Sulfamoylbenzamide derivatives are small molecular core protein allosteric modulators (CpAMs) that bind to the heteroaryldihydropyrimidine (HAP) pocket between the core protein dimer-dimer interfaces. CpAM binding alters the kinetics and pathway of capsid assembly and can result in the formation of morphologically "normal" capsids devoid of viral pregenomic RNA (pgRNA) and DNA polymerase. In order to investigate the mechanism underlying CpAM inhibition of pgRNA encapsidation, we developed an immunoblotting assay that can resolve core protein based on its phosphorylation status and demonstrated, for the first time, that core protein is hyperphosphorylated in free dimers and empty capsids from both mock-treated and CpAM-treated cells but is hypophosphorylated in pgRNA- and DNA-containing nucleocapsids. Interestingly, inhibition of pgRNA encapsidation by a heat shock protein 90 (HSP90) inhibitor prevented core protein dephosphorylation. Moreover, core proteins with point mutations at the wall of the HAP pocket, V124A and V124W, assembled empty capsids and nucleocapsids with altered phosphorylation status. The results thus suggest that core protein dephosphorylation occurs in the assembly of pgRNA and that interference with the interaction between core protein subunits at dimer-dimer interfaces during nucleocapsid assembly alters not only capsid structure, but also core protein dephosphorylation. Hence, inhibition of pgRNA encapsidation by CpAMs might be due to disruption of core protein dephosphorylation during nucleocapsid assembly. IMPORTANCE Dynamic phosphorylation of HBV core protein regulates multiple steps of viral replication. However, the regulatory function was mainly investigated by phosphomimetic mutagenesis, which disrupts the natural dynamics of core protein phosphorylation/dephosphorylation. Development of an immunoblotting assay capable of resolving hyper- and hypophosphorylated core proteins allowed us to track the phosphorylation status of core proteins existing as free dimers and the variety of intracellular capsids and to investigate the role of core protein phosphorylation/dephosphorylation in viral replication. Here, we found that disruption of core protein interaction at dimer-dimer interfaces during nucleocapsid assembly (by CpAMs or mutagenesis) inhibited core protein dephosphorylation and pgRNA packaging. Our work has thus revealed a novel function of core protein dephosphorylation in HBV replication and the mechanism by which CpAMs, a class of compounds that are currently in clinical trials for treatment of chronic hepatitis B, induce the assembly of empty capsids. Copyright © 2018 American Society for Microbiology.

Evaluation of medium-chain-length polyhydroxyalkanoate production by Pseudomonas putida LS46 using biodiesel by-product streams.

PubMed

Fu, Jilagamazhi; Sharma, Umesh; Sparling, Richard; Cicek, Nazim; Levin, David B

2014-07-01

Medium-chain-length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas putida LS46 was analyzed in shake-flask-based batch reactions, using pure chemical-grade glycerol (PG), biodiesel-derived "waste" glycerol (WG), and biodiesel-derived "waste" free fatty acids (WFA). Cell growth, substrate consumption, mcl-PHA accumulation within the cells, and the monomer composition of the synthesized biopolymers were monitored. The patterns of mcl-PHA synthesis in P. putida LS46 cells grown on PG and WG were similar but differed from that of cells grown with WFA. Polymer accumulation in glycerol-based cultures was stimulated by nitrogen limitation and plateaued after 48 h in both PG and WG cultures, with a total accumulation of 17.9% cell dry mass and 16.3% cell dry mass, respectively. In contrast, mcl-PHA synthesis was independent of nitrogen concentration in P. putida LS46 cells cultured with WFA, which accumulated to 29% cell dry mass. In all cases, the mcl-PHAs synthesized consisted primarily of 3-hydroxyoctanoate (C(8)) and 3-hydroxydecanoate (C(10)). WG and WFA supported similar or greater cell growth and mcl-PHA accumulation than PG under the experimental conditions used. These results suggest that biodiesel by-product streams could be used as low-cost carbon sources for sustainable mcl-PHA production.

Antibody Conjugation Approach Enhances Breadth and Potency of Neutralization of Anti-HIV-1 Antibodies and CD4-IgG

PubMed Central

Gavrilyuk, Julia; Ban, Hitoshi; Uehara, Hisatoshi; Sirk, Shannon J.; Saye-Francisco, Karen; Cuevas, Angelica; Zablowsky, Elise; Oza, Avinash; Seaman, Michael S.; Burton, Dennis R.

2013-01-01

Broadly neutralizing antibodies PG9 and PG16 effectively neutralize 70 to 80% of circulating HIV-1 isolates. In this study, the neutralization abilities of PG9 and PG16 were further enhanced by bioconjugation with aplaviroc, a small-molecule inhibitor of virus entry into host cells. A novel air-stable diazonium hexafluorophosphate reagent that allows for rapid, tyrosine-selective functionalization of proteins and antibodies under mild conditions was used to prepare a series of aplaviroc-conjugated antibodies, including b12, 2G12, PG9, PG16, and CD4-IgG. The conjugated antibodies blocked HIV-1 entry through two mechanisms: by binding to the virus itself and by blocking the CCR5 receptor on host cells. Chemical modification did not significantly alter the potency of the parent antibodies against nonresistant HIV-1 strains. Conjugation did not alter the pharmacokinetics of a model IgG in blood. The PG9-aplaviroc conjugate was tested against a panel of 117 HIV-1 strains and was found to neutralize 100% of the viruses. PG9-aplaviroc conjugate IC50s were lower than those of PG9 in neutralization studies of 36 of the 117 HIV-1 strains. These results support this new approach to bispecific antibodies and offer a potential new strategy for combining HIV-1 therapies. PMID:23427154

Effect of doublecortin on self-renewal and differentiation in brain tumor stem cells

PubMed Central

Santra, Manoranjan; Santra, Sutapa; Buller, Ben; Santra, Kastuv; Nallani, Ankita; Chopp, Michael

2011-01-01

Analysis of Affymetrix Probe data from glioma patient samples in conjuction with patient Kaplan-Meier Survival Plot indicate that expression of a glioma suppressor gene doublecortin (DCX) favors glioma patient survival. From neurosphere formation in culture, Time-Lapse Microscopy video recording and tumor xenograft, we show that DCX synthesis significantly reduces self-renewal of brain tumor stem cells (BTSCs) in human primary glioma (YU-PG, HF66) cells from surgically-removed human glioma specimens and U87 cells in vitro and in vivo. Time-Lapse Microscopic video recording revealed that double transfection of YU-PG, HF66 and U87 cells with DCX and neurabin II caused incomplete cell cycle with failure of cytokinesis, i.e. endomitosis by dividing into three daughter cells from one mother BTSC. Activation of c-jun NH2-terminal kinase 1 (JNK1) after simvastatin (10nM) treatment of DCX+neurabin II+ BTSCs from YU-PG, HF66 and U87 cells induced terminal differentiation into neuron-like cells. TUNEL staining data demonstrated that JNK1 activation also induced apoptosis only in double transfected BTSCs with DCX and neurabin II, but not in single transfected BTSCs from YU-PG, HF66 and U87 cells. Western blot analysis showed that procaspase-3 was induced after DCX transfection and activated after simvastatin treatment in YU-PG, HF66 and U87 BTSCs. Sequential immunoprecipitation and Western blot data revealed that DCX synthesis blocked protein phosphatase-1 (PP1)/caspase-3 protein-protein interaction and increased PP1-DCX interaction. These data demonstrate that DCX synthesis induces apoptosis in BTSCs via a novel JNK1/neurabin II/DCX/PP1/caspase-3 pathway. PMID:21477071

Effect of doublecortin on self-renewal and differentiation in brain tumor stem cells.

PubMed

Santra, Manoranjan; Santra, Sutapa; Buller, Ben; Santra, Kastuv; Nallani, Ankita; Chopp, Michael

2011-07-01

Analysis of microarray probe data from glioma patient samples, in conjunction with patient Kaplan-Meier survival plots, indicates that expression of a glioma suppressor gene doublecortin (DCX) favors glioma patient survival. From neurosphere formation in culture, time-lapse microscopic video recording, and tumor xenograft, we show that DCX synthesis significantly reduces self-renewal of brain tumor stem cells (BTSC) in human primary glioma (YU-PG, HF66) cells from surgically removed human glioma specimens and U87 cells in vitro and in vivo. Time-lapse microscopic video recording revealed that double transfection of YU-PG, HF66, and U87 cells with DCX and neurabin II caused incomplete cell cycle with failure of cytokinesis, that is, endomitosis by dividing into three daughter cells from one mother BTSC. Activation of c-jun NH2-terminal kinase 1 (JNK1) after simvastatin (10 nM) treatment of DCX(+) neurabin II(+) BTSC from YU-PG, HF66, and U87 cells induced terminal differentiation into neuron-like cells. dUTP nick end labeling data indicated that JNK1 activation also induced apoptosis only in double transfected BTSC with DCX and neurabin II, but not in single transfected BTSC from YU-PG, HF66, and U87 cells. Western blot analysis showed that procaspase-3 was induced after DCX transfection and activated after simvastatin treatment in YU-PG, HF66, and U87 BTSC. Sequential immunoprecipitation and Western blot data revealed that DCX synthesis blocked protein phosphatase-1 (PP1)/caspase-3 protein-protein interaction and increased PP1-DCX interaction. These data show that DCX synthesis induces apoptosis in BTSC through a novel JNK1/neurabin II/DCX/PP1/caspase-3 pathway. © 2011 Japanese Cancer Association.

Salmon cartilage proteoglycan suppresses mouse experimental colitis through induction of Foxp3{sup +} regulatory T cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mitsui, Toshihito; Department of Digestive Surgery, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori 036-8562; Sashinami, Hiroshi

Research highlights: {yields} Salmon proteoglycan suppresses IL-10{sup -/-} cell transfer-induced colitis progression. {yields} Salmon proteoglycan suppresses Th1- and Th17-related factors in colitis mice. {yields} Salmon proteoglycan enhances Foxp3 expression. -- Abstract: Proteoglycans (PGs) are complex glycohydrates which are widely distributed in extracellular matrix (ECM). PGs are involved in the construction of ECM, cell proliferation and differentiation. ECM components are involved in transduction of proinflammatory responses, but it is still unknown whether PGs are involved in inflammatory response. In this study, we investigated the effect of PG extracted from salmon cartilage on the progression of experimental colitis-induced in severe combined immunodeficiencymore » mice by cell transfer from interleukin-10 (IL-10){sup -/-} mice. IL-10{sup -/-} cell-transferred mice showed weight loss, colon shortening and histological appearance of mild colitis. Daily oral administration of PG attenuated the clinical progression of colitis in a dose-dependent manner. Colitis-induced mice showed the elevated expression of IFN-{gamma}, IL-12, TNF-{alpha}, IL-21, IL-23p19, IL-6, IL-17A and retinoic acid-related orphan receptor {gamma}t (ROR{gamma}t) in lamina propria mononuclear cells (LPMCs) and oral administration of PG suppressed the expression of these factors. Conversely, expression of Foxp3 that induces CD4{sup +}CD25{sup +} regulatory T cells in LPMCs was enhanced by PG administration. These findings suggested that salmon PG attenuated the progression of colitis due to suppression of inflammatory response by enhancement of regulatory T cell induction.« less

Mixed endocrine gastric tumors associated with hypergastrinemia of antral origin.

PubMed Central

Larsson, L. I.; Rehfeld, J. F.; Stockbrügger, R.; Blohme, G.; Schöön, I. M.; Lundqvist, G.; Kindblom, L. G.; Säve-Söderberg, J.; Grimelius, L.; Olbe, L.

1978-01-01

A patient with atrophic gastritis and excessively raised serum gastrin concentrations (4000 to 5000 pg/ml) was found to have multiple polypous tumors of the gastric corpus mucosa. Following gastrectomy, serum gastrin concentrations decreased to undetectable levels. The tumors consisted of a mixed population of endocrine cells. The majority of tumor cells were of the ECL type, but, in addition, enterochromaffin cells of various subtypes as well as agranular cells were found. The tumors were locally invasive and invaded the walls of submucosal blood vessels. The surrounding mucosa showed a severe atrophic gastritis with intestinalization and contained numerous goblet cells, enterochromaffin cells, and cholecystokinin cells. Cholecystokinin cells do not occur in the normal oxyntic mucosa. Hence, the observation of this cell type in intestinalized gastric epithelium suggests that "intestinalization also is associated with changes in endocrine cell populations. Gastrin has been shown to affect the function of the ECL cells. Indications for a trophic action of gastrin on these cells have been obtained. It is discussed whether greatly raised serum gastrin levels in patients with atrophic gastritis may be associated with increased risks for the development of certain types of gastric tumors. Images Figure 4 Figure 5 Figure 6 Figure 7 Figure 1 Figure 2 Figure 3 PMID:696807

Coordination of peptidoglycan synthesis and outer membrane constriction during Escherichia coli cell division

PubMed Central

Gray, Andrew N; Egan, Alexander JF; van't Veer, Inge L; Verheul, Jolanda; Colavin, Alexandre; Koumoutsi, Alexandra; Biboy, Jacob; Altelaar, A F Maarten; Damen, Mirjam J; Huang, Kerwyn Casey; Simorre, Jean-Pierre; Breukink, Eefjan; den Blaauwen, Tanneke; Typas, Athanasios; Gross, Carol A; Vollmer, Waldemar

2015-01-01

To maintain cellular structure and integrity during division, Gram-negative bacteria must carefully coordinate constriction of a tripartite cell envelope of inner membrane, peptidoglycan (PG), and outer membrane (OM). It has remained enigmatic how this is accomplished. Here, we show that envelope machines facilitating septal PG synthesis (PBP1B-LpoB complex) and OM constriction (Tol system) are physically and functionally coordinated via YbgF, renamed CpoB (Coordinator of PG synthesis and OM constriction, associated with PBP1B). CpoB localizes to the septum concurrent with PBP1B-LpoB and Tol at the onset of constriction, interacts with both complexes, and regulates PBP1B activity in response to Tol energy state. This coordination links PG synthesis with OM invagination and imparts a unique mode of bifunctional PG synthase regulation by selectively modulating PBP1B cross-linking activity. Coordination of the PBP1B and Tol machines by CpoB contributes to effective PBP1B function in vivo and maintenance of cell envelope integrity during division. DOI: http://dx.doi.org/10.7554/eLife.07118.001 PMID:25951518

Recognition of microbial molecular patterns and stimulation of prophenoloxidase activation by a β-1,3-glucanase-related protein in Manduca sexta larval plasma

PubMed Central

Wang, Yang; Sumathipala, Niranji; Rayaprolu, Subrahmanyam; Jiang, Haobo

2011-01-01

Detection of pathogenic invaders is the essential first step of a successful defense response in multicellular organisms. In this study, we have identified a new member of the β-1,3-glucanase-related protein superfamily from the tobacco hornworm Manduca sexta. This protein, designated microbe binding protein (MBP), is 61% identical in sequence to Bombyx mori Gram-negative bacteria binding protein, but only 34-36% identical to M. sexta β-1,3-glucan recognition protein-1 and 2. Its mRNA levels were strongly up-regulated in hemocytes and fat body of immune challenged larvae, along with an increase in concentration of the plasma protein. We expressed M. sexta MBP in a baculovirus-insect cell system. The purified protein associated with intact bacteria and fungi. It specifically bound to lipoteichoic acid, lipopolysaccharide, diaminopimelic acid-type peptidoglycans (DAP-PGs) from Escherichia coli and Bacillus subtilis, but less so to laminarin or Lys-type PG from Staphylococcus aureus. The complex binding pattern was influenced by other plasma factors and additional microbial surface molecules. After different amounts of MBP had been incubated with larval plasma on ice, a concentration-dependent increase in phenoloxidase (PO) activity occurred in the absence of any microbial elicitor. The activity increase was also observed in the mixture of plasma and a bacterial or fungal cell wall component. The prophenoloxidase (proPO) activation became more prominent when DAP-PGs, Micrococcus luteus Lys-PG, or lipoteichoic acid was included in the mixture of MBP and plasma. Statistic analysis suggested that a synergistic enhancement of proPO activation was caused by an interaction between MBP and these elicitors, but not S. aureus Lys-PG, lipopolysaccharide, curdlan, or laminarin. These data indicate that M. sexta MBP is a component of the surveillance mechanism and, by working together with other pattern recognition molecules and serine proteinases, triggers the proPO activation system. PMID:21296155

Elevated Plasma Levels of sIL-2R in Complex Regional Pain Syndrome: A Pathogenic Role for T-Lymphocytes?

PubMed Central

Stronks, Dirk L.; Dik, Willem A.; Schreurs, Marco W. J.

2017-01-01

The immune system has long been thought to be involved in the pathophysiology of complex regional pain syndrome (CRPS). However, not much is known about the role of the immune system and specifically T-cells in the onset and maintenance of this disease. In this study, we aimed to evaluate T-cell activity in CRPS by comparing blood soluble interleukin-2 receptor (sIL-2R) levels between CRPS patients and healthy controls. CRPS patients had statistically significant elevated levels of sIL-2R as compared to healthy controls (median sIL-2R levels: 4151 pg/ml (Q3 − Q1 = 5731 pg/ml − 3546 pg/ml) versus 1907 pg/ml (Q3 − Q1: 2206 pg/ml − 1374 pg/ml), p < 0.001, resp.). Furthermore, sIL-2R level seems to be a good discriminator between CRPS patients and healthy controls with a high sensitivity (90%) and specificity (89.5%). Our finding indicates increased T-cell activity in patients with CRPS. This finding is of considerable relevance as it could point towards a T-cell-mediated inflammatory process in this disease. This could pave the way for new anti-inflammatory therapies in the treatment of CRPS. Furthermore, sIL-2R could be a promising new marker for determining inflammatory disease activity in CRPS. PMID:28634419

The Phytotoxin Coronatine Induces Abscission-Related Gene Expression and Boll Ripening during Defoliation of Cotton

PubMed Central

Tian, Xiaoli; Duan, Liusheng; Zhang, Mingcai; Tan, Weiming; Xu, Dongyong; Li, Zhaohu

2014-01-01

Defoliants can increase machine harvest efficiency of cotton (Gossypium hirusutum L.), prevent lodging and reduce the time from defoliation to harvest. Coronatine (COR) is a chlorosis-inducing non-host-specific phytotoxin that induces leaf and/or fruit abscission in some crops. The present study investigates how COR might induce cotton leaf abscission by modulating genes involved in cell wall hydrolases and ACC (ethylene precursor) in various cotton tissues. The effects of COR on cotton boll ripening, seedcotton yield, and seed development were also studied. After 14 d of treatment with COR, cells within the leaf abscission zone (AZ) showed marked differentiation. Elevated transcripts of GhCEL1, GhPG and GhACS were observed in the AZs treated with COR and Thidiazuron (TDZ). The relative expression of GhCEL1 and GhACS in TDZ treated plants was approximately twice that in plants treated with COR for 12 h. However, only GhACS expression increased in leaf blade and petiole. There was a continuous increase in the activity of hydrolytic enzymes such as cellulase (CEL) and polygalacturonase (PG), and ACC accumulation in AZs following COR and TDZ treatments, but there was greater increase in ACC activity of COR treated boll crust, indicating that COR had greater ripening effect than TDZ. Coronatine significantly enhanced boll opening without affecting boll weight, lint percentage and seed quality. Therefore, COR can be a potential cotton defoliant with different physiological mechanism of action from the currently used TDZ. PMID:24845465

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duenas, Maria Emilia; Klein, Adam T.; Alexander, Liza E.

Metabolism in plants is compartmentalized among different tissues, cells and subcellular organelles. Mass spectrometry imaging (MSI) with matrix-assisted laser desorption ionization (MALDI) has recently advanced to allow for the visualization of metabolites at single-cell resolution. Here we applied 5- and 10 μm high spatial resolution MALDI-MSI to the asymmetric Kranz anatomy of Zea mays (maize) leaves to study the differential localization of two major anionic lipids in thylakoid membranes, sulfoquinovosyldiacylglycerols (SQDG) and phosphatidylglycerols (PG). The quantification and localization of SQDG and PG molecular species, among mesophyll (M) and bundle sheath (BS) cells, are compared across the leaf developmental gradient frommore » four maize genotypes (the inbreds B73 and Mo17, and the reciprocal hybrids B73 × Mo17 and Mo17 × B73). SQDG species are uniformly distributed in both photosynthetic cell types, regardless of leaf development or genotype; however, PG shows photosynthetic cell-specific differential localization depending on the genotype and the fatty acyl chain constituent. Overall, 16:1-containing PGs primarily contribute to the thylakoid membranes of M cells, whereas BS chloroplasts are mostly composed of 16:0-containing PGs. Furthermore, PG 32:0 shows genotype-specific differences in cellular distribution, with preferential localization in BS cells for B73, but more uniform distribution between BS and M cells in Mo17. Maternal inheritance is exhibited within the hybrids, such that the localization of PG 32:0 in B73 × Mo17 is similar to the distribution in the B73 parental inbred, whereas that of Mo17 × B73 resembles the Mo17 parent. As a result, this study demonstrates the power of MALDI-MSI to reveal unprecedented insights on metabolic outcomes in multicellular organisms at single-cell resolution.« less

Modification of response functions of cat visual cortical cells by spatially congruent perturbing stimuli.

PubMed

Kabara, J F; Bonds, A B

2001-12-01

Responses of cat striate cortical cells to a drifting sinusoidal grating were modified by the superimposition of a second, perturbing grating (PG) that did not excite the cell when presented alone. One consequence of the presence of a PG was a shift in the tuning curves. The orientation tuning of all 41 cells exposed to a PG and the spatial frequency tuning of 83% of the 23 cells exposed to a PG showed statistically significant dislocations of both the response function peak and center of mass from their single grating values. As found in earlier reports, the presence of PGs suppressed responsiveness. However, reductions measured at the single grating optimum orientation or spatial frequency were on average 1.3 times greater than the suppression found at the peak of the response function modified by the presence of the PG. Much of the loss in response seen at the single grating optimum is thus a result of a shift in the tuning function rather than outright suppression. On average orientation shifts were repulsive and proportional (approximately 0.10 deg/deg) to the angle between the perturbing stimulus and the optimum single grating orientation. Shifts in the spatial frequency response function were both attractive and repulsive, resulting in an overall average of zero. For both simple and complex cells, PGs generally broadened orientation response function bandwidths. Similarly, complex cell spatial frequency response function bandwidths broadened. Simple cell spatial frequency response functions usually did not change, and those that did broadened only 4% on average. These data support the hypothesis that additional sinusoidal components in compound stimuli retune cells' response functions for orientation and spatial frequency.

Abdominal Subcutaneous Fat: A Favorable or Nonfunctional Fat Depot for Glucose Metabolism in Chinese Adults?

PubMed

Hou, Xuhong; Chen, Peizhu; Hu, Gang; Wei, Li; Jiao, Lei; Wang, Hongmei; Liang, Yebei; Bao, Yuqian; Jia, Weiping

2018-06-01

The objective of this study was to assess the associations of abdominal visceral and subcutaneous adipose tissue with blood glucose and beta-cell function. In this study, 11,223 participants without known diabetes were selected for this cross-sectional analysis. Visceral and subcutaneous fat area (VFA and SFA) were measured by magnetic resonance imaging. An oral glucose tolerance test was conducted, and beta-cell function was evaluated. Men had significantly larger VFA but smaller SFA than women. After controlling for age, linear regression showed that SFA was adversely associated with 0-minute, 30-minute, and 2-hour plasma glucose (PG) and early-, first- and second-phase disposition indices (DIs). After further adjustment for BMI and VFA, some associations of SFA with PG indices and DIs disappeared, while the other associations became significantly weaker in men (2-hour PG: 0.05 and DI 2nd : -0.05) or were reversed in women (0-minute, 30-minute, and 2-hour PG: from -0.07 to -0.04; DI 1st : 0.04, P < 0.05). After adjustment for age, BMI, and SFA, VFA was significantly and adversely associated with PG indices and DIs, with the largest standardized regression coefficients with 2-hour PG. The associations of SFA with blood glucose and beta-cell function were clinically insignificant in Chinese adults. VFA had the strongest association with 2-hour PG. © 2018 The Obesity Society.

Membrane selectivity and disordering mechanism of antimicrobial peptide protegrin-1

NASA Astrophysics Data System (ADS)

Ishitsuka, Yuji

Protegrin-1 (PG-1) is a beta-sheet antimicrobial peptide (AMP), a class of peptides innate to various organisms and functions as a defense agent against harmful microorganisms by means of membrane disordering. Characteristic chemical and structural properties of AMPs allow selective interaction against invaders' cell membranes. Despite their enormous biomedical potential, progress towards developing them into therapeutic agents has been hampered by a lack of insight into their mechanism of action. AMP insertion assays using Langmuir monolayers reveal that both electrostatic properties of the lipid head group as well as the packing density of the lipid tail group play important roles in determining the membrane selectivity of AMPs. These results help elucidate how the AMP selectively targets the cell membrane of microorganisms over the cell membrane of the host. In addition, these results also explain the higher hemolytic ability of PG-1 against human red blood cells (RBCs) compared to the hemolytic ability of PG-1 against sheep and pig RBCs. Synchrotron X-ray reflectivity shows that PG-1 penetrates into the lipid layer. Grazing incidence X-ray diffraction and fluorescence microscopy indicate that the insertion of PG-1 disorders tail group packing. Membrane selectivity and insertion location information of AMPs with different primary sequence and secondary structure have been obtained by using a truncated version of PG-1: PC-17, and an alpha-helical AMP, LL-37, respectively. The similarity of the membrane disordering process across these various peptides motivated us to test the membrane disordering effect of molecules designed to mimic these peptides. Peptide-mimics based on meta-phenylene ethynylenes demonstrate similar membrane disordering effects, showing that the potency of AMPs is derived from their overall chemical and structural properties, rather than exact peptide sequence. Atomic force microscopy (AFM) was used to directly image first, the PG-1 concentration-dependent membrane thinning effect, and second, the PG-1-induced structural transformation of a contiguous supported bilayer patch into a porous one. Our results point the membrane disordering mechanism of PG-1 towards the carpet/toroidal model of membrane disordering. Ongoing AMP-related projects are also discussed.

Functional differences in the acinar cells of the murine major salivary glands.

PubMed

Kondo, Y; Nakamoto, T; Jaramillo, Y; Choi, S; Catalan, M A; Melvin, J E

2015-05-01

In humans, approximately 90% of saliva is secreted by the 3 major salivary glands: the parotid (PG), the submandibular (SMG), and the sublingual glands (SLG). Even though it is known that all 3 major salivary glands secrete saliva by a Cl(-)-dependent mechanism, salivary secretion rates differ greatly among these glands. The goal of this study was to gain insight into the properties of the ion-transporting pathways in acinar cells that might account for the differences among the major salivary glands. Pilocarpine-induced saliva was simultaneously collected in vivo from the 3 major salivary glands of mice. When normalized by gland weight, the amount of saliva secreted by the PG was more than 2-fold larger than that obtained from the SMG and SLG. At the cellular level, carbachol induced an increase in the intracellular [Ca(2+)] that was more than 2-fold larger in PG and SMG than in SLG acinar cells. Carbachol-stimulated Cl(-) efflux and the protein levels of the Ca(2+)-activated Cl(-) channel TMEM16A, the major apical Cl(-) efflux pathway in salivary acinar cells, were significantly greater in PG compared with SMG and SLG. In addition, we evaluated the transporter activity of the Na(+)-K(+)-2Cl(-) cotransporters (NKCC1) and anion exchangers (AE), the 2 primary basolateral Cl(-) uptake mechanisms in acinar cells. The SMG NKCC1 activity was about twice that of the PG and more than 12-fold greater than that of the SLG. AE activity was similar in PG and SLG, and both PG and SLG AE activity was about 2-fold larger than that of SMG. In summary, the salivation kinetics of the 3 major glands are distinct, and these differences can be explained by the unique functional properties of each gland related to Cl(-) movement, including the transporter activities of the Cl(-) uptake and efflux pathways, and intracellular Ca(2+) mobilization. © International & American Associations for Dental Research 2015.

Evidence for a Common Toolbox Based on Necrotrophy in a Fungal Lineage Spanning Necrotrophs, Biotrophs, Endophytes, Host Generalists and Specialists

PubMed Central

Andrew, Marion; Barua, Reeta; Short, Steven M.; Kohn, Linda M.

2012-01-01

The Sclerotiniaceae (Ascomycotina, Leotiomycetes) is a relatively recently evolved lineage of necrotrophic host generalists, and necrotrophic or biotrophic host specialists, some latent or symptomless. We hypothesized that they inherited a basic toolbox of genes for plant symbiosis from their common ancestor. Maintenance and evolutionary diversification of symbiosis could require selection on toolbox genes or on timing and magnitude of gene expression. The genes studied were chosen because their products have been previously investigated as pathogenicity factors in the Sclerotiniaceae. They encode proteins associated with cell wall degradation: acid protease 1 (acp1), aspartyl protease (asps), and polygalacturonases (pg1, pg3, pg5, pg6), and the oxalic acid (OA) pathway: a zinc finger transcription factor (pac1), and oxaloacetate acetylhydrolase (oah), catalyst in OA production, essential for full symptom production in Sclerotinia sclerotiorum. Site-specific likelihood analyses provided evidence for purifying selection in all 8 pathogenicity-related genes. Consistent with an evolutionary arms race model, positive selection was detected in 5 of 8 genes. Only generalists produced large, proliferating disease lesions on excised Arabidopsis thaliana leaves and oxalic acid by 72 hours in vitro. In planta expression of oah was 10–300 times greater among the necrotrophic host generalists than necrotrophic and biotrophic host specialists; pac1 was not differentially expressed. Ability to amplify 6/8 pathogenicity related genes and produce oxalic acid in all genera are consistent with the common toolbox hypothesis for this gene sample. That our data did not distinguish biotrophs from necrotrophs is consistent with 1) a common toolbox based on necrotrophy and 2) the most conservative interpretation of the 3-locus housekeeping gene phylogeny – a baseline of necrotrophy from which forms of biotrophy emerged at least twice. Early oah overexpression likely expands the host range of necrotrophic generalists in the Sclerotiniaceae, while specialists and biotrophs deploy oah, or other as-yet-unknown toolbox genes, differently. PMID:22253834

Natural killer cells mediate pathophysiology in response to reduced uterine perfusion pressure.

PubMed

Elfarra, Jamil; Amaral, Lorena M; McCalmon, Maggie; Scott, Jeremy D; Cunningham, Mark W; Gnam, Ashley; Ibrahim, Tarek; LaMarca, Babbette; Cornelius, Denise C

2017-12-01

Preeclampsia is associated with hypertension, small-for-gestational-age babies, and increased cytolytic natural killer (NK) cells. The specific role of cytolytic NK cells in the pathophysiology of preeclampsia has not been clearly defined. We hypothesized that R educed U terine P erfusion P ressure (RUPP) stimulates proliferation and cytolytic activation of NK cells, and that reducing NK cells in RUPP would prevent hypertension, intrauterine growth restriction, and inflammation in response to placental ischemia. RUPP was induced on gestation day (GD) 14 in pregnant rats. NK cells were depleted by i.p. administration of anti-asialo GM1 antibody on GDs 15 and 17. Placental and circulating NK cells were quantified via flow cytometry, mean arterial pressure (MAP), fetal weights, and cytokines were measured on GD 19. Total placental NK cells were 7.4 ± 2% of gated cells in normal pregnant (NP; n =10) and 16.5 ± 3% of gated cells in RUPP ( n =10) rats. Furthermore, cytolytic placental NK cells also increased in RUPP. Depletion of NK cells in RUPP (RUPP + anti-ASGM1) significantly improved MAP and fetal weights. MAP was 108 ± 2 mmHg in NP, 125 ± 2 mmHg in RUPP, and 112 ± 2 mmHg in RUPP + anti-ASGM1 ( n =12). Fetal weight was 2.32 ± 0.05 in NP, 1.8 ± 0.04g in RUPP, and increased to 2.0 ± 0.04g in RUPP + anti-ASGM1. Placental interferon-γ (IFN-γ) was 40.4 ± 5.2 pg/mg in NP, 72.17 ± 3.2 pg/mg in RUPP, and 44.0 ± 6.5 pg/mg in RUPP + anti-ASGM1 ( P <0.05). Placental tumor necrosis factor-α (TNF-α) was 17.9 ± 1.7 pg/mg in NP, 23.9 ± 2.2 pg/mg in RUPP, and 12.9 ± 2.3 pg/mg in RUPP + anti-ASGM1 ( P <0.05). Depletion of NK cells significantly lowered MAP, intrauterine growth restriction, and inflammation in RUPP rats indicating that cytolytic NK cells are important in preeclampsia pathophysiology. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Change of Peripheral Blood Treg/Thl7 in Cognitive Impairment with Chronic Renal Failure Patients.

PubMed

Wang, Jie; Li, Xue-Bin; Huang, Peng; Huang, Mei-Ying; Gu, Xian-Jun

2018-01-01

To investigate the changes in peripheral blood Treg/Th17 cell balance and its significance in patients with chronic renal failure (CRF) and cognitive impairment. A total of 71 patients with CRF were enrolled as a study group. The patients were divided into a cognitive impairment group and a normal cognitive function group according to the Mini-Mental State Examination (MMSE). Peripheral blood Treg and Th17 cells were analyzed by flow cytometry and their relevant cytokines (IL-17, IL-10 and TGF-β) and other biochemical indicators, including C-reactive protein (CRP) and IL-6, were determined by ELISA. Thepatients with both CRF and cognitive impairment were older than the cognitive normal groups. Peripheral blood Treg cells by Flow cytometry (the CRF cognitive impairment group 5.57±1.3%, CRF group with normal cognitive function 7.5 ± 0.9% and normal control group 9.7 ± 1.7%,P<0.05) and its related cytokines (IL-10 and TGF-β) by ELISA detection were lower in the group with cognitive impairment than in the group without cognitive impairment ( IL-10, 7.4±4.2 pg/mL, 13.8±3.9 pg/mL, 18.3±3.2 pg/mL; TGF-β 335.6±175.3 pg/mL, 512.7 ± 114.6 pg/mL, 953.8±373.4 pg/mL P < 0.05, respectively).However, Th17 cell numbers (the CRF cognitive impairment group 3.3 ± 0.7%, CRF group with normal cognitive function2.2 ± 0.5% and normal control group 1.5 ± 0.3%),and cytokine levels (IL-17, IL-6 and CRP) were higher in the group with cognitive impairment IL-6 (21.3 ± 5.1 pg/mL), IL-17 (18.5 ± 4.2 pg/mL) and CRP (20.3 ± 5.9 mg/L) in the CRF group with cognitive impairment when compared with the CRF group and normal cognitive function (12.2 ± 4.5 pg/mL, 12.1 ± 3.7 pg/mL and 13.5 ± 4.6 mg/L, respectively) or the normal control group (9.2 ± 5.8 pg/mL, 7.4 ± 2.6 pg/mL and 3.2 ± 1.3 mg/L, respectively, P<0.05). The frequencies of Treg in patients with CRF were positively correlated with the MMSE scores ((r = 0.518, P < 0.05), but the Th17 numbers were negatively correlated (r = -0.435, P < 0.05). An imbalance of peripheral blood Treg/Th17 cells is associated with cognitive impairment in patients with CRF. © 2018 The Author(s). Published by S. Karger AG, Basel.

Fractionation, partial characterization and bioactivity of water-soluble polysaccharides and polysaccharide-protein complexes from Pleurotus geesteranus.

PubMed

Zhang, Mei; Zhu, Lin; Cui, Steve W; Wang, Qi; Zhou, Ting; Shen, Hengsheng

2011-01-01

Fractionation and purification of mushroom polysaccharides is a critical process for mushroom clinical application. After a hot-water treatment, the crude Pleurotus geesteranus (PG) was further fractionated into four fractions (PG-1, -2, -3, -4) using gradient precipitation with water and ammonia sulphate. By controlling the initial polymer concentration and ratio of solvents, this process produced PG fractions with high chemical uniformity and narrow Mw distribution without free proteins. Structurally, PG-1 and PG-2 are pure homopolysaccharide mainly composed of glucose; and PG-3 and PG-4 are heteropolysaccharide-protein complexes. PG-2, a high M(w) fraction mainly composed of glucose presented significant cytotoxicity at the concentration of 200 and 100 μg/ml to human breast cancer cells. Here, we report a new mushroom polysaccharides extraction and fractionation method, with which we produced four fractions of PG with PG-2 appearing effective anti-tumour activity. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

Glyphosate catabolism by Pseudomonas sp

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shinabarger, D.L.

1986-01-01

The pathway for the degradation of glyphosate (N-phosphonomethylglycine) by Pseudomonas sp. PG2982 has been determined using metabolic radiolabeling experiments. Radiorespirometry experiments utilizing (3-/sup 14/C) glyphosate revealed that approximately 50-59% of the C3 carbon was oxidized to CO/sub 2/. Fractionation of stationary phase cells labeled with (3-/sup 14/C)glyphosate revealed that from 45-47% of the assimilated C3 carbon is distributed to proteins and that amino acids methionine and serine are highly labeled. The nucleic acid bases adenine and guanine received 90% of the C3 label that was incorporated into nucleic acids, and the only pyrimidine base labeled was thymine. Pulse labeling ofmore » PG2982 cells with (3-/sup 14/C)glyphosate revealed that (3-/sup 14/C)sarcosine is an intermediate in glyphosate degradation. Examination of crude extracts prepared from PG2982 cells revealed the presence of an enzyme that oxidizes sarcosine to glycine and formaldehyde. These results indicate that the first step in glyphosate degradation by PG2982 is cleavage of the carbon-phosphorus bond, resulting in the release of sarcosine and a phosphate group. The phosphate group is utilized as a source of phosphorus, and the sarcosine is degraded to glycine and formaldehyde. Phosphonate utilization by Pseudomonas sp. PG2982 was investigated. Each of the ten phosphonates tested were utilized as a sole source of phosphorus by PG2982. Representative compounds tested included alkylphosphonates, 1-amino-substituted alkylphosphonates, amino-terminal phosphonates, and an arylphosphonate. PG2982 cultures degraded phenylphosphonate to benzene and produced methane from methylphosphonate. The data indicate that PG2982 is capable of cleaving the carbon-phosphorus bond of several structurally different phosphonates.« less

49 CFR 173.27 - General requirements for transportation by aircraft.

Code of Federal Regulations, 2014 CFR

2014-10-01

... Fuel cells cartridges (UN3478, UN3479), see § 173.230 of this part. Class 3 PG I: Forbidden PG II: 0.5L... material is 1.0 L. For Fuel cell cartridges containing flammable liquids (UN3473), see § 173.230 of this...., UN3134) is 1 kg. For fuel cell cartridges containing water reactive substances (UN3476), see § 173.230 of...

49 CFR 173.27 - General requirements for transportation by aircraft.

Code of Federal Regulations, 2012 CFR

2012-10-01

... Fuel cells cartridges (UN3478, UN3479), see § 173.230 of this part. Class 3 PG I: Forbidden PG II: 0.5L... material is 1.0 L. For Fuel cell cartridges containing flammable liquids (UN3473), see § 173.230 of this...., UN3134) is 1 kg. For fuel cell cartridges containing water reactive substances (UN3476), see § 173.230 of...

49 CFR 173.27 - General requirements for transportation by aircraft.

Code of Federal Regulations, 2013 CFR

2013-10-01

... Fuel cells cartridges (UN3478, UN3479), see § 173.230 of this part. Class 3 PG I: Forbidden PG II: 0.5L... material is 1.0 L. For Fuel cell cartridges containing flammable liquids (UN3473), see § 173.230 of this...., UN3134) is 1 kg. For fuel cell cartridges containing water reactive substances (UN3476), see § 173.230 of...

Computational fluid dynamics (CFD) simulations of molten steel flow patterns and particle-wall adhesion in continuous casting of steels

NASA Astrophysics Data System (ADS)

Mohammadi-Ghaleni, Mahdi

The Sun has long been the most important energy source for planet Earth. Sunlight offers the potential to function as a source of clean, renewable energy; photovoltaic (PV) cells have been designed to tap into this abundant solar energy to generate electricity. Organic photovoltaic (OPV) devices show promise as technologies capable of lightweight, low cost and flexible alternatives to traditional silicon PV but the nature of conjugated organic and polymeric semiconductors have limited performance and, therefore, application. However, recent advances have shown that the addition of pristine graphene (PG) to the active layer of OPV devices can yield three-fold performance improvements in blends of P3HT (poly(3-hexylthiophene-2,5-diyl) & PCBM (phenyl C 61 butyric acid methyl ester) and, later, in all-polymer blends of P3HT & F8BT (poly(9,9-dioctylfluorene-alt-benzothiadiazole). In both OPV systems, increased performance is believed to be due to high charge carrier mobility imparted by the PG additive to the composite active layer blend. In this work, the effect of addition of PG to the active layer blend of P3HT & PCPDTBT (poly[2,6-(4,4-bis-(2-ethylhexyl)-4H-cyclopenta [2,1-b;3,4-b']dithiophene)-alt-4,7(2,1,3-benzothiadiazole)]) systems was investigated. PV devices were designed, fabricated and tested using standard processing methods and testing procedures. Although PG increased OPV device performance relative to samples without PG, power conversion efficiency (eta) on an absolute scale was lower than expected despite the otherwise complementary properties of these materials. Based on the literature, the low performance of these devices was hypothesized to result from non-ideal active layer morphology, lacking charge carried percolation pathways to the electrodes. Small angle neutron scattering (SANS) was employed to probe the active layer morphology in polymer blend films similar to the active layers of the cells. Deuterated P3HT (d-P3HT) was used to exploit the large scattering length density (SLD) contrast between hydrogen and deuterium. Rigorous analysis of the SANS data allowed the nanostructure to be determined and a model of disk-like d-P3HT crystallites dispersed in a matrix of the amorphous polymers was constructed. This structure shows limited interfacial area for exciton dissociation and exhibits a lack of charge percolation pathways to the electrodes. Morphological insight offered by SANS analysis along with literature review allowed higher performance all-polymer photovoltaic cells to be designed and tested using the same semiconducting polymers. By introducing a co-solvent and modifying the thermal annealing procedure, significant performance gains were realized for subsequent devices. The increased performance observed following the change in procedure is believed to be due to enhanced active layer morphology and formation of a bulk heterojunction (BHJ) structure, to be studied in future work. Although there is room for further performance gains in P3HT-PCPDTBT devices as well as application to other OPV systems in future work, the methods, results and discussion presented here highlight the importance of structure-property relationships in all-polymer photovoltaic cells.

CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours

PubMed Central

Costa-Cabral, Sara; Brough, Rachel; Konde, Asha; Aarts, Marieke; Campbell, James; Marinari, Eliana; Riffell, Jenna; Bardelli, Alberto; Torrance, Christopher; Lord, Christopher J.; Ashworth, Alan

2016-01-01

Activating KRAS mutations are found in approximately 20% of human cancers but no RAS-directed therapies are currently available. Here we describe a novel, robust, KRAS synthetic lethal interaction with the cyclin dependent kinase, CDK1. This was discovered using parallel siRNA screens in KRAS mutant and wild type colorectal isogenic tumour cells and subsequently validated in a genetically diverse panel of 26 colorectal and pancreatic tumour cell models. This established that the KRAS/CDK1 synthetic lethality applies in tumour cells with either amino acid position 12 (p.G12V, pG12D, p.G12S) or amino acid position 13 (p.G13D) KRAS mutations and can also be replicated in vivo in a xenograft model using a small molecule CDK1 inhibitor. Mechanistically, CDK1 inhibition caused a reduction in the S-phase fraction of KRAS mutant cells, an effect also characterised by modulation of Rb, a master control of the G1/S checkpoint. Taken together, these observations suggest that the KRAS/CDK1 interaction is a robust synthetic lethal effect worthy of further investigation. PMID:26881434

Endogenous Molecules Induced by a Pathogen-Associated Molecular Pattern (PAMP) Elicit Innate Immunity in Shrimp

PubMed Central

Chen, Yu-Yuan; Chen, Jiann-Chu; Lin, Yong-Chin; Kitikiew, Suwaree; Li, Hui-Fang; Bai, Jia-Chin; Tseng, Kuei-Chi; Lin, Bo-Wei; Liu, Po-Chun; Shi, Yin-Ze; Kuo, Yi-Hsuan; Chang, Yu-Hsuan

2014-01-01

Invertebrates rely on an innate immune system to combat invading pathogens. The system is initiated in the presence of cell wall components from microbes like lipopolysaccharide (LPS), β-1,3-glucan (βG) and peptidoglycan (PG), altogether known as pathogen-associated molecular patterns (PAMPs), via a recognition of pattern recognition protein (PRP) or receptor (PRR) through complicated reactions. We show herein that shrimp hemocytes incubated with LPS, βG, and PG caused necrosis and released endogenous molecules (EMs), namely EM-L, EM-β, and EM-P, and found that shrimp hemocytes incubated with EM-L, EM-β, and EM-P caused changes in cell viability, degranulation and necrosis of hemocytes, and increased phenoloxidase (PO) activity and respiratory burst (RB) indicating activation of immunity in vitro. We found that shrimp receiving EM-L, EM-β, and EM-P had increases in hemocyte count and other immune parameters as well as higher phagocytic activity toward a Vibrio pathogen, and found that shrimp receiving EM-L had increases in proliferation cell ratio and mitotic index of hematopoietic tissues (HPTs). We identified proteins of EMs deduced from SDS-PAGE and LC-ESI-MS/MS analyses. EM-L and EM-P contained damage-associated molecular patterns (DAMPs) including HMGBa, HMGBb, histone 2A (H2A), H2B, and H4, and other proteins including proPO, Rab 7 GPTase, and Rab 11 GPTase, which were not observed in controls (EM-C, hemocytes incubated in shrimp salt solution). We concluded that EMs induced by PAMPs contain DAMPs and other immune molecules, and they could elicit innate immunity in shrimp. Further research is needed to identify which individual molecule or combined molecules of EMs cause the results, and determine the mechanism of action in innate immunity. PMID:25517999

Excretion of extracellular lipids by Streptococcus mutans BHT and FA-1.

PubMed Central

Cabacungan, E; Pieringer, R A

1980-01-01

Streptococcus mutans BHT and FA-1, when grown to log phase on chemically defined medium containing [14C]glycerol, excreted 15% of the total biosynthesized 14C-lipid into the medium. When grown to early stationary phase, 28 to 33% of the 14C-lipid was found in the medium. The radioactive lipids of these varieties of S. mutans were identified as diacylglycerol, diglucosyl diacylglycerol (DGD), monoglucosyl diacylglycerol, diphosphatidylglycerol, phosphatidylglycerol (PG), and smaller amounts of two other lipids tentatively were identified as amino acyl-PG and glycerol phosphoryl-DGD. All lipids were found as extracellular and intracellular components from cells grown to either log or stationary phase. However, there were some shifts in the relative percentage of these lipids as the cells changed from log to stationary phase. For example, the intracellular lipid content of log-phase S. mutans BHT was composed of 49% PG and 19% DGD, but these percents shifted to 18% PG and 57% DGD when the cells were grown to stationary phase. However, the extracellular lipids of this organism contained 50 to 60% PG and 20% DGD in both log and stationary phases. PMID:7380539

A PCR-aided transcript titration assay (PATTY) to measure topoisomerase I gene expression in human tumor specimens.

PubMed

Meersma, G J; Bakker, M; Groen, H J; Van der Zee, A G; Jensen, P B; Giaccone, G; De Vries, E G; Smit, E F

1999-01-01

Topoisomerase I (topo I) inhibitors are promising anticancer agents with demonstrated activity against a wide range of solid tumors. Quantitative information on topol mRNA levels in tumor biopsies may predict response to topo I inhibitors. A polymerase chain reaction aided transcript titration assay (PATTY) was developed to allow quantitation of topol mRNA in small samples. Concentrations of topol mRNA in total RNA samples were estimated by RT-PCR analysis in a human small cell lung cancer (SCLC) cell line (GLC,) and its topotecan (GL2C/SK and F) and camptothecin (GL2C/Campt) resistant sublines, human non-small cell lung cancer (NSCLC) and ovarian carcinoma samples. Topol PATTY showed a decreased topo I mRNA level in GLC2/SK and F (4.5 pg/100 ng total RNA) and GLC,/Campt (2.2 pg/100 ng total RNA), respectively, compared to the parent cell line GLC2 (5.4 pg/100 ng total RNA). Topol protein levels as measured by Western blotting were compatible with topol mRNA levels. Median (range) topol mRNA levels were 3.23 (2.33-5.10) pg/100 ng total RNA in resected NSCLC specimen (n = 6), and 2.03 (0.54-0.95) pg/100 ng total RNA in resected ovarian cancer specimen (n = 6). We conclude that topol PATTY is a new assay that quantitates topol mRNA levels in cell lines and small tumor samples.

Angiotensin 1-7 Is a Negative Modulator of Aldosterone Secretion In Vitro and In Vivo.

PubMed

Shefer, Gabi; Marcus, Yonit; Knoll, Esther; Dolkart, Oleg; Foichtwanger, Shulamit; Nevo, Nava; Limor, Rona; Stern, Naftali

2016-08-01

Angiotensin (1-7) [Ang 1-7] is a 7 amino acid peptide generated predominantly from Ang II by the action of Ang-converting enzyme 2. We previously showed that Ang 1-7 reduced plasma aldosterone and plasma renin activity in high fructose-fed rats, and that the reduction in circulating aldosterone seemed to accord a parallel reduction in plasma renin activity. Here, we tested the possibility that Ang 1-7 affects aldosterone secretion acting directly in glomerulosa cells. First, as detected by immunofluorescence, the receptor for Ang 1-7, Mas1 is localized predominantly at the rat adrenal subcapsular region. Second, in isolated rat glomerulosa cells incubates, Ang 1-7 attenuated the aldosterone response to Ang II, with the strongest effect seen on Ang II (10(-9) M) (control 22±2.5 pg/10(5) cells; Ang II [10(-9) M] 189±11 pg/10(5) cells; Ang II [10(-9) M]+Ang 1-7 [10(-6) M] 33±3.6 pg/10(5) cells; P<0.001) and the largest effect on adrenocorticotropic hormone (10(-8) M) (control 30±3.4 pg/10(5) cells; ACTH [10(-8) M] 409±32.5 pg/10(5) cells; ACTH [10(-8) M]+Ang 1-7 [10(-6) M] 280±12.5 pg/10(5) cells; P<0.001). In contrast, Ang 1-7 did not affect the aldosterone response to potassium (K(+)). In rats subjected to a low-salt diet for 7 days, continuous infusion of Ang 1-7 (576 μg/kg per day) resulted in a lesser rise in aldosterone (salt deplete+Ang 1-7, 16.4±4.8 ng/dL) compared with rats receiving vehicle (salt deplete+vehicle, 27.6±5.3 ng/dL; P<0.01) but did not modify plasma renin activity. Taken together, these results indicate that Ang 1-7 can act as a negative modulator of aldosterone secretion in vitro and in vivo. © 2016 American Heart Association, Inc.

Research with parthenogenetic stem cells will help decide whether a safer clinical use is possible.

PubMed

Muñoz, M; Penarossa, G; Caamaño, J N; Díez, C; Brevini, T A L; Gómez, E

2015-04-01

The derivation and use of parthenogenetic stem cells (pESCs) are envisaged as a reliable alternative to conventional embryonic stem cells. Similar to embryonic stem cells in their proliferation, expression of pluripotency markers and capacity to multilineage differentiation, pESCs are at a lower risk of immune rejection within stem cell-based therapeutics. Moreover, pESCs represent an important model system to study the effect of paternally imprinted genes on cell differentiation. However, currently available information about the genetic and epigenetic behaviour of pESCs is limited. Thus, a detailed look at the biology of parthenogenetic (PG) embryos and PG-derived cell lines would allow gaining insight into the full potential of pESC in biotechnology. In this commentary article we review some features related to the biology of PG embryos and pESCs. In addition, novel traits on bovine pESCs (bpESCs) are discussed. Copyright © 2013 John Wiley & Sons, Ltd.

Higher biomolecules yield in phytoplankton under copper exposure.

PubMed

Silva, Jaqueline Carmo; Echeveste, Pedro; Lombardi, Ana Teresa

2018-05-30

Copper is an important metal for industry, and its toxic threshold in natural ecosystems has increased since the industrial revolution. As an essential nutrient, it is required in minute amounts, being toxic in slightly increased concentrations, causing great biochemical transformation in microalgae. This study aimed at investigating the physiology of Scenedesmus quadricauda, a cosmopolitan species, exposed to copper concentrations including those that trigger intracellular biochemical modifications. The Cu exposure concentrations tested ranged from 0.1 to 25 µM, thus including environmentally important levels. Microalgae cultures were kept under controlled environmental conditions and monitored daily for cell density, in vivo chlorophyll a, and photosynthetic quantum yield (Φ M ). After 24 h growth, free Cu 2+ ions were determined, and after 96 h, cellular Cu concentration, total carbohydrates, proteins, lipids, and cell volume were determined. The results showed that both free Cu 2+ ions and cellular Cu increased with Cu increase in culture medium. Microalgae cell abundance and in vivo chlorophyll a were mostly affected at 2.5 µM Cu exposure (3.8 pg Cu cell -1 ) and above. Approximately 31% decrease of photosynthetic quantum yield was obtained at the highest Cu exposure concentration (25 µM; 25 pg Cu cell -1 ) in comparison with the control. However, at environmentally relevant copper concentrations (0.5 µM Cu; 0.4 pg Cu cell -1 ) cell volume increased in comparison with the control. Considering biomolecules accumulation per unit cell volume, the highest carbohydrates and proteins yield was obtained at 1.0 µM Cu (1.1 pg Cu cell -1 ), while for lipids higher Cu was necessary (2.5 µM Cu; 3.8 pg Cu cell -1 ). This study is a contribution to the understanding of the effects of environmentally significant copper concentrations in the physiology of S. quadricauda, as well as to biotechnological approach to increase biomolecule yield in microalgae production. Copyright © 2018 Elsevier Inc. All rights reserved.

High spatial resolution mass spectrometry imaging reveals the genetically programmed, developmental modification of the distribution of thylakoid membrane lipids among individual cells of maize leaf

DOE PAGES

Duenas, Maria Emilia; Klein, Adam T.; Alexander, Liza E.; ...

2016-11-17

Metabolism in plants is compartmentalized among different tissues, cells and subcellular organelles. Mass spectrometry imaging (MSI) with matrix-assisted laser desorption ionization (MALDI) has recently advanced to allow for the visualization of metabolites at single-cell resolution. Here we applied 5- and 10 μm high spatial resolution MALDI-MSI to the asymmetric Kranz anatomy of Zea mays (maize) leaves to study the differential localization of two major anionic lipids in thylakoid membranes, sulfoquinovosyldiacylglycerols (SQDG) and phosphatidylglycerols (PG). The quantification and localization of SQDG and PG molecular species, among mesophyll (M) and bundle sheath (BS) cells, are compared across the leaf developmental gradient frommore » four maize genotypes (the inbreds B73 and Mo17, and the reciprocal hybrids B73 × Mo17 and Mo17 × B73). SQDG species are uniformly distributed in both photosynthetic cell types, regardless of leaf development or genotype; however, PG shows photosynthetic cell-specific differential localization depending on the genotype and the fatty acyl chain constituent. Overall, 16:1-containing PGs primarily contribute to the thylakoid membranes of M cells, whereas BS chloroplasts are mostly composed of 16:0-containing PGs. Furthermore, PG 32:0 shows genotype-specific differences in cellular distribution, with preferential localization in BS cells for B73, but more uniform distribution between BS and M cells in Mo17. Maternal inheritance is exhibited within the hybrids, such that the localization of PG 32:0 in B73 × Mo17 is similar to the distribution in the B73 parental inbred, whereas that of Mo17 × B73 resembles the Mo17 parent. As a result, this study demonstrates the power of MALDI-MSI to reveal unprecedented insights on metabolic outcomes in multicellular organisms at single-cell resolution.« less

Subchronic Infection of Porphyromonas gingivalis and Tannerella forsythia Stimulates an Immune Response but Not Arthritis in Experimental Murine Model

PubMed Central

Hernández-Aguas, Jorday; Montiel-Hernández, José Luis; Ruiz-Ramos, Rosa Velia; Escamilla García, Erandi; Guzmán-García, Mario Alberto; Ayón-Haro, Esperanza Raquel; Garza-Elizondo, Mario Alberto

2017-01-01

Studies have proposed that Porphyromonas gingivalis (Pg) and Tannerella forsythia (Tf) promote a nonspecific inflammatory response that could produce systemic disease. Oral inoculation of Pg and Tf on the immune and arthritis response was evaluated in BALB/C mice divided into four groups: (1) sham; (2) food contaminated with Pg/Tf; (3) complete Freund's adjuvant (CFA) + Pg/Tf; and (4) CFA alone. CFA was administered subcutaneously on days 1 and 14. The arthritis response was monitored for 21 days after day 14 of CFA administration. IL-1β and IL-6 were determined in serum. T cell activation was evaluated by CD25 in salivary lymph nodes or mouse spleen. Pad inflammation appeared by day 19 in the CFA group, but animals with bacteria inoculation presented a delay. A significant increase in IL-6 was found in Groups 3 and 4, but not with respect to IL-1β. We observed an increase in CD25 in cells derived from cervical nodes and in animals with bacteria inoculation and CFA. A local immune response was observed in mice inoculated with Pg and Tf (T cell activation); a systemic response was observed with CFA. Since pad inflammation was delayed by bacterial inoculation this suggests that local T cell activation could decrease pad inflammation. PMID:28676826

Microvascular Autonomic Composites

DTIC Science & Technology

2012-01-06

thermogravimetric analysis (TGA) was employed. The double wall allowed for increased thermal stability of the microcapsules, which was...fluorescent nanoparticles (Berfield et al. 2006). Digital Image Correlation (DIC) is a data analysis method, which applies a mathematical...Theme IV: Experimental Assessment & Analysis 2.4.1 Optical diagnostics for complex microfluidic systems pg. 50 2.4.2 Fluorescent thermometry

First report of the toxin profile of Dinophysis sacculus Stein from LC-MS analysis of laboratory cultures.

PubMed

Riobó, P; Reguera, B; Franco, J M; Rodríguez, F

2013-12-15

Dinophysis sacculus is associated with DSP outbreaks especially in the Mediterranean Sea and is supposed to be mildly toxic based on few toxin results from field samples. First report of LC-MS analysis of D. sacculus cultures from Galicia (NW Spain) showed moderate amounts of OA (7.8 pg cell(-1)) comparable to those found in Dinophysis acuminata from the same region, PTX2 (13.2 pg cell(-1)) and trace amounts of DTX1 (0.8 pg OA equiv. cell(-1)). The contribution of D. sacculus to DSP outbreaks in the Galician Northern Rías should not be underestimated. Copyright © 2013 Elsevier Ltd. All rights reserved.

[Bone marrow mesenchymal stem cells suppress E coli-induced bacterial prostatitis in rats].

PubMed

Han, Guang-wei; Liu, Cheng-cheng; Gao, Wen-hong; Cui, Dong; Yi, Shan-hong

2015-04-01

To investigate the inhibitory effect of bone marrow mesenchymal stem cells (BMSCs) on E coliinduced prostatitis in rats. BMSCs were isolated, cultured and amplified by the attached choice method. Fifty SD rats were randomized into five groups of equal number: normal control, acute bacterial prostatitis (ABP) , chronic bacterial prostatitis (CBP), ABP + BMSCs, and CBP + BMSCs, and the animals in the latter four groups were injected with E. coli into both sides of the prostate under ultrasound guidance for 1 - 14 days to induce ABP and for 4 - 12 weeks to induce CBP. The control rats were injected with the same amount of PBS. Two weeks after injection of BMSCs into the prostates, pathomorphological changes in the prostate were observed under the light microscope and the mRNA and protein levels of IL-1β and TNF-α determined by RT-PCR and ELISA, respectively, followed by statistical analysis with SPSS 18.0. Histopathological evaluation showed typical pathological inflammatory changes in the prostates of the rats in the ABP and CBP groups, including glandular structural changes, interstitial edema, inflammatory cell infiltration, and fibrous hyperplasia, which were all remarkably relieved after treated with BMSCs. The mRNA and protein levels of IL-β ([0.829 ± 0.121] and [271.75 ± 90.59] pg/ml) and TNF-α ([0.913 ± 0. 094] and [105.78 ± 19. 05] pg/ml) in the ABP and those of IL-1β ([0. 975 ± 0. 114] and [265. 31 ± 71. 34] pg/ml) and TNF-α ([0. 886 ± 0. 084] and [107. 45 ± 26. 11 ] pg/ml) in the CBP groups were significantly higher than those in the control rats ([0. 342 ± 0.087] and [45.76 17. 99] pg/ml, P <0. 05); ([0.247 ± 0.054] and ([19.42 ± 7. 75] pg/ml, P <0. 01) as well as than those in the ABP + BMSCs ([0. 433 ± 0. 072] and [51. 34 ± 22. 13] pg/ml, P < 0. 05 ) ; ( [0. 313 ± 0. 076] and [28. 38 ± 8. 78] pg/ml, P < 0. 01) and the CBP + BMSCs group ([0.396 ± 0.064] and [56.37 ± 21.22] pg/ml, P <0.05); ([0.417 ± 0.068] and [29.21 ± 10.22] pg/ml, P <0.01). Injection of BMSCs can reduce E coli-induced prostatic inflammation reaction, which.may be associated with its reduction of inflammatory cell infiltration and the expressions of IL-1β and TNF-α in the prostate tissue.

A Comprehensive Review of Punica granatum (Pomegranate) Properties in Toxicological, Pharmacological, Cellular and Molecular Biology Researches

PubMed Central

Rahimi, Hamid Reza; Arastoo, Mohammad; Ostad, Seyed Nasser

2012-01-01

Punica granatum (Pg), commonly known as pomegranate (Pg), is a member of the monogeneric family, Punicaceae, and is mainly found in Iran which is considered to be its primary centre of origin. Pg and its chemical components possess various pharmacological and toxicological properties including antioxidant, anti-inflammatory (by inhibiting pro-inflammatory cytokines), anti-cancer and anti-angiogenesis activities. They also show inhibitory effects on invasion/motility, cell cycle, apoptosis, and vital enzymes such as cyclooxygenase (COX), lipooxygenase (LOX), cytochrome P450 (CYP450), phospholipase A2 (PLA2), ornithine decarboxylase (ODC), carbonic anhydrase (CA), 17beta-hydroxysteroid dehydrogenase (17β-HSDs) and serine protease (SP). Furthermore, they can stimulate cell differentiation and possess anti-mutagenic effects. Pg can also interfere with several signaling pathways including PI3K/AKT, mTOR, PI3K, Bcl-X, Bax, Bad, MAPK, ERK1/2, P38, JNK, and caspase. However, the exact mechanisms for its pharmacological and toxicological properties remain to be unclear and need further evaluation. These properties strongly suggest a wide range use of Pg for clinical applications. This review will discuss the areas for which Pg has shown therapeutic properties in different mechanisms. PMID:24250463

Activation of postsynaptic GABAB receptors modulates the bursting pattern and synaptic activity of olfactory bulb juxtaglomerular neurons.

PubMed

Karpuk, Nikolay; Hayar, Abdallah

2008-01-01

Olfactory bulb glomeruli are formed by a network of three major types of neurons collectively called juxtaglomerular (JG) cells, which include external tufted (ET), periglomerular (PG), and short axon (SA) cells. There is solid evidence that gamma-aminobutyric acid (GABA) released from PG neurons presynaptically inhibits glutamate release from olfactory nerve terminals via activation of GABA(B) receptors (GABA(B)-Rs). However, it is still unclear whether ET cells have GABA(B)-Rs. We have investigated whether ET cells have functional postsynaptic GABA(B)-Rs using extracellular and whole cell recordings in olfactory bulb slices. In the presence of fast synaptic blockers (CNQX, APV, and gabazine), the GABA(B)-R agonist baclofen either completely inhibited the bursting or reduced the bursting frequency and increased the burst duration and the number of spikes/burst in ET cells. In the presence of fast synaptic blockers and tetrodotoxin, baclofen induced an outward current in ET cells, suggesting a direct postsynaptic effect. Baclofen reduced the frequency and amplitude of spontaneous EPSCs in PG and SA cells. In the presence of sodium and potassium channel blockers, baclofen reduced the frequency of miniature EPSCs, which were inhibited by the calcium channel blocker cadmium. All baclofen effects were reversed by application of the GABA(B)-R antagonist CGP55845. We suggest that activation of GABA(B)-Rs directly inhibits ET cell bursting and decreases excitatory dendrodendritic transmission from ET to PG and SA cells. Thus the postsynaptic GABA(B)-Rs on ET cells may play an important role in shaping the activation pattern of the glomeruli during olfactory coding.

Clopidogrel, a P2Y12 Receptor Antagonist, Potentiates the Inflammatory Response in a Rat Model of Peptidoglycan Polysaccharide-Induced Arthritis

PubMed Central

Rico, Mario C.; Dela Cadena, Raul A.; Kunapuli, Satya P.

2011-01-01

The P2Y12 receptor plays a crucial role in the regulation of platelet activation by several agonists, which is irreversibly antagonized by the active metabolite of clopidogrel, a widely used anti-thrombotic drug. In this study, we investigated whether reduction of platelet reactivity leads to reduced inflammatory responses using a rat model of erosive arthritis. We evaluated the effect of clopidogrel on inflammation in Lewis rats in a peptidoglycan polysaccharide (PG-PS)-induced arthritis model with four groups of rats: 1) untreated, 2) clopidogrel-treated, 3) PG-PS-induced, and 4) PG-PS-induced and clopidogrel-treated. There were significant differences between the PG-PS+clopidogrel group when compared to the PG-PS group including: increased joint diameter and clinical manifestations of inflammation, elevated plasma levels of pro-inflammatory cytokines (IL-1 beta, interferon (IFN) gamma, and IL-6), an elevated neutrophil blood count and an increased circulating platelet count. Plasma levels of IL-10 were significantly lower in the PG-PS+clopidogrel group compared to the PG-PS group. Plasma levels of platelet factor 4 (PF4) were elevated in both the PG-PS and the PG-PS+clopidogrel groups, however PF4 levels showed no difference upon clopidogrel treatment, suggesting that the pro- inflammatory effect of clopidogrel may be due to its action on cells other than platelets. Histology indicated an increase in leukocyte infiltration at the inflammatory area of the joint, increased pannus formation, blood vessel proliferation, subsynovial fibrosis and cartilage erosion upon treatment with clopidogrel in PG-PS-induced arthritis animals. In summary, animals treated with clopidogrel showed a pro-inflammatory effect in the PG-PS-induced arthritis animal model, which might not be mediated by platelets. Elucidation of the mechanism of clopidogrel-induced cell responses is important to understand the role of the P2Y12 receptor in inflammation. PMID:22028806

Modified end-to-side double-layer open pancreaticogastrostomy after Whipple procedure: surgical tips for a safe anastomosis.

PubMed

Dalla Valle, Raffaele; Rossini, Matteo; Lamecchi, Laura; Iaria, Maurizio

2018-03-01

Pancreatic fistula (PF) remains the Achilles' heel of pancreaticoduodenectomy (PD). Pancreaticogastrostomy (PG) appears to be associated with a lower risk of postoperative leak according to recent evidence. We started to fashion PG, especially in soft pancreas, modifying the original technique described by Bassi. At our institution, 105 PD procedures were carried out from January 2011 to December 2016; pancreatic-enteric continuity was restored by PG in 35 cases. Superior mesenteric/portal vein resection/reconstruction was necessary in three patients. A total of 34/35 patients underwent PG with an open anterior gastrostomy approach. Briefly, our double-layer PG anastomosis (illustrated by a video) starts with a posterior row of interrupted absorbable 4/0 monofilament sutures including the gastric serosa and the pancreatic capsule. It is essential to mobilize the left pancreas for 4-5 cm and to shape the posterior gastrostomy shorter than the pancreatic stump. After a wide anterior auxiliary gastrostomy the pancreas is invaginated into the stomach and an interrupted row of sutures between the posterior gastric wall (full-thickness) and the body of the pancreatic stump is fashioned. The anterior gastrostomy is closed with an absorbable running suture. Finally, a further layer of sutures is applied over the posterior suture line between the gastric serosa and the pancreatic capsule. The 90-day postoperative mortality was nihil. No biliary leakage was detected and the overall PF rate was 11.4% (4/35) according to the ISGPF study group. Only one patient suffered a grade B PF (in this case, PG was carried out only through a posterior gastrostomy), whereas three patients had a minor (grade A) PF. Our modified PG proved to be safe and easy to perform, while it carried excellent outcomes even in the setting of soft pancreas. Despite the limited number of cases, such modified PG appears promising, particularly for pancreatic remnants at higher risk of PF.

Copper-phospholipid interaction at cell membrane model hydrophobic surfaces.

PubMed

Mlakar, Marina; Cuculić, Vlado; Frka, Sanja; Gašparović, Blaženka

2018-04-01

Detailed investigation of Cu (II) binding with natural lipid phosphatidylglycerol (PG) in aqueous solution was carried out by voltammetric measurements at the mercury drop electrode, complemented by monolayer studies in a Langmuir trough and electrophoretic measurements, all used as models for hydrophobic cell membranes. Penetration of copper ions into the PG layer was facilitated by the formation of hydrophilic Cu-Phenanthroline (Phen) complex in the subphase, followed by the mixed ligand Cu-Phen-PG complex formation at the hydrophobic interface. Electrophoretic measurements indicated a comparatively low abundance of the formed mixed ligand complex within the PG vesicles, resulting it the zeta potential change of +0.83mV, while monolayer studies confirmed their co-existence at the interface. The Cu-Phen-PG complex was identified in the pH range from 6 to 9. The stoichiometry of the complex ([PhenCuOHPG]), as well as its stability and kinetics of formation, were determined at the mercury drop electrode. Cu-Phen-PG reduces quasireversibly at about -0.7V vs. Ag/AgCl including reactant adsorption, followed by irreversible mixed complex dissociation, indicating a two-electron transfer - chemical reaction (EC mechanism). Consequently, the surface concentration (γ) of the adsorbed [PhenCuOHPG] complex at the hydrophobic electrode surface was calculated to be (3.35±0.67)×10 -11 molcm -2 . Information on the mechanism of Cu (II) - lipid complex formation is a significant contribution to the understanding of complex processes at natural cell membranes. Copyright © 2017 Elsevier B.V. All rights reserved.

Positive and negative regulatory regions control the spatial distribution of polygalacturonase transcription in tomato fruit pericarp.

PubMed Central

Montgomery, J; Pollard, V; Deikman, J; Fischer, R L

1993-01-01

The tomato fruit consists of a thick, fleshy pericarp composed predominantly of highly vacuolated parenchymatous cells, which surrounds the seeds. During ripening, the activation of gene expression results in dramatic biochemical and physiological changes in the pericarp. The polygalacturonase (PG) gene, unlike many fruit ripening-induced genes, is not activated by the increase in ethylene hormone concentration associated with the onset of ripening. To investigate ethylene concentration-independent gene transcription in ripe tomato fruit, we analyzed the expression of chimeric PG promoter-beta-glucuronidase (GUS) reporter gene fusions in transgenic tomato plants. We determined that a 1.4-kb PG promoter directs ripening-regulated transcription in outer pericarp but not in inner pericarp cells, with a sharp boundary of PG promoter activity located midway through the pericarp. Promoter deletion analysis indicated that a minimum of three promoter regions influence the spatial regulation of PG transcription. A positive regulatory region from -231 to -134 promotes gene transcription in the outer pericarp of ripe fruit. A second positive regulatory region from -806 to -443 extends gene activity to the inner pericarp. However, a negative regulatory region from -1411 to -1150 inhibits gene transcription in the inner pericarp. DNase I footprint analysis showed that nuclear proteins in unripe and ripe fruit interact with DNA sequences within each of these three regulatory regions. Thus, temporal and spatial control of PG transcription is mediated by the interaction of negative and positive regulatory promoter elements, resulting in gene activity in the outer pericarp but not the inner pericarp of ripe tomato fruit. The expression pattern of PG suggests that, although they are morphologically similar, there is a fundamental difference between the parenchymatous cells within the inner and outer pericarp. PMID:8400876

Pro-inflammatory Analysis of Macrophages in Contact with Titanium Particles and Porphyromonas gingivalis.

PubMed

Dodo, Cindy Goes; Meirelles, Luiz; Aviles-Reyes, Alejandro; Ruiz, Karina Gonzalez Silvério; Abranches, Jacqueline; Cury, Altair Antoninha Del Bel

2017-01-01

During insertion of titanium dental implants, particles may shear from the implant to the periimplant region causing osteolysis, and their association with bacteria can exacerbate the inflammatory reaction. However, the association of a high invasive bacterium from the oral cavity, Porphyromonas gingivalis (Pg), and titanium particles remains unknown. This study evaluated pro-inflammatory reaction of human macrophages in contact with micro and nanoparticles of titanium associated with Porphyromonas gingivalis lipopolysaccharide (PgLPS). THP-1 cell were used and treated for 12, 24 and 48 h following 6 groups: Control(C), PgLPS (L); Microparticles (M); Nanoparticles (N); PgLPS and microparticles (LM); PgLPS and nanoparticles (LN). The following assays were carried out: i) cell viability using MTS, ii) cell morphology by SEM and iii) expression of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) by qRT-PCR and ELISA. For statistics two-way ANOVA followed by Tukey's test was used (p<0.05). After treatment, cells presented similar viability and morphology demonstrating that the treatments were not able to induce cell death. Gene expression was significantly higher for TNF-α and IL1-β after 12 h, and for IL-6 after 24 h in the N and LN groups. Cytokine production over time was an ascending curve for TNF-α with the peak at 48 h and IL1-β and IL-6 had a straight line among the time points, although cells from N group presented a significant production of IL-6 at 48 h. In conclusion, these results suggest that titanium nanoparticles stimulate stronger pro-inflammatory response in macrophages, independent of their association with LPS from P.gingivalis.

The algicidal mechanism of prodigiosin from Hahella sp. KA22 against Microcystis aeruginosa.

PubMed

Yang, Ke; Chen, Qiuliang; Zhang, Danyang; Zhang, Huajun; Lei, Xueqian; Chen, Zhangran; Li, Yi; Hong, Yaling; Ma, Xiaohong; Zheng, Wei; Tian, Yun; Zheng, Tianling; Xu, Hong

2017-08-10

In recent years, Microcystis aeruginosa blooms have occurred throughout the world, causing huge economic losses and destroying aquatic ecosystems. It is necessary to develop effective and ecofriendly methods to control M. aeruginosa blooms. Here, we report a high algicidal activity of prodigiosin (PG) against M. aeruginosa as well as the algicidal mechanism. PG showed high algicidal activity against M. aeruginosa, with a 50% lethal dose (LD 50 ) of 5.87 μg/mL in 72 h. A combination of methods, including propidium iodide and Annexin V-fluorescein staining assays and light and electron microscopy indicated the existence of two modes of cell death with features similar to those in eukaryotic programmed cell death: necrotic-like and apoptotic-like. Biochemical and physiological analyses showed that PG generates reactive oxygen species (ROS), which induce lipid peroxidation, damage the membrane system and destroy the function of the photosystem. A proteomics analysis revealed that many proteins were differentially expressed in response to PG stress and that most of these proteins were involved in important metabolic processes, which may trigger necrotic-like or apoptotic-like cell death. The present study sheds light on the multiple toxicity mechanisms of PG on M. aeruginosa and its potential for controlling the occurrence of M. aeruginosa blooms in lakes.

Proliferation of smooth muscle cells stimulated by Porphyromonas gingivalis is inhibited by apple polyphenol.

PubMed

Inaba, Hiroaki; Tagashira, Motoyuki; Kanda, Tomomasa; Amano, Atsuo

2011-11-01

Porphyromonas gingivalis (Pg) is thought to be involved in the progression of occlusive arterial lesions, whereas vascular smooth muscle cell (SMC) proliferation is considered to be involved in occlusive arterial disease. We previously showed that bacteremia caused by Pg infection induced proliferation of mouse aortic SMCs. Furthermore, human SMCs stimulated with human plasma incubated with Pg showed a marked transformation from the contractile to proliferative phenotype. In the present study, we examine the involvement of Pg gingipains and fimbriae in induction of the SMC transformation and proliferation, and effective inhibitors. Pg strains including gingipain- and fimbria-null mutants were incubated in human plasma, after which the bacteria were removed and the supernatants were added to cultured SMCs. To evaluate the effects of inhibitors, Pg organisms were incubated in plasma in the presence of apple polyphenol (AP), epigallocatechin gallate, KYT-1 (Arg-gingipain inhibitor), and KYT-36 (Lys-gingipain inhibitor). Plasma supernatants from wild-type and fimbria-mutant cultures markedly stimulated cellular proliferation, whereas those containing gingipain-null mutants showed negligible effects. SMC proliferation was also induced by plasma treated with trypsin. Furthermore, plasma supernatants cultured in the presence of KYT-1/KYT-36 and AP showed significant inhibitory effects on SMC proliferation, whereas cultures with epigallocatechin gallate did not. Our results suggest that Pg gingipains are involved in the induction of SMC transformation and proliferation, whereas this was inhibited by AP.

Hypersensitive Ethylene Signaling and ZMdPG1 Expression Lead to Fruit Softening and Dehiscence

PubMed Central

Li, Min; Zhang, Yanmin; Zhang, Zongying; Ji, Xiaohao; Zhang, Rui; Liu, Daliang; Gao, Liping; Zhang, Jing; Wang, Biao; Wu, Yusen; Wu, Shujing; Chen, Xiaoliu; Feng, Shouqian; Chen, Xuesen

2013-01-01

‘Taishanzaoxia’ fruit rapid softening and dehiscence during ripening stage and this process is very sensitive to endogenous ethylene. In this study, we cloned five ethylene signal transcription factors (ZMdEIL1, ZMdEIL2, ZMdEIL3, ZMdERF1 and ZMdERF2) and one functional gene, ZMdPG1, encoding polygalacturonase that could loose the cell connection which associated with fruit firmness decrease and fruit dehiscence to illustrate the reasons for this specific fruit phenotypic and physiological changes. Expression analysis showed that ZMdERF1 and ZMdEIL2 transcription were more abundant in ‘Taishanzaoxia’ softening fruit and dehiscent fruit and their expression was inhibited by an ethylene inhibitor 1-methylcyclopropene. Therefore, ZMdERF1 and ZMdEIL2 expression were responses to endogenous ethylene and associated with fruit softening and dehiscence. ZMdPG1 expression was induced when fruit softening and dehiscence but this induction can be blocked by 1-MCP, indicating that ZMdPG1 was essential for fruit softening and dehiscence and its expression was mediated by the endogenously occurred ethylene. ZMdPG1 overexpression in Arabidopsis led to silique early dehiscence while suppressing ZMdPG1 expression by antisense ZMdPG1 prevented silique naturally opening. The result also suggested that ZMdPG1 related with the connection between cells that contributed to fruit softening and dehiscence. ZMdERF1 was more closely related with ethylene signaling but it was not directly regulated the ZMdPG1, which might be regulated by the synergic pattern of ethylene transcription factors because of both the ZMdERF1 and ZMdERF2 could interact with ZMdEIL2. PMID:23527016

D2-40 negative pyogenic granuloma-like Kaposi's sarcoma: Diagnostic features and histogenetic hypothesis of an uncommon skin tumor in HIV-negative patients.

PubMed

Cabibi, D; Giannone, A G; Guarnotta, C; Schillaci, O; Franco, V

2015-07-01

Pyogenic granuloma-like Kaposi's sarcoma (PGLKS) is a recently described skin tumor showing features both of pyogenic granuloma (PG) and Kaposi's sarcoma (KS). The differential diagnosis is often challenging. We reviewed a series of 50 PG and 23 Ks located on distal extremities with the aid of an immunohistochemical panel comprising CD34, CD31, FVIII, SMA, D2-40, HHV8. After revision, 6/50 PG lesions previously diagnosed as PG, showed positive immunostaining for LNA1-HHV8 and focal positivity for CD31 and FVIII in the endothelial cells of the proliferating vessels, with some SMA positive pericytes. D2-40, a marker of lymphatic endothelium positive in KS, stained negatively. These lesions were renamed PGLKS. Of note, in our series, PGLKS represented the only form of KS localized in the hand; all the patients were HIV-negative, older than PG patients, with a prevalence for male gender. PGLKS and PG need a different management and a follow-up is advisable for PGLKS, as for the other variants of KS. To date, D2-40 negative immunostaining has not yet been reported in PGLKS and should not lead to a misdiagnosis of PG. The morphological similarities with PG and the immunohistochemical findings, showing a defective phenotype of the neoplastic cells, suggest a histogenetic hypothesis in which D2-40 negative PGLKS could represent an early stage of HHV8 infection of a pre-existing PG, whose vessels loose progressively their blood vascular markers but have not still acquired the lymphatic ones. Copyright © 2015 Elsevier GmbH. All rights reserved.

Pyrogallol, an absorbable microbial gallotannins-metabolite and mango polyphenols (Mangifera Indica L.) suppress breast cancer ductal carcinoma in situ proliferation in vitro.

PubMed

Nemec, Matthew J; Kim, Hyemee; Marciante, Alexandria B; Barnes, Ryan C; Talcott, Stephen T; Mertens-Talcott, Susanne U

2016-09-14

Mango is rich in bioactive absorbable polyphenols, but also contains considerable amounts of unabsorbable gallotannins at varying degrees of polymerization. Gallotannins are not absorbable upon consumption and have rarely been considered in the discussion of health benefits of polyphenols. Therefore, the objective of this study was to investigate the anti-proliferative activities of the major microbial metabolite of gallotannins, pyrogallol (PG) and a low molecular weight fraction of mango (Mangifera Indica L.) polyphenols (ML) and involved pathways including the AKT/mTOR signaling axis in an in situ breast cancer cell line, MCF10DCIS.COM. Fluorouracil (5-FU), a widely used genotoxic cancer therapeutic, was used a positive control and in combination with ML and PG to assess potential interactions. Concentrations that were non-cytotoxic in non-cancer cells were identified in non-cancer mammary fibroblasts (MCF-12F) and only non-cytotoxic dietarily relevant concentrations were selected for the investigation in MCF10DCIS.COM cancer cells. In addition to proliferation and viability, mRNA and expression of total and phosphorylated protein were investigated. Results show that both, ML and PG significantly reduced proliferation in MCF10DCIS.COM, but did not significantly reduce viability following a 48 h exposure. ML significantly reduced mRNA expression of mTOR and HIF-1α, while PG significantly reduced mRNA of IGF-1R, AKT, mTOR and HIF-1α. ML and PG reduced total protein expression of IGF-1R, IR, AKT, mTOR, and P70S6K. In addition, PG reduced IRS protein. Both treatments also had an effect on phosphorylated protein levels, with PG significantly reducing IGF-1R, AKT, and P70S6K levels. ML had a similar effect and significantly decreased IR, AKT, and P70S6K phosphorylation levels. Within the low concentration-range, ML and PG did not interact with the cytotoxic activities of 5-FU. Overall, the AKT/mTOR signaling axis appears to be implicated as causal in decreased proliferation induced by diet-relevant concentrations of ML and PG.

Acute myelobalstic leukemia and hypercalcemia. A case of probable ectopic parathyroid hormone production.

PubMed

Zidar, B L; Shadduck, R K; Winkelstein, A; Zeigler, Z; Hawker, C D

1976-09-23

We studied a patient with acute myeloblastic leukemia, hypercalcemia, hypophosphatemia and inappropriately elevated serum parathyroid hormone levels to define the mechanism of the hypercalcemia. On six occasions during two years, hypercalcemia occurred in conjunction with relapses of leukmia. Each time, serum calcium decreased to normal levels in parallel with reduction of the leukemic mass. During two periods of hypercalcemia, immunoreactive parathyroid hormone values were abnormally high. In addition, hormone was detected in vitro after short-term incubation of the leukemic cells (after 24 hours, the patient's cells produced 129 pg of PTH per milliliter, whereas myeloblasts from a normocalcemic patient with leukemia produced only 33 pg). In freeze-thawing experiments, 39 pg of parathyroid hormone was released form 1 x 108 of the patient's myeloblasts; no hormone was released from the normocalcemia cells. These findings suggest that the hypercalcemia resulted from ectopic parathyroid hormone production by leukemic cells.

An update on polygalacturonase-inhibiting protein (PGIP), a leucine-rich repeat protein that protects crop plants against pathogens

PubMed Central

Kalunke, Raviraj M.; Tundo, Silvio; Benedetti, Manuel; Cervone, Felice; De Lorenzo, Giulia; D'Ovidio, Renato

2015-01-01

Polygalacturonase inhibiting proteins (PGIPs) are cell wall proteins that inhibit the pectin-depolymerizing activity of polygalacturonases secreted by microbial pathogens and insects. These ubiquitous inhibitors have a leucine-rich repeat structure that is strongly conserved in monocot and dicot plants. Previous reviews have summarized the importance of PGIP in plant defense and the structural basis of PG-PGIP interaction; here we update the current knowledge about PGIPs with the recent findings on the composition and evolution of pgip gene families, with a special emphasis on legume and cereal crops. We also update the information about the inhibition properties of single pgip gene products against microbial PGs and the results, including field tests, showing the capacity of PGIP to protect crop plants against fungal, oomycetes and bacterial pathogens. PMID:25852708

Isolation and Identification of Acholeplasma sp. from the Mud Crab, Scylla serrata

PubMed Central

Chen, Ji-Gang; Lou, Dan; Yang, Ji-Fang

2011-01-01

For the first time, a mollicute-like organism (MLO) was cultured from moribund mud crabs (Scylla serrata) during an outbreak of clearwater disease in Zhejiang Province, China. The MLO displayed a fried-egg colony morphology in culture, did not possess a cell wall, and was not retained by 0.45 μm and 0.2 μm filters. It was able to ferment glucose, sucrose, lactose, and maltose, but it did not utilize arginine and urea. The MLO grew in the absence of bovine serum and was not susceptible to digitonin. Sequence analysis of the 16S rRNA gene revealed that this MLO had 99% identity with Acholeplasma laidlawii PG-8A, which indicates that the organism isolated from mud crabs is a member of the genus Acholeplasma. PMID:21808652

Functional Assessment of Skeletal Muscle Regeneration Utilizing Homologous Extracellular Matrix as Scaffolding

DTIC Science & Technology

2010-01-01

as a biologic scaffold material. Biomaterials 28, 3587, 2007. 24. Conconi, M.T., De Coppi, P., Bellini, S., Zara , G., Sabatti, M., Marzaro, M., Zanon...full-thickness ab- dominal wall defects. Tissue Eng 12, 1929, 2006. 26. Gamba, P.G., Conconi, M.T., Lo Piccolo, R., Zara , G., Spi- nazzi, R., and

Lactoferricin mediates anabolic and anti-catabolic effects in the intervertebral disc.

PubMed

Kim, Jae-Sung; Ellman, Michael B; An, Howard S; Yan, Dongyao; van Wijnen, Andre J; Murphy, Gillian; Hoskin, David W; Im, Hee-Jeong

2012-04-01

Lactoferricin (LfcinB) antagonizes biological effects mediated by angiogenic and catabolic growth factors, in addition to pro-inflammatory cytokines and chemokines in human endothelial cells and tumor cells. However, the effect of LfcinB on intervertebral disc (IVD) cell metabolism has not yet been investigated. Using bovine nucleus pulposus (NP) cells, we analyzed the effect of LfcinB on proteoglycan (PG) accumulation, PG synthesis, and anabolic gene expression. We assessed expression of genes for matrix-degrading enzymes such as matrix metalloproteases (MMPs) and a disintegrin-like and metalloprotease with thrombospondin motifs (ADAMTS family), as well as their endogenous inhibitors, tissue inhibitor of metalloproteases (TIMPs). In order to understand the specific molecular mechanisms by which LfcinB exerts its biological effects, we investigated intracellular signaling pathways in NP cells. LfcinB increased PG accumulation mainly via PG synthesis in a dose-dependent manner. Simultaneously, LfcinB dose-dependently downregulated catabolic enzymes. LfcinB's anti-catabolic effects were further demonstrated by a dose-dependent increase in multiple TIMP family members. Our results demonstrate that ERK and/or p38 mitogen-activated protein kinase pathways are the key signaling cascades that exert the biological effects of LfcinB in NP cells, regulating transcription of aggrecan, SOX-9, TIMP-1, TIMP-2, TIMP-3, and iNOS. Our results suggest that LfcinB has anabolic and potent anti-catabolic biological effects on bovine IVD cells that may have considerable promise in the treatment of disc degeneration in the future. Copyright © 2011 Wiley Periodicals, Inc.

Hypoxia-responsive ERFs involved in postdeastringency softening of persimmon fruit.

PubMed

Wang, Miao-Miao; Zhu, Qing-Gang; Deng, Chu-Li; Luo, Zheng-Rong; Sun, Ning-Jing; Grierson, Donald; Yin, Xue-Ren; Chen, Kun-Song

2017-11-01

Removal of astringency by endogenously formed acetaldehyde, achieved by postharvest anaerobic treatment, is of critical importance for many types of persimmon fruit. Although an anaerobic environment accelerates de-astringency, it also has the deleterious effect of promoting excessive softening, reducing shelf life and marketability. Some hypoxia-responsive ethylene response factors (ERFs) participate in anaerobic de-astringency, but their role in accelerated softening was unclear. Undesirable rapid softening induced by high CO 2 (95%) was ameliorated by adding the ethylene inhibitor 1-MCP (1 μL/L), resulting in reduced astringency while maintaining firmness, suggesting that CO 2 -induced softening involves ethylene signalling. Among the hypoxia-responsive genes, expression of eight involved in fruit cell wall metabolism (Dkβ-gal1/4, DkEGase1, DkPE1/2, DkPG1, DkXTH9/10) and three ethylene response factor genes (DkERF8/16/19) showed significant correlations with postdeastringency fruit softening. Dual-luciferase assay indicated that DkERF8/16/19 could trans-activate the DkXTH9 promoter and this interaction was abolished by a mutation introduced into the C-repeat/dehydration-responsive element of the DkXTH9 promoter, supporting the conclusion that these DkERFs bind directly to the DkXTH9 promoter and regulate this gene, which encodes an important cell wall metabolism enzyme. Some hypoxia-responsive ERF genes are involved in deastringency and softening, and this linkage was uncoupled by 1-MCP. Fruit of the Japanese cultivar 'Tonewase' provide a model for altered anaerobic response, as they lost astringency yet maintained firmness after CO 2 treatment without 1-MCP and changes in cell wall enzymes and ERFs did not occur. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Tissue specific localization of pectin-Ca²⁺ cross-linkages and pectin methyl-esterification during fruit ripening in tomato (Solanum lycopersicum).

PubMed

Hyodo, Hiromi; Terao, Azusa; Furukawa, Jun; Sakamoto, Naoya; Yurimoto, Hisayoshi; Satoh, Shinobu; Iwai, Hiroaki

2013-01-01

Fruit ripening is one of the developmental processes accompanying seed development. The tomato is a well-known model for studying fruit ripening and development, and the disassembly of primary cell walls and the middle lamella, such as through pectin de-methylesterified by pectin methylesterase (PE) and depolymerization by polygalacturonase (PG), is generally accepted to be one of the major changes that occur during ripening. Although many reports of the changes in pectin during tomato fruit ripening are focused on the relation to softening of the pericarp or the Blossom-end rot by calcium (Ca²⁺) deficiency disorder, the changes in pectin structure and localization in each tissues during tomato fruit ripening is not well known. In this study, to elucidate the tissue-specific role of pectin during fruit development and ripening, we examined gene expression, the enzymatic activities involved in pectin synthesis and depolymerisation in fruit using biochemical and immunohistochemical analyses, and uronic acids and calcium (Ca)-bound pectin were determined by secondary ion-microprobe mass spectrometry. These results show that changes in pectin properties during fruit development and ripening have tissue-specific patterns. In particular, differential control of pectin methyl-esterification occurs in each tissue. Variations in the cell walls of the pericarp are quite different from that of locular tissues. The Ca-binding pectin and hairy pectin in skin cell layers are important for intercellular and tissue-tissue adhesion. Maintenance of the globular form and softening of tomato fruit may be regulated by the arrangement of pectin structures in each tissue.

Tissue Specific Localization of Pectin–Ca2+ Cross-Linkages and Pectin Methyl-Esterification during Fruit Ripening in Tomato (Solanum lycopersicum)

PubMed Central

Hyodo, Hiromi; Terao, Azusa; Furukawa, Jun; Sakamoto, Naoya; Yurimoto, Hisayoshi; Satoh, Shinobu; Iwai, Hiroaki

2013-01-01

Fruit ripening is one of the developmental processes accompanying seed development. The tomato is a well-known model for studying fruit ripening and development, and the disassembly of primary cell walls and the middle lamella, such as through pectin de-methylesterified by pectin methylesterase (PE) and depolymerization by polygalacturonase (PG), is generally accepted to be one of the major changes that occur during ripening. Although many reports of the changes in pectin during tomato fruit ripening are focused on the relation to softening of the pericarp or the Blossom-end rot by calcium (Ca2+) deficiency disorder, the changes in pectin structure and localization in each tissues during tomato fruit ripening is not well known. In this study, to elucidate the tissue-specific role of pectin during fruit development and ripening, we examined gene expression, the enzymatic activities involved in pectin synthesis and depolymerisation in fruit using biochemical and immunohistochemical analyses, and uronic acids and calcium (Ca)-bound pectin were determined by secondary ion-microprobe mass spectrometry. These results show that changes in pectin properties during fruit development and ripening have tissue-specific patterns. In particular, differential control of pectin methyl-esterification occurs in each tissue. Variations in the cell walls of the pericarp are quite different from that of locular tissues. The Ca-binding pectin and hairy pectin in skin cell layers are important for intercellular and tissue–tissue adhesion. Maintenance of the globular form and softening of tomato fruit may be regulated by the arrangement of pectin structures in each tissue. PMID:24236073

Peptidoglycan Branched Stem Peptides Contribute to Streptococcus pneumoniae Virulence by Inhibiting Pneumolysin Release

PubMed Central

Greene, Neil G.; Narciso, Ana R.; Filipe, Sergio R.; Camilli, Andrew

2015-01-01

Streptococcus pneumoniae (the pneumococcus) colonizes the human nasopharynx and is a significant pathogen worldwide. Pneumolysin (Ply) is a multi-functional, extracellular virulence factor produced by this organism that is critical for pathogenesis. Despite the absence of any apparent secretion or cell surface attachment motifs, Ply localizes to the cell envelope of actively growing cells. We sought to characterize the consequences of this surface localization. Through functional assays with whole cells and subcellular fractions, we determined that Ply activity and its release into the extracellular environment are inhibited by peptidoglycan (PG) structure. The ability of PG to inhibit Ply release was dependent on the stem peptide composition of this macromolecule, which was manipulated by mutation of the murMN operon that encodes proteins responsible for branched stem peptide synthesis. Additionally, removal of choline-binding proteins from the cell surface significantly reduced Ply release to levels observed in a mutant with a high proportion of branched stem peptides suggesting a link between this structural feature and surface-associated choline-binding proteins involved in PG metabolism. Of clinical relevance, we also demonstrate that a hyperactive, mosaic murMN allele associated with penicillin resistance causes decreased Ply release with concomitant increases in the amount of branched stem peptides. Finally, using a murMN deletion mutant, we observed that increased Ply release is detrimental to virulence during a murine model of pneumonia. Taken together, our results reveal a novel role for branched stem peptides in pneumococcal pathogenesis and demonstrate the importance of controlled Ply release during infection. These results highlight the importance of PG composition in pathogenesis and may have broad implications for the diverse PG structures observed in other bacterial pathogens. PMID:26114646

Peptidoglycan Branched Stem Peptides Contribute to Streptococcus pneumoniae Virulence by Inhibiting Pneumolysin Release.

PubMed

Greene, Neil G; Narciso, Ana R; Filipe, Sergio R; Camilli, Andrew

2015-06-01

Streptococcus pneumoniae (the pneumococcus) colonizes the human nasopharynx and is a significant pathogen worldwide. Pneumolysin (Ply) is a multi-functional, extracellular virulence factor produced by this organism that is critical for pathogenesis. Despite the absence of any apparent secretion or cell surface attachment motifs, Ply localizes to the cell envelope of actively growing cells. We sought to characterize the consequences of this surface localization. Through functional assays with whole cells and subcellular fractions, we determined that Ply activity and its release into the extracellular environment are inhibited by peptidoglycan (PG) structure. The ability of PG to inhibit Ply release was dependent on the stem peptide composition of this macromolecule, which was manipulated by mutation of the murMN operon that encodes proteins responsible for branched stem peptide synthesis. Additionally, removal of choline-binding proteins from the cell surface significantly reduced Ply release to levels observed in a mutant with a high proportion of branched stem peptides suggesting a link between this structural feature and surface-associated choline-binding proteins involved in PG metabolism. Of clinical relevance, we also demonstrate that a hyperactive, mosaic murMN allele associated with penicillin resistance causes decreased Ply release with concomitant increases in the amount of branched stem peptides. Finally, using a murMN deletion mutant, we observed that increased Ply release is detrimental to virulence during a murine model of pneumonia. Taken together, our results reveal a novel role for branched stem peptides in pneumococcal pathogenesis and demonstrate the importance of controlled Ply release during infection. These results highlight the importance of PG composition in pathogenesis and may have broad implications for the diverse PG structures observed in other bacterial pathogens.

Comparative study on in vivo response of porous calcium carbonate composite ceramic and biphasic calcium phosphate ceramic.

PubMed

He, Fupo; Ren, Weiwei; Tian, Xiumei; Liu, Wei; Wu, Shanghua; Chen, Xiaoming

2016-07-01

In a previous study, robust calcium carbonate composite ceramics (CC/PG) were prepared by using phosphate-based glass (PG) as an additive, which showed good cell response. In the present study the in vivo response of porous CC/PG was compared to that of porous biphasic calcium phosphate ceramics (BCP), using a rabbit femoral critical-size grafting model. The materials degradation and bone formation processes were evaluated by general observation, X-ray radiography, micro-computed tomography, and histological examination. The results demonstrated excellent biocompatibility and osteoconductivity, and progressive degradation of CC/PG and BCP. Although the in vitro degradation rate of CC/PG was distinctly faster than that of BCP, at 4week post-implantation, the bone generation and material degradation of CC/PG were less than those of BCP. Nevertheless, at postoperative week 8, the increment of bone formation and material degradation of CC/PG was pronouncedly larger than that of BCP. These results show that CC/PG is a potential resorbable bone graft aside from the traditional synthetic ones. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of nasal septum perforation repair on nasal airflow: An analysis using computational fluid dynamics on preoperative and postoperative three-dimensional models.

PubMed

Nomura, Tsutomu; Ushio, Munetaka; Kondo, Kenji; Kikuchi, Shigeru

2018-10-01

The purpose of this research is to examine the changes in nasal airflow dynamics before and after the nasal perforation repair. Three dimensional (3D) models of the nasal cavity before and after septal perforation repair was reconstructed using preoperative and postoperative computed tomography (CT) images of a patient. The numerical simulation was carried out using ANSYS CFX V15.0. Pre- and post-operative models were compared by their velocity, pressure (P), pressure gradient (PG), wall shear (WS), shear strain rate (SSR) and turbulence kinetic energy (TKE) in three plains. In the post-operative state, the cross flows disappeared. In preoperative state, there were areas showing high PG, WS, SSR at the posterior border of the perforation, which exactly correspond to the area showing erosive mucosa on endoscopic inspection of the patient. In postoperative state, such high PG, WS and SSR areas disappeared. High TKEs also disappeared after surgery. The effects of septal perforation repair on airflow dynamics were evaluated using computer fluid dynamics (CFD). High WS, PG and SSR observed at the edge of the septal perforation may be related to the clinical symptom such as nasal bleeding and pain. TKE was considered to cause nasal symptom. Copyright © 2018 Elsevier B.V. All rights reserved.

Cathepsin S Is Involved in Th17 Differentiation Through the Upregulation of IL-6 by Activating PAR-2 after Systemic Exposure to Lipopolysaccharide from Porphyromonas gingivalis.

PubMed

Dekita, Masato; Wu, Zhou; Ni, Junjun; Zhang, Xinwen; Liu, Yicong; Yan, Xu; Nakanishi, Hiroshi; Takahashi, Ichiro

2017-01-01

Positive links have been found between periodontitis and numerous diseases in humans via persistent inflammation throughout the body. However, the main factors responsible for maintaining this pro-inflammatory condition are poorly understood. The spleen, the largest secondary immune organ, is a central hub regulating the immune response/inflammation due to the dendritic cell (DC) response to CD4 + T cell subtype differentiation, and lysosomal proteinase cathepsin S (CatS) is known to be involved in DC functions. In the present study, we found that CatS-induced IL-6 production by splenic DCs subsequently promotes Th17 differentiation, in response to systemic exposure to lipopolysaccharide derived from Porphyromonas gingivalis (PgLPS). The population of CD11c + DCs was significantly increased in the splenic marginal zone (MZ) locally of wild-type (DBA/2) mice with splenomegaly but not in that of CatS deficient ( CatS -/- ) mice after systemic exposure to PgLPS for 7 consecutive days (5 mg/kg/day, intraperitoneal). Similarly, the population of Th17 + CD4 + T cells was also significantly increased in the splenic MZ of wild-type mice but not in that of CatS -/- mice after PgLPS exposure. Furthermore, the increase in the Th17 + CD4 + T cell population paralleled increases in the levels of CatS and IL-6 in CD11c + cells in the splenic MZ. In isolated primary splenic CD11c + cells, the mRNA expression and the production of IL-6 was dramatically increased in wild-type mice but not in CatS - /- mice after direct stimulation with PgLPS (1 μg/ml), and this PgLPS-induced increase in the IL-6 expression was completely abolished by pre-treatment with Z-Phe-Leu-COCHO (Z-FL), the specific inhibitor of CatS. The PgLPS activated protease-activated receptor (PAR) 2 in the isolated splenic CD11c + cells was also significantly inhibited by CatS deficiently. In addition, the PgLPS - induced increase in the IL-6 production by splenic CD11c + cells was completely abolished by pre-treatment with FSLLRY-NH 2 , a PAR2 antagonist, as well as Akti, a specific inhibitor of Akt. These findings indicate that CatS plays a critical role in driving splenic DC-dependent Th17 differentiation through the upregulation of IL-6 by activating PAR2 after exposure to components of periodontal bacteria. Therefore, CatS-specific inhibitors may be effective in alleviating periodontitis-related immune/inflammation.

Combinatory annotation of cell membrane receptors and signalling pathways of Bombyx mori prothoracic glands

PubMed Central

Moulos, Panagiotis; Samiotaki, Martina; Panayotou, George; Dedos, Skarlatos G.

2016-01-01

The cells of prothoracic glands (PG) are the main site of synthesis and secretion of ecdysteroids, the biochemical products of cholesterol conversion to steroids that shape the morphogenic development of insects. Despite the availability of genome sequences from several insect species and the extensive knowledge of certain signalling pathways that underpin ecdysteroidogenesis, the spectrum of signalling molecules and ecdysteroidogenic cascades is still not fully comprehensive. To fill this gap and obtain the complete list of cell membrane receptors expressed in PG cells, we used combinatory bioinformatic, proteomic and transcriptomic analysis and quantitative PCR to annotate and determine the expression profiles of genes identified as putative cell membrane receptors of the model insect species, Bombyx mori, and subsequently enrich the repertoire of signalling pathways that are present in its PG cells. The genome annotation dataset we report here highlights modules and pathways that may be directly involved in ecdysteroidogenesis and aims to disseminate data and assist other researchers in the discovery of the role of such receptors and their ligands. PMID:27576083

Serum Gastrin in Chronic Gastritis

PubMed Central

Korman, M. G.; Strickland, R. G.; Hansky, J.

1971-01-01

Fasting gastrin levels in serum were measured in 49 patients with different types of chronic gastritis and in matched controls. In 15 patients with established pernicious anaemia the mean (± S.E. of mean) level of gastrin was greatly raised (699 ± 99 pg/ml). In 17 patients with chronic atrophic gastritis, seropositive for parietal cell antibody but with adequate vitamin-B12 absorption, the level was also raised (476 ± 74 pg/ml). By contrast, in “simple” atrophic gastritis seronegative for parietal cell antibody the gastrin levels were significantly lower for both diffuse atrophic gastritis (129 ± 31 pg/ml) and multifocal gastritis (14 ± 4 pg/ml). These levels were similar to those in the controls (46 ± 7 pg/ml). The mechanism of the raised gastrin levels remains uncertain, but neither achlorhydria nor in vivo action of the parietal cell antibody wholly accounted for the hypergastrinaemia. We conclude that hypergastrinaemia is characteristic of gastritis associated with autoimmune reactions to gastric antigens and pernicious anaemia and that a raised serum gastrin is a useful marker of the type of gastritis that tends to progress to the gastric lesion of pernicious anaemia. The findings suggest that this type of gastritis is an essentially different disease from “simple” atrophic gastritis, and the differences in gastrin levels may be due to sparing of the antral mucosa in the autoimmune type but not in “simple” gastritis. PMID:5550864

Revalidation of the genus Chiloguembelitria Hofker: Implications for the evolution of early Danian planktonic foraminifera

NASA Astrophysics Data System (ADS)

Arenillas, Ignacio; Arz, José A.; Gilabert, Vicente

2017-10-01

Guembelitria is the only planktonic foraminiferal genus whose survival from the mass extinction event of the Cretaceous/Paleogene (K/Pg) boundary has been clearly proven. The evolution of Guembelitria after the K/Pg boundary led to the appearance of two guembelitriid lineages in the early Danian: one biserial, represented by Woodringina and culminating in Chiloguembelina, and the other trochospiral, represented by Trochoguembelitria and culminating in Globoconusa. We have re-examined the genus Chiloguembelitria, another guembelitriid descended from Guembelitria and whose taxonomic validity had been questioned, it being considered a junior synonym of the latter. Nevertheless, Chiloguembelitria differs from Guembelitria mainly in the wall texture (pustulate to rugose vs. pore-mounded) and the position of the aperture (umbilical-extraumbilical to extraumbilical vs. umbilical). Chiloguembelitria shares its wall texture with Trochoguembelitria and some of the earliest specimens of Woodringina, suggesting that it played an important role in the evolution of early Danian guembelitriids, as it seems to be the most immediate ancestor of both trochospiral and biserial lineages. Morphological and morphostatistical analyses of Chiloguembelitria discriminate at least five species: Chg. danica, Chg. irregularis, and three new species: Chg. hofkeri, Chg. trilobata and Chg. biseriata.

Assessment of Growth Factors Secreted by Human Breastmilk Mesenchymal Stem Cells.

PubMed

Kaingade, Pankaj Mahipatrao; Somasundaram, Indumathi; Nikam, Amar Babaso; Sarang, Shabari Amit; Patel, Jagdish Shantilal

2016-01-01

Human breastmilk is a dynamic, multifaceted biological fluid containing nutrients, bioactive substances, and growth factors. It is effective in supporting growth and development of an infant. As breastmilk has been found to possess mesenchymal stem cells, the importance of the components of breastmilk and their physiological roles is increasing day by day. The present study was intended to identify the secretions of growth factors, mainly vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), from human breastmilk mesenchymal stem cells under basal conditions of in vitro cell culture using synthetic media and human cord serum. The growth factors were analyzed with the enzyme-linked immunosorbent assay technique. The cultured mesenchymal stem cells of breastmilk without serum revealed significant differences in secretions of the VEGF and HGF growth factors (8.55 ± 2.26402 pg/mL and 230.8 ± 45.9861 pg/mL, respectively) compared with mesenchymal stem cells of breastmilk with serum (21.31 ± 4.69 pg/mL and 2,404.42 ± 481.593 pg/mL, respectively). Results obtained from our study demonstrate that both VEGF and HGF are secreted in vitro by human breastmilk mesenchymal stem cells. The roles of VEGF and HGF in surfactant secretion, pulmonary maturation, and neonatal maturity have been well established. Thus, we emphasize that breastmilk-derived MSCs could be a potent therapeutic source in treating neonatal diseases. Besides, due to its immense potency, the study also emphasizes the importance of breastfeeding, which is promoted by organizations like the World Heatlh Organization and UNICEF.

[Growth Factors and Interleukins in Amniotic Membrane Tissue Homogenate].

PubMed

Stachon, T; Bischoff, M; Seitz, B; Huber, M; Zawada, M; Langenbucher, A; Szentmáry, N

2015-07-01

Application of amniotic membrane homogenate eye drops may be a potential treatment alternative for therapy resistant corneal epithelial defects. The purpose of this study was to determine the concentrations of epidermal growth factor (EGF), fibroblast growth factor basic (bFGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), interleukin-6 (IL-6) and interleukin-8 (IL-8) in amniotic membrane homogenates. Amniotic membranes of 8 placentas were prepared and thereafter stored at - 80 °C using the standard methods of the LIONS Cornea Bank Saar-Lor-Lux, Trier/Westpfalz. Following defreezing, amniotic membranes were cut in two pieces and homogenized in liquid nitrogen. One part of the homogenate was prepared in cell-lysis buffer, the other part was prepared in PBS. The tissue homogenates were stored at - 20 °C until enzyme-linked immunosorbent assay (ELISA) analysis for EGF, bFGF, HGF, KGF, IL-6 and IL-8 concentrations. Concentrations of KGF, IL-6 and IL-8 were below the detection limit using both preparation techniques. The EGF concentration in tissue homogenates treated with cell-lysis buffer (2412 pg/g tissue) was not significantly different compared to that of tissue homogenates treated with PBS (1586 pg/g tissue, p = 0.72). bFGF release was also not significantly different using cell-lysis buffer (3606 pg/g tissue) or PBS treated tissue homogenates (4649 pg/g tissue, p = 0.35). HGF release was significantly lower using cell-lysis buffer (23,555 pg/g tissue), compared to PBS treated tissue (47,766 pg/g tissue, p = 0.007). Containing EGF, bFGF and HGF, and lacking IL-6 and IL-8, the application of amniotic membrane homogenate eye drops may be a potential treatment alternative for therapy-resistant corneal epithelial defects. Georg Thieme Verlag KG Stuttgart · New York.

Functional properties of LRRK2 mutations in Taiwanese Parkinson disease.

PubMed

Chang, Kuo-Hsuan; Chen, Chiung-Mei; Lin, Chih-Hsin; Chang, Wen-Teng; Jiang, Pei-Ru; Hsiao, Ya-Chin; Wu, Yih-Ru; Lee-Chen, Guey-Jen

2017-03-01

Leucine-rich repeat kinase 2 (LRRK2) is a large protein encoding multiple functional domains. Mutations within different LRRK2 domains have been considered to be involved in the development of Parkinson disease by different mechanisms. Our previous study found three LRRK2 mutations-p.R767H, p.S885N, and p.R1441H-in Taiwanese patients with Parkinson disease. We evaluated the functional properties of LRRK2 p.R767H, p.S885N, and p.R1441H mutations by overexpressing them in human embryonic kidney 293 and neuroblastoma SK-N-SH cells. The common p.G2019S mutation in the kinase domain was included for comparison. In 293 cells, overexpressed p.R1441H-but not p.R767H, p.S885N, or p.G2019-increased GTP binding affinity to prolong the active state. Overexpressed p.R1441H and p.G2019S generated inclusions in 293 cells. In SK-N-SH cells, the α-synuclein was coexpressed with wild type as well as mutated p.R767H, p.S885N, p.R1441H, and p.G2019 LRRK2 proteins. Part of the perinuclear inclusions formed by p.R1441H and p.G2019S were colocalized with α-synuclein. Additionally, p.S885N and p.R1441H mutations caused reduced interaction between LRRK2 and ARHGEF7, a putative guanine nucleotide exchange factor for LRRK2, whereas this interaction was well preserved in p.R767H and p.G2019S mutations. Our study suggests that p.R1441H protein facilitates the formation of intracellular inclusions, compromises GTP hydrolysis by increasing its affinity for GTP, and reduces its interaction with ARHGEF7. Copyright © 2016. Published by Elsevier B.V.

Nutrient-Dependent Endocycling in Steroidogenic Tissue Dictates Timing of Metamorphosis in Drosophila melanogaster

PubMed Central

Ohhara, Yuya; Kobayashi, Satoru

2017-01-01

Many animals have an intrinsic growth checkpoint during juvenile development, after which an irreversible decision is made to upregulate steroidogenesis, triggering the metamorphic juvenile-to-adult transition. However, a molecular process underlying such a critical developmental decision remains obscure. Here we show that nutrient-dependent endocycling in steroidogenic cells provides the machinery necessary for irreversible activation of metamorphosis in Drosophila melanogaster. Endocycle progression in cells of the prothoracic gland (PG) is tightly coupled with the growth checkpoint, and block of endocycle in PG cells causes larval developmental arrest due to reduction in biosynthesis of the steroid hormone ecdysone. Moreover, inhibition of the nutrient sensor target of rapamycin (TOR) in the PG during the checkpoint period causes endocycle inhibition and developmental arrest, which can be rescued by inducing additional rounds of endocycles by Cyclin E. We propose that a TOR-mediated cell cycle checkpoint in steroidogenic tissue provides a systemic growth checkpoint for reproductive maturation. PMID:28121986

Inhibition of Estrogen-Induced Growth of Breast Cancer by Targeting Mitrochondrial Oxidants

DTIC Science & Technology

2007-04-01

expected estradiol induced oxidants production in MCF-7 cells in dose dependent manner (Fig. 1). 0 50 100 150 200 250 300 DMSO 100pg 10ng 100ng...dose dependent manner. This is in agreement with previous findings (Foster et al., 2001). 0 50 100 150 200 250 300 DMSO 100pg/ml 10ng/ml 100ng/ml C...significantly inhibited E2-induced cell growth by as much as 50 % after a 72 h treatment. The reduction of E2-induced cell growth observed with NAC and

Optical dissection of odor information processing in vivo using GCaMPs expressed in specified cell types of the olfactory bulb

PubMed Central

Wachowiak, Matt; Economo, Michael N.; Díaz-Quesada, Marta; Brunert, Daniela; Wesson, Daniel W.; White, John. A.; Rothermel, Markus

2013-01-01

Understanding central processing requires precise monitoring of neural activity across populations of identified neurons in the intact brain. Here we used recently-optimized variants of the genetically-encoded calcium sensor GCaMP (GCaMP3 and GCaMPG5G) to image activity among genetically- and anatomically-defined neuronal populations in the olfactory bulb (OB), including two types of GABA-ergic interneurons (periglomerular (PG) and short axon (SA) cells) and OB output neurons (mitral/tufted (MT) cells) projecting to piriform cortex. We first established that changes in neuronal spiking can be accurately related to GCaMP fluorescence changes via a simple quantitative relationship over a large dynamic range. We next used in vivo two-photon imaging from individual neurons and epifluorescence signals reflecting population-level activity to investigate the spatiotemporal representation of odorants across these neuron types in anesthetized and awake mice. Under anesthesia, individual PG and SA cells showed temporally simple responses and little spontaneous activity, while MT cells were spontaneously active and showed diverse temporal responses. At the population level, response patterns of PG, SA and MT cells were surprisingly similar to those imaged from sensory inputs, with shared odorant-specific topography across the dorsal OB and inhalation-coupled temporal dynamics. During wakefulness, PG and SA cell responses increased in magnitude but remained temporally simple while those of MT cells changed to complex spatiotemporal patterns reflecting restricted excitation and widespread inhibition. These results point to multiple circuit elements with distinct roles in transforming odor representations in the OB and provide a framework for further dissecting early olfactory processing using optical and genetic tools. PMID:23516293

Effects of nasal septum perforation repair surgery on three-dimensional airflow: an evaluation using computational fluid dynamics.

PubMed

Nomura, Tsutomu; Ushio, Munetaka; Kondo, Kenji; Yamasoba, Tatsuya

2015-11-01

The purpose of this research is to determine the cause of nasal perforation symptoms and to predict post-operative function after nasal perforation repair surgery. A realistic three-dimensional (3D) model of the nose with a septal perforation was reconstructed using a computed tomography (CT) scan from a patient with nasal septal defect. The numerical simulation was carried out using ANSYS CFX V13.0. Pre- and post-operative models were compared by their velocity, pressure gradient (PG), wall shear (WS), shear strain rate (SSR) and turbulence kinetic energy in three plains. In the post-operative state, the crossflows had disappeared, and stream lines bound to the olfactory cleft area had appeared. After surgery, almost all of high-shear stress areas were disappeared comparing pre-operative model. In conclusion, the effects of surgery to correct nasal septal perforation were evaluated using a three-dimensional airflow evaluation. Following the surgery, crossflows disappeared, and WS, PG and SSR rate were decreased. A high WS.PG and SSR were suspected as causes of nasal perforation symptoms.

Polymeric Nano-Micelles as Novel Cargo-Carriers for LY2157299 Liver Cancer Cells Delivery.

PubMed

Hanafy, Nemany Abdelhamid Nemany; Quarta, Alessandra; Ferraro, Marzia Maria; Dini, Luciana; Nobile, Concetta; De Giorgi, Maria Luisa; Carallo, Sonia; Citti, Cinzia; Gaballo, Antonio; Cannazza, Giuseppe; Rinaldi, Rosaria; Giannelli, Gianluigi; Leporatti, Stefano

2018-03-06

LY2157299 (LY), which is very small molecule bringing high cancer diffusion, is a pathway antagonist against TGFβ. LY dosage can be diluted by blood plasma, can be captured by immune system or it might be dissolved during digestion in gastrointestinal tract. The aim of our study is to optimize a "nano-elastic" carrier to avoid acidic pH of gastrointestinal tract, colon alkaline pH, and anti-immune recognition. Polygalacturonic acid (PgA) is not degradable in the gastrointestinal tract due to its insolubility at acidic pH. To avoid PgA solubility in the colon, we have designed its conjugation with Polyacrylic acid (PAA). PgA-PAA conjugation has enhanced their potential use for oral and injected dosage. Following these pre-requisites, novel polymeric nano-micelles derived from PgA-PAA conjugation and loading LY2157299 are developed and characterized. Efficacy, uptake and targeting against a hepatocellular carcinoma cell line (HLF) have also been demonstrated.

Panax ginseng extract antagonizes the effect of DKK-1-induced catagen-ike changes of hair follicles

PubMed Central

Lee, Yonghee; Kim, Su Na; Hong, Yong Deog; Park, Byung Cheol; Na, Yongjoo

2017-01-01

It is well known that Panax ginseng (PG) has various pharmacological effects such as anti-aging and anti-inflammation. In a previous study, the authors identified that PG extract induced hair growth by means of a mechanism similar to that of minoxidil. In the present study, the inhibitory effect of PG extract on Dickkopf-1 (DKK-1)-induced catagen-like changes in hair follicles (HFs) was investigated in addition to the underlying mechanism of action. The effects of PG extract on cell proliferation, anti-apoptotic effect, and hair growth were observed using cultured outer root sheath (ORS) keratinocytes and human HFs with or without DKK-1 treatment. The PG extract significantly stimulated proliferation and inhibited apoptosis, respectively, in ORS keratinocytes. PG extract treatment affected the expression of apoptosis-related genes Bcl-2 and Bax. DKK-1 inhibited hair growth, and PG extract dramatically reversed the effect of DKK-1 on ex vivo human hair organ culture. PG extract antagonizes DKK-1-induced catagen-like changes, in part, through the regulation of apoptosis-related gene expression in HFs. These findings suggested that PG extract may reduce hair loss despite the presence of DKK-1, a strong catagen inducer via apoptosis. PMID:28849028

Peptidoglycan Hydrolases of Escherichia coli

PubMed Central

van Heijenoort, Jean

2011-01-01

Summary: The review summarizes the abundant information on the 35 identified peptidoglycan (PG) hydrolases of Escherichia coli classified into 12 distinct families, including mainly glycosidases, peptidases, and amidases. An attempt is also made to critically assess their functions in PG maturation, turnover, elongation, septation, and recycling as well as in cell autolysis. There is at least one hydrolytic activity for each bond linking PG components, and most hydrolase genes were identified. Few hydrolases appear to be individually essential. The crystal structures and reaction mechanisms of certain hydrolases having defined functions were investigated. However, our knowledge of the biochemical properties of most hydrolases still remains fragmentary, and that of their cellular functions remains elusive. Owing to redundancy, PG hydrolases far outnumber the enzymes of PG biosynthesis. The presence of the two sets of enzymes acting on the PG bonds raises the question of their functional correlations. It is difficult to understand why E. coli keeps such a large set of PG hydrolases. The subtle differences in substrate specificities between the isoenzymes of each family certainly reflect a variety of as-yet-unidentified physiological functions. Their study will be a far more difficult challenge than that of the steps of the PG biosynthesis pathway. PMID:22126997

Biochemical evaluation of the anticancer potential of the polyamine-based nanocarrier Nano11047.

PubMed

Murray-Stewart, Tracy; Ferrari, Elena; Xie, Ying; Yu, Fei; Marton, Laurence J; Oupicky, David; Casero, Robert A

2017-01-01

Synthesizing polycationic polymers directly from existing drugs overcomes the drug-loading limitations often associated with pharmacologically inert nanocarriers. We recently described nanocarriers formed from a first-generation polyamine analogue, bis(ethyl)norspermine (BENSpm), that could simultaneously target polyamine metabolism while delivering therapeutic nucleic acids. In the current study, we describe the synthesis and evaluation of self-immolative nanocarriers derived from the second-generation polyamine analogue PG-11047. Polyamines are absolutely essential for proliferation and their metabolism is frequently dysregulated in cancer. Through its effects on polyamine metabolism, PG-11047 effectively inhibits tumor growth in cancer cell lines of multiple origins as well as in human tumor mouse xenografts. Promising clinical trials have been completed verifying the safety and tolerance of this rotationally restricted polyamine analogue. We therefore used PG-11047 as the basis for Nano11047, a biodegradable, prodrug nanocarrier capable of targeting polyamine metabolism. Following exposure of lung cancer cell lines to Nano11047, uptake and intracellular degradation into the parent compound PG-11047 was observed. The release of PG-11047 highly induced the polyamine catabolic enzyme activities of spermidine/spermine N1-acetyltransferase (SSAT) and spermine oxidase (SMOX). By contrast, the activity of ornithine decarboxylase (ODC), a rate-limiting enzyme in polyamine biosynthesis and a putative oncogene, was decreased. Consequently, intracellular levels of the natural polyamines were depleted concurrent with tumor cell growth inhibition. This availability of Nano11047 as a novel drug form and potential nucleic acid delivery vector will potentially benefit and encourage future clinical studies.

The Effect of Coumestrol on Progesterone and Prostaglandin Production in the Mare: In Vitro and In Vivo Studies.

PubMed

Szóstek, Anna Z; Sadowska, Agnieszka; Piotrowska-Tomala, Katarzyna K; Botelho, Marta; Fradinho, Maria João; Rebordão, Maria Rosa; Ferreira-Dias, Graça M; Skarzynski, Dariusz J

2016-09-01

Coumestrol (Cou) is a plant-derived phytoestrogen that induces various pathologies in the female reproductive tract. Although effects of phytoestrogens on reproductive function in other species are well documented, their influence on progesterone (P 4 ) and prostaglandin (PG) secretion in the mare is unknown. The aim of this study was to determine if Cou directly affects P 4 and PG concentrations (in vivo) and endometrial PG secretion (in vitro) in the mare. In experiment 1, the mares (n = 4) were fed for 14 days on a diet containing increasing proportions of alfalfa pellets (250 g-1 kg/day). An additional 4 mares were fed a standard diet (control group). Sequential blood samples were obtained for 8 h after feeding on Days 13 and 14 (1 kg/day alfalfa pellets). Feeding the mares alfalfa pellets up-regulated PGE 2 and 13,14-dihydro-15-ketoprostaglandin F 2alpha (PGFM) and down-regulated P 4 in the blood plasma compared to those in the control group (P < 0.05). In experiment 2, epithelial and stromal cells were exposed to E 2 (10 -9 M) or Cou (10 -8 M) for 24 h. In the in vitro study, Cou increased PG secretion in epithelial and stromal cells (P < 0.05). In both types of endometrial cells, Cou up-regulated PTGS-2 protein expression (P < 0.05). Moreover, PGES and PGFS proteins were up-regulated by Cou in epithelial cells (P < 0.01). These results indicate that Cou can disturb reproductive function by affecting reproductive hormone secretion and altering the endometrial milieu through PG stimulation. Coumestrol therefore may impair physiologic regulation of the estrous cycle and early pregnancy. © 2016 by the Society for the Study of Reproduction, Inc.

Interleukin-6 and granulocyte-macrophage colony-stimulating factor in apical periodontitis: correlation with clinical and histologic findings of the involved teeth.

PubMed

Radics, T; Kiss, C; Tar, I; Márton, I J

2003-02-01

Apical periodontitis is characterized by the presence of immunocompetent cells producing a wide variety of inflammatory mediators. Releasing cytokines with long-range action, such as interleukin-6 (IL-6) and granulocyte-macrophage colony-stimulating factor (GM-CSF), apical periodontitis may induce changes in remote organs of the host. This study quantified the levels of IL-6 and GM-CSF in symptomatic and asymptomatic human periradicular lesions. Lesions were also characterized by size and histologic findings. Tissue samples were homogenized and supernatants were assayed using an enzyme-linked immunosorbent assay (ELISA). Correlations between cytokine levels and characteristic features (as single variables) of the lesions were analysed. There was a trend for higher levels of IL-6 and GM-CSF in symptomatic than in asymptomatic lesions, but the difference was not significant. Levels also tended to be higher in large than in small lesions, in polymorphonuclear (PMN) cell-rich than in PMN cell-poor samples, and in epithelialized than in non-epithelialized lesions. Significantly higher levels of IL-6 (778.1 +/- 220.5 pg/microg) and GM-CSF (363.3 +/- 98.4 pg/microg) were found in samples coincidentally possessing symptomatic and epithelialized features than in asymptomatic, small, PMN cell-poor, non-epithelialized lesions (IL-6: 45.2 +/- 13.1 pg/microg and GM-CSF: 135.1 +/- 26.4 pg/microg). These results suggest that symptomatic lesions containing epithelial cells represent an immunologically active stage of apical periodontitis, whereas asymptomatic, small, PMN cell-poor, non-epithelialized lesions represent healing apical lesions.

Acellular therapeutic approach for heart failure: in vitro production of extracellular vesicles from human cardiovascular progenitors.

PubMed

El Harane, Nadia; Kervadec, Anaïs; Bellamy, Valérie; Pidial, Laetitia; Neametalla, Hany J; Perier, Marie-Cécile; Lima Correa, Bruna; Thiébault, Léa; Cagnard, Nicolas; Duché, Angéline; Brunaud, Camille; Lemitre, Mathilde; Gauthier, Jeanne; Bourdillon, Alexandra T; Renault, Marc P; Hovhannisyan, Yeranuhi; Paiva, Solenne; Colas, Alexandre R; Agbulut, Onnik; Hagège, Albert; Silvestre, Jean-Sébastien; Menasché, Philippe; Renault, Nisa K E

2018-05-21

We have shown that extracellular vesicles (EVs) secreted by embryonic stem cell-derived cardiovascular progenitor cells (Pg) recapitulate the therapeutic effects of their parent cells in a mouse model of chronic heart failure (CHF). Our objectives are to investigate whether EV released by more readily available cell sources are therapeutic, whether their effectiveness is influenced by the differentiation state of the secreting cell, and through which mechanisms they act. The total EV secreted by human induced pluripotent stem cell-derived cardiovascular progenitors (iPSC-Pg) and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) were isolated by ultracentrifugation and characterized by Nanoparticle Tracking Analysis, western blot, and cryo-electron microscopy. In vitro bioactivity assays were used to evaluate their cellular effects. Cell and EV microRNA (miRNA) content were assessed by miRNA array. Myocardial infarction was induced in 199 nude mice. Three weeks later, mice with left ventricular ejection fraction (LVEF) ≤ 45% received transcutaneous echo-guided injections of iPSC-CM (1.4 × 106, n = 19), iPSC-Pg (1.4 × 106, n = 17), total EV secreted by 1.4 × 106 iPSC-Pg (n = 19), or phosphate-buffered saline (control, n = 17) into the peri-infarct myocardium. Seven weeks later, hearts were evaluated by echocardiography, histology, and gene expression profiling, blinded to treatment group. In vitro, EV were internalized by target cells, increased cell survival, cell proliferation, and endothelial cell migration in a dose-dependent manner and stimulated tube formation. Extracellular vesicles were rich in miRNAs and most of the 16 highly abundant, evolutionarily conserved miRNAs are associated with tissue-repair pathways. In vivo, EV outperformed cell injections, significantly improving cardiac function through decreased left ventricular volumes (left ventricular end systolic volume: -11%, P < 0.001; left ventricular end diastolic volume: -4%, P = 0.002), and increased LVEF (+14%, P < 0.0001) relative to baseline values. Gene profiling revealed that EV-treated hearts were enriched for tissue reparative pathways. Extracellular vesicles secreted by iPSC-Pg are effective in the treatment of CHF, possibly, in part, through their specific miRNA signature and the associated stimulation of distinct cardioprotective pathways. The processing and regulatory advantages of EV could make them effective substitutes for cell transplantation.

The interleukin-1, interleukin-2, interleukin-6 and tumour necrosis factor alpha serological levels in localised and systemic sclerosis.

PubMed

Alecu, M; Geleriu, L; Coman, G; Gălăţescu, L

1998-01-01

Serological level of interleukin-1 (IL-1), Interleukin-2 (IL-2), Interleukin-6 (IL-6) and tumour necrosis factor (TNF) alpha was investigated in 26 patients with scleroderma, divided into three lots, by the extension and the progress of the disease. Determinations were performed by ELISA in attack and in remission (after treatment with prednison). Normal values: IL-1 (0-5 pg/ml), IL-2 (0-5 pg/ml), IL-6 (5-15 pg/ml), TNF (0-16 pg/ml). Lot A. Results obtained at the first determination showed that IL-1 is elevated in 4 cases (10-15 pg/ml), IL-2 in 5 cases (10-32 pg/ml), IL-6 in 5 cases (15-42 pg/ml) and TNF in 4 cases (18-34 pg/ml). In the second determination IL-1 was increased in 1 case (8 pg/ml), IL-2 in 1 case (9 pg/ml), IL-6 in 2 cases (12 pg/ml) and TNF was normal. Lot B. In the first determination IL-1 was elevated in 5 cases (8-12 pg/ml), IL-2 in 5 cases (10-15 pg/ml), IL-6 in 7 cases (16-20 pg/ml) and TNF was raised in 3 cases (18-25 pg/ml). At the second determination IL-1 showed normal values in all the cases, IL-2 was raised in 2 cases (10 pg/ml), IL-6 in 2 cases (12.15 pg/ml), TNF in 1 case (20 pg/ml). Lot C. In the first determination there were raised values in 4 cases for IL-1 (6-8 pg/ml), 3 cases for IL-2 (10-18 pg/ml), 5 cases for IL-6 (18-20 pg/ml), 2 cases for TNF (20 pg/ml). At the second determination IL-2 was elevated in 1 case (10 pg/ml), IL-6 in 1 case (15 pg/ml). We consider that in scleroderma there is a disturbance of the investigated cytokines due to the activation and involvement of the secretory cells into the pathogenesis of the disease. The increase of the serological levels of IL-1, IL-2, IL-6 and TNF depends on the extension of the lesions and the clinical and biological activity periods of the disease. The absence of the increase of the serological levels does not exclude their activity at the lesional site.

Infection of Brachypodium distachyon by Formae Speciales of Puccinia graminis: Early Infection Events and Host-Pathogen Incompatibility

PubMed Central

Figueroa, Melania; Alderman, Stephen; Garvin, David F.; Pfender, William F.

2013-01-01

Puccinia graminis causes stem rust, a serious disease of cereals and forage grasses. Important formae speciales of P. graminis and their typical hosts are P. graminis f. sp. tritici (Pg-tr) in wheat and barley, P. graminis f. sp. lolii (Pg-lo) in perennial ryegrass and tall fescue, and P. graminis f. sp. phlei-pratensis (Pg-pp) in timothy grass. Brachypodium distachyon is an emerging genetic model to study fungal disease resistance in cereals and temperate grasses. We characterized the P. graminis-Brachypodium pathosystem to evaluate its potential for investigating incompatibility and non-host resistance to P. graminis. Inoculation of eight Brachypodium inbred lines with Pg-tr, Pg-lo or Pg-pp resulted in sporulating lesions later accompanied by necrosis. Histological analysis of early infection events in one Brachypodium inbred line (Bd1-1) indicated that Pg-lo and Pg-pp were markedly more efficient than Pg-tr at establishing a biotrophic interaction. Formation of appressoria was completed (60–70% of germinated spores) by 12 h post-inoculation (hpi) under dark and wet conditions, and after 4 h of subsequent light exposure fungal penetration structures (penetration peg, substomatal vesicle and primary infection hyphae) had developed. Brachypodium Bd1-1 exhibited pre-haustorial resistance to Pg-tr, i.e. infection usually stopped at appressorial formation. By 68 hpi, only 0.3% and 0.7% of the Pg-tr urediniospores developed haustoria and colonies, respectively. In contrast, development of advanced infection structures by Pg-lo and Pg-pp was significantly more common; however, Brachypodium displayed post-haustorial resistance to these isolates. By 68 hpi the percentage of urediniospores that only develop a haustorium mother cell or haustorium in Pg-lo and Pg-pp reached 8% and 5%, respectively. The formation of colonies reached 14% and 13%, respectively. We conclude that Brachypodium is an apt grass model to study the molecular and genetic components of incompatiblity and non-host resistance to P. graminis. PMID:23441218

Ecological response to collapse of the biological pump following the mass extinction at the Cretaceous-Paleogene boundary

NASA Astrophysics Data System (ADS)

Vellekoop, Johan; Woelders, Lineke; Açikalin, Sanem; Smit, Jan; van de Schootbrugge, Bas; Yilmaz, Ismail Ö.; Brinkhuis, Henk; Speijer, Robert P.

2017-02-01

It is commonly accepted that the mass extinction associated with the Cretaceous-Paleogene (K-Pg) boundary (˜ 66 Ma) is related to the environmental effects of a large extraterrestrial impact. The biological and oceanographic consequences of the mass extinction are, however, still poorly understood. According to the Living Ocean model, the biological crisis at the K-Pg boundary resulted in a long-term reduction of export productivity in the early Paleocene. Here, we combine organic-walled dinoflagellate cyst (dinocyst) and benthic foraminiferal analyses to provide new insights into changes in the coupling of pelagic and benthic ecosystems. To this end, we perform dinocyst and benthic foraminiferal analyses on the recently discovered Tethyan K-Pg boundary section at Okçular, Turkey, and compare the results with other K-Pg boundary sites in the Tethys. The post-impact dominance of epibenthic morphotypes and an increase of inferred heterotrophic dinocysts in the early Paleocene at Okçular are consistent with published records from other western Tethyan sites. Together, these records indicate that during the early Paleocene more nutrients remained available for the Tethyan planktonic community, whereas benthic communities were deprived of food. Hence, in the post-impact phase the reduction of export productivity likely resulted in enhanced recycling of nutrients in the upper part of the water column, all along the western Tethyan margins.

Fragments of the V1/V2 domain of HIV-1 glycoprotein 120 engineered for improved binding to the broadly neutralizing PG9 antibody.

PubMed

Morales, Javier F; Yu, Bin; Perez, Gerardo; Mesa, Kathryn A; Alexander, David L; Berman, Phillip W

2016-09-01

The V1/V2 domain of the HIV-1 envelope protein gp120 possesses two important epitopes: a glycan-dependent epitope recognized by the prototypic broadly neutralizing monoclonal antibody (bN-mAb), PG9, as well as an epitope recognized by non-neutralizing antibodies that has been associated with protection from HIV infection in the RV144 HIV vaccine trial. Because both of these epitopes are poorly immunogenic in the context of full length envelope proteins, immunization with properly folded and glycosylated fragments (scaffolds) represents a potential way to enhance the immune response to these specific epitopes. Previous studies showed that V1/V2 domain scaffolds could be produced from a few selected isolates, but not from many of the isolates that would be advantageous in a multivalent vaccine. In this paper, we used a protein engineering approach to improve the conformational stability and antibody binding activity of V1/V2 domain scaffolds from multiple diverse isolates, including several that were initially unable to bind the prototypic PG9 bN-mAb. Significantly, this effort required replicating both the correct glycan structure as well as the β-sheet structure required for PG9 binding. Although scaffolds incorporating the glycans required for PG9 binding (e.g., mannose-5) can be produced using glycosylation inhibitors (e.g., swainsonine), or mutant cell lines (e.g. GnTI(-) 293 HEK), these are not practical for biopharmaceutical production of proteins intended for clinical trials. In this report, we describe engineered glycopeptide scaffolds from three different clades of HIV-1 that bind PG9 with high affinity when expressed in a wildtype cell line suitable for biopharmaceutical production. The mutations that improved PG9 binding to scaffolds produced in normal cells included amino acid positions outside of the antibody contact region designed to stabilize the β-sheet and turn structures. The scaffolds produced address three major problems in HIV vaccine development: (1) improving antibody responses to poorly immunogenic epitopes in the V1/V2 domain; (2) eliminating antibody responses to highly immunogenic (decoy) epitopes outside the V1/V2 domain; and (3) enabling the production of V1/V2 scaffolds in a cell line suitable for biopharmaceutical production. Copyright © 2016 Elsevier Ltd. All rights reserved.

Proteome-wide analysis and CXCL4 as a biomarker in systemic sclerosis.

PubMed

van Bon, Lenny; Affandi, Alsya J; Broen, Jasper; Christmann, Romy B; Marijnissen, Renoud J; Stawski, Lukasz; Farina, Giuseppina A; Stifano, Giuseppina; Mathes, Allison L; Cossu, Marta; York, Michael; Collins, Cindy; Wenink, Mark; Huijbens, Richard; Hesselstrand, Roger; Saxne, Tore; DiMarzio, Mike; Wuttge, Dirk; Agarwal, Sandeep K; Reveille, John D; Assassi, Shervin; Mayes, Maureen; Deng, Yanhui; Drenth, Joost P H; de Graaf, Jacqueline; den Heijer, Martin; Kallenberg, Cees G M; Bijl, Marc; Loof, Arnoud; van den Berg, Wim B; Joosten, Leo A B; Smith, Vanessa; de Keyser, Filip; Scorza, Rafaella; Lunardi, Claudio; van Riel, Piet L C M; Vonk, Madelon; van Heerde, Waander; Meller, Stephan; Homey, Bernhard; Beretta, Lorenzo; Roest, Mark; Trojanowska, Maria; Lafyatis, Robert; Radstake, Timothy R D J

2014-01-30

Plasmacytoid dendritic cells have been implicated in the pathogenesis of systemic sclerosis through mechanisms beyond the previously suggested production of type I interferon. We isolated plasmacytoid dendritic cells from healthy persons and from patients with systemic sclerosis who had distinct clinical phenotypes. We then performed proteome-wide analysis and validated these observations in five large cohorts of patients with systemic sclerosis. Next, we compared the results with those in patients with systemic lupus erythematosus, ankylosing spondylitis, and hepatic fibrosis. We correlated plasma levels of CXCL4 protein with features of systemic sclerosis and studied the direct effects of CXCL4 in vitro and in vivo. Proteome-wide analysis and validation showed that CXCL4 is the predominant protein secreted by plasmacytoid dendritic cells in systemic sclerosis, both in circulation and in skin. The mean (±SD) level of CXCL4 in patients with systemic sclerosis was 25,624±2652 pg per milliliter, which was significantly higher than the level in controls (92.5±77.9 pg per milliliter) and than the level in patients with systemic lupus erythematosus (1346±1011 pg per milliliter), ankylosing spondylitis (1368±1162 pg per milliliter), or liver fibrosis (1668±1263 pg per milliliter). CXCL4 levels correlated with skin and lung fibrosis and with pulmonary arterial hypertension. Among chemokines, only CXCL4 predicted the risk and progression of systemic sclerosis. In vitro, CXCL4 down-regulated expression of transcription factor FLI1, induced markers of endothelial-cell activation, and potentiated responses of toll-like receptors. In vivo, CXCL4 induced the influx of inflammatory cells and skin transcriptome changes, as in systemic sclerosis. Levels of CXCL4 were elevated in patients with systemic sclerosis and correlated with the presence and progression of complications, such as lung fibrosis and pulmonary arterial hypertension. (Funded by the Dutch Arthritis Association and others.).

Proteome-wide Analysis and CXCL4 as a Biomarker in Systemic Sclerosis

PubMed Central

van Bon, L.; Affandi, A.J.; Broen, J.; Christmann, R.B.; Marijnissen, R.J.; Stawski, L.; Farina, G.A.; Stifano, G.; Mathes, A.L.; Cossu, M.; York, M.; Collins, C.; Wenink, M.; Huijbens, R.; Hesselstrand, R.; Saxne, T.; DiMarzio, M.; Wuttge, D.; Agarwal, S.K.; Reveille, J.D.; Assassi, S.; Mayes, M.; Deng, Y.; Drenth, J.P.H.; de Graaf, J.; den Heijer, M.; Kallenberg, C.G.M.; Bijl, M.; Loof, A.; van den Berg, W.B.; Joosten, L.A.B.; Smith, V.; de Keyser, F.; Scorza, R.; Lunardi, C.; van Riel, P.L.C.M.; Vonk, M.; van Heerde, W.; Meller, S.; Homey, B.; Beretta, L.; Roest, M.; Trojanowska, M.; Lafyatis, R.; Radstake, T.R.D.J.

2014-01-01

Background Plasmacytoid dendritic cells have been implicated in the pathogenesis of systemic sclerosis through mechanisms beyond the previously suggested production of type I interferon. Methods We isolated plasmacytoid dendritic cells from healthy persons and from patients with systemic sclerosis who had distinct clinical phenotypes. We then performed proteome-wide analysis and validated these observations in five large cohorts of patients with systemic sclerosis. Next, we compared the results with those in patients with systemic lupus erythematosus, ankylosing spondylitis, and hepatic fibrosis. We correlated plasma levels of CXCL4 protein with features of systemic sclerosis and studied the direct effects of CXCL4 in vitro and in vivo. Results Proteome-wide analysis and validation showed that CXCL4 is the predominant protein secreted by plasmacytoid dendritic cells in systemic sclerosis, both in circulation and in skin. The mean (±SD) level of CXCL4 in patients with systemic sclerosis was 25,624±2652 pg per milliliter, which was significantly higher than the level in controls (92.5±77.9 pg per milliliter) and than the level in patients with systemic lupus erythematosus (1346±1011 pg per milliliter), ankylosing spondylitis (1368±1162 pg per milliliter), or liver fibrosis (1668±1263 pg per milliliter). CXCL4 levels correlated with skin and lung fibrosis and with pulmonary arterial hypertension. Among chemokines, only CXCL4 predicted the risk and progression of systemic sclerosis. In vitro, CXCL4 downregulated expression of transcription factor FLI1, induced markers of endothelial-cell activation, and potentiated responses of toll-like receptors. In vivo, CXCL4 induced the influx of inflammatory cells and skin transcriptome changes, as in systemic sclerosis. Conclusions Levels of CXCL4 were elevated in patients with systemic sclerosis and correlated with the presence and progression of complications, such as lung fibrosis and pulmonary arterial hypertension. (Funded by the Dutch Arthritis Association and others.) PMID:24350901

CHCHD10 mutations p.R15L and p.G66V cause motoneuron disease by haploinsufficiency.

PubMed

Brockmann, Sarah J; Freischmidt, Axel; Oeckl, Patrick; Müller, Kathrin; Ponna, Srinivas K; Helferich, Anika M; Paone, Christoph; Reinders, Jörg; Kojer, Kerstin; Orth, Michael; Jokela, Manu; Auranen, Mari; Udd, Bjarne; Hermann, Andreas; Danzer, Karin M; Lichtner, Peter; Walther, Paul; Ludolph, Albert C; Andersen, Peter M; Otto, Markus; Kursula, Petri; Just, Steffen; Weishaupt, Jochen H

2018-02-15

Mutations in the mitochondrially located protein CHCHD10 cause motoneuron disease by an unknown mechanism. In this study, we investigate the mutations p.R15L and p.G66V in comparison to wild-type CHCHD10 and the non-pathogenic variant p.P34S in vitro, in patient cells as well as in the vertebrate in vivo model zebrafish. We demonstrate a reduction of CHCHD10 protein levels in p.R15L and p.G66V mutant patient cells to approximately 50%. Quantitative real-time PCR revealed that expression of CHCHD10 p.R15L, but not of CHCHD10 p.G66V, is already abrogated at the mRNA level. Altered secondary structure and rapid protein degradation are observed with regard to the CHCHD10 p.G66V mutant. In contrast, no significant differences in expression, degradation rate or secondary structure of non-pathogenic CHCHD10 p.P34S are detected when compared with wild-type protein. Knockdown of CHCHD10 expression in zebrafish to about 50% causes motoneuron pathology, abnormal myofibrillar structure and motility deficits in vivo. Thus, our data show that the CHCHD10 mutations p.R15L and p.G66V cause motoneuron disease primarily based on haploinsufficiency of CHCHD10. © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Effects of different cytokines on immune responses of rainbow trout in a virus DNA vaccination model

PubMed Central

Cao, Yongsheng; Zhang, Qiya; Xu, Liming; Li, Shaowu; Wang, Di; Zhao, Jingzhuang; Liu, Hongbai; Feng, Jian; Lu, Tongyan

2017-01-01

Seven rainbow trout cytokine genes (interleukin (IL)-2, IL-8, IL-15, IL-17, IL-1β, intracellular interferon (iIFN) 1a, and IFN-γ2) were evaluated for their adjuvant effects on a DNA vaccine, called pG, containing the glycoprotein gene of infectious hematopoietic necrosis virus (IHNV). Distinct DNA constructs in expression plasmid pcDNA3.1 encoding a cytokine gene were generated. Immunofluorescence assays in rainbow trout gonadal cells demonstrated successful protein expression from all these constructs. Subsequently, fish were immunized with pG alone or together with a cytokine expression plasmid. Results showed that each cytokine plasmids at an appropriate dose showed notable effects on immune gene expression. IL-17 and IFN-γ2 can enhance early specific IgM response. All cytokines, except IL-8, can benefit initial neutralizing antibody (NAb) titers. At 35 days post immunization (dpi), NAb titers of fish immunized with pG and IL-2, iIFN1a, or IFN-γ2 plasmids remained at high levels (1:160). NAb titers of fish immunized with pG alone decreased to 1:40. IL-8 or IL-1β can enhance antigen-specific proliferative T-cell responses at 14 dpi. At 28 dpi, coinjection of pG with IL-2, IL-8, IL-15, or IL-17 plasmids induced considerably stronger lymphocyte proliferation than that with injection of pG alone. All cytokine plasmids delivered with pG plasmid enhanced protection of trout against IHNV-mediated mortality. These results indicate that the type and dose of trout cytokine genes injected into fish affect quality of immune response to DNA vaccination. PMID:29348820

Characterization of the Porphyromonas gingivalis conjugative transposon CTnPg1: determination of the integration site and the genes essential for conjugal transfer.

PubMed

Naito, Mariko; Sato, Keiko; Shoji, Mikio; Yukitake, Hideharu; Ogura, Yoshitoshi; Hayashi, Tetsuya; Nakayama, Koji

2011-07-01

In our previous study, extensive genomic rearrangements were found in two strains of the Gram-negative anaerobic bacterium Porphyromonas (Por.) gingivalis, and most of these rearrangements were associated with mobile genetic elements such as insertion sequences and conjugative transposons (CTns). CTnPg1, identified in Por. gingivalis strain ATCC 33277, was the first complete CTn reported for the genus Porphyromonas. In the present study, we found that CTnPg1 can be transferred from strain ATCC 33277 to another Por. gingivalis strain, W83, at a frequency of 10(-7) to 10(-6). The excision of CTnPg1 from the chromosome in a donor cell depends on an integrase (Int; PGN_0094) encoded in CTnPg1, whereas CTnPg1 excision is independent of PGN_0084 (a DNA topoisomerase I homologue; Exc) encoded within CTnPg1 and recA (PGN_1057) on the donor chromosome. Intriguingly, however, the transfer of CTnPg1 between Por. gingivalis strains requires RecA function in the recipient. Sequencing analysis of CTnPg1-integrated sites on the chromosomes of transconjugants revealed that the consensus attachment (att) sequence is a 13 bp sequence, TTTTCNNNNAAAA. We further report that CTnPg1 is able to transfer to two other bacterial species, Bacteroides thetaiotaomicron and Prevotella oralis. In addition, CTnPg1-like CTns are located in the genomes of other oral anaerobic bacteria, Porphyromonas endodontalis, Prevotella buccae and Prevotella intermedia, with the same consensus att sequence. These results suggest that CTns in the CTnPg1 family are widely distributed among oral anaerobic Gram-negative bacteria found in humans and play important roles in horizontal gene transfer among these bacteria.

Identification of Cell Wall Synthesis Regulatory Genes Controlling Biomass Characteristics and Yield in Rice (Oryza Sativa)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peng, Zhaohua PEng; Ronald, Palmela; Wang, Guo-Liang

This project aims to identify the regulatory genes of rice cell wall synthesis pathways using a cell wall removal and regeneration system. We completed the gene expression profiling studies following the time course from cell wall removal to cell wall regeneration in rice suspension cells. We also completed, total proteome, nuclear subproteome and histone modification studies following the course from cell wall removal and cell wall regeneration process. A large number of differentially expressed regulatory genes and proteins were identified. Meanwhile, we generated RNAi and over-expression transgenic rice for 45 genes with at least 10 independent transgenic lines for eachmore » gene. In addition, we ordered T-DNA and transposon insertion mutants for 60 genes from Korea, Japan, and France and characterized the mutants. Overall, we have mutants and transgenic lines for over 90 genes, exceeded our proposed goal of generating mutants for 50 genes. Interesting Discoveries a) Cell wall re-synthesis in protoplasts may involve a novel cell wall synthesis mechanism. The synthesis of the primary cell wall is initiated in late cytokinesis with further modification during cell expansion. Phragmoplast plays an essential role in cell wall synthesis. It services as a scaffold for building the cell plate and formation of a new cell wall. Only one phragmoplast and one new cell wall is produced for each dividing cell. When the cell wall was removed enzymatically, we found that cell wall re-synthesis started from multiple locations simultaneously, suggesting that a novel mechanism is involved in cell wall re-synthesis. This observation raised many interesting questions, such as how the starting sites of cell wall synthesis are determined, whether phragmoplast and cell plate like structures are involved in cell wall re-synthesis, and more importantly whether the same set of enzymes and apparatus are used in cell wall re-synthesis as during cytokinesis. Given that many known cell wall synthesis pathway genes are induced by removal of cell wall, some cell wall synthesis apparatus must be shared in both cases. The cell wall re-synthesis mechanism may have broad application because our preliminary assay indicates that the cell wall characteristics are highly different from those produced during cytokinesis. A thorough understanding on the regulation of cell wall re-synthesis may lead to improvement of cell wall characteristics. b) Removal of cell wall results in chromatin decondensation Another interesting observation was that removal of cell wall was associated with substantial chromatin change. Our DNA DAPI stain, chromatin MNase digestion, histone modification proteomics, protein differential expression analysis, and DNA oligo array studies all supported that substantial chromatin change was associated with removal of cell wall treatment. It is still under investigation if the chromatin change is associated with activation of cell wall synthesis genes, in which chromatin remodeling is required. Another possibility is that the cell wall is required for stabilizing the chromatin structure in plant cells. Given that spindle fiber is directly connected with both chromatin structure and cell wall synthesis, it is possible that there is an intrinsic connection between cell wall and chromatin.« less

Design of polymer-biopolymer-hydroxyapatite biomaterials for bone tissue engineering: Through molecular control of interfaces

NASA Astrophysics Data System (ADS)

Verma, Devendra

In this dissertation, novel biomaterials are designed for bone biomaterials and bone tissue engineering applications. Novel biomaterials of hydroxyapatite with synthetic and natural polymers have been fabricated using a combination of processing routes. Initially, we investigated hydroxyapatite-polycaprolactone-polyacrylic acid composites and observed that minimal interfacial interactions between polymer and mineral led to inadequate improvement in the mechanical properties. Bioactivity experiments on these composites showed that the presence of functional groups, such as carboxylate groups, influence bioactivity of the composites. We have developed and investigated composites of hydroxyapatite with chitosan and polygalacturonic acid (PgA). Chitosan and PgA are biocompatible, biodegradable, and also electrostatically complementary to each other. This strategy led to significant improvement in mechanical properties of new composites. The nanostructure analysis using atomic force microscopy revealed a multilevel organization in these composites. Enhancement in mechanical response was attributed to stronger interfaces due to strong electrostatic interaction between oppositely charged chitosan and PgA. Further analysis using the Rietveld method showed that biopolymers have marked impact on hydroxyapatite crystal growth and also on its crystal structure. Significant changes were observed in the lattice parameters of hydroxyapatite synthesized by following biomineralization method (organics mediated mineralization). For scaffold preparation, chitosan and PgA were mixed first, and then, nano-hydroxyapatite was added. Oppositely charged polyelectrolytes, such as chitosan and PgA, spontaneously form complex upon mixing. The poly-electrolyte complex exists as nano-sized particles. Chitosan/PgA scaffolds with and without hydroxyapatite were prepared by the freeze drying method. By controlling the rate of cooling and concentration, we have produced both fibrous and sheet-containing scaffolds. Hydroxyapatite-containing chitosan/PgA scaffolds maintained their structural integrity under wet conditions. These scaffolds showed extremely porous (97.4%) and interconnected architecture. These scaffolds also promoted cell adhesion, proliferation and differentiation, Osteoblast cells formed nodular structure on thin films and scaffold. Mineralization of these nodules was confirmed by alizarin red S staining. Even after 20 days of seeding, all the cells were found alive. Our results indicated that chitosan-PgA-hydroxyapatite composite scaffolds have high potential for bone tissue engineering. This dissertation represents a comprehensive study on design of novel bone biomaterials through tailoring of interfaces in nanocomposites of polymers, biopolymer and hydroxyapatite.

Transcriptomics-based identification of WRKY genes and characterization of a salt and hormone-responsive PgWRKY1 gene in Panax ginseng.

PubMed

Nuruzzaman, Mohammed; Cao, Hongzhe; Xiu, Hao; Luo, Tiao; Li, Jijia; Chen, Xianghui; Luo, Junli; Luo, Zhiyong

2016-02-01

WRKY proteins belong to a transcription factor (TF) family and play dynamic roles in many plant processes, including plant responses to abiotic and biotic stresses, as well as secondary metabolism. However, no WRKY gene in Panax ginseng C.A. Meyer has been reported to date. In this study, a number of WRKY unigenes from methyl jasmonate (MeJA)-treated adventitious root transcriptome of this species were identified using next-generation sequencing technology. A total of 48 promising WRKY unigenes encoding WRKY proteins were obtained by eliminating wrong and incomplete open reading frame (ORF). Phylogenetic analysis reveals 48 WRKY TFs, including 11 Group I, 36 Group II, and 1 Group III. Moreover, one MeJA-responsive unigene designated as PgWRKY1 was cloned and characterized. It contains an entire ORF of 1077 bp and encodes a polypeptide of 358 amino acid residues. The PgWRKY1 protein contains a single WRKY domain consisting of a conserved amino acid sequence motif WRKYGQK and a C2H2-type zinc-finger motif belonging to WRKY subgroup II-d. Subcellular localization of PgWRKY1-GFP fusion protein in onion and tobacco epidermis cells revealed that PgWRKY1 was exclusively present in the nucleus. Quantitative real-time polymerase chain reaction analysis demonstrated that the expression of PgWRKY1 was relatively higher in roots and lateral roots compared with leaves, stems, and seeds. Importantly, PgWRKY1 expression was significantly induced by salicylic acid, abscisic acid, and NaCl, but downregulated by MeJA treatment. These results suggested that PgWRKY1 might be a multiple stress-inducible gene responding to hormones and salt stresses. © The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

A comparative study of the adhesion of biosynthesized gold and conjugated gold/prodigiosin nanoparticles to triple negative breast cancer cells.

PubMed

Dozie-Nwachukwu, S O; Obayemi, J D; Danyuo, Y; Anuku, N; Odusanya, O S; Malatesta, K; Soboyejo, W O

2017-08-17

This paper explores the adhesion of biosynthesized gold nanoparticles (AuNPs) and gold (Au) nanoparticle/prodigiosin (PG) drug nanoparticles to breast cancer cells (MDA-MB-231 cells). The AuNPs were synthesized in a record time (less than 30 s) from Nauclea latifolia leaf extracts, while the PG was produced via bacterial synthesis with Serratia marcescens sp. The size distributions and shapes of the resulting AuNPs were characterized using transmission electron microscopy (TEM), while the resulting hydrodynamic diameters and polydispersity indices were studied using dynamic light scattering (DLS). Atomic Force Microscopy (AFM) was used to study the adhesion between the synthesized gold nanoparticles (AuNPs)/LHRH-conjugated AuNPs and triple negative breast cancer cells (MDA-MB-231 cells), as well as the adhesion between LHRH-conjugated AuNP/PG drug and MDA-MB-231 breast cancer cells. The adhesion forces between LHRH-conjugated AuNPs and breast cancer cells are shown to be five times greater than those between AuNPs and normal breast cells. The increase in adhesion is shown to be due to the over-expression of LHRH receptors on the surfaces of MDA-MB-231 breast cancer cells, which was revealed by confocal immuno-fluorescence microscopy. The implications of the results are then discussed for the selective and specific targeting and treatment of triple negative breast cancer.

Glycyrrhizin restores the impaired IL-12 production in thermally injured mice.

PubMed

Utsunomiya, T; Kobayashi, M; Ito, M; Herndon, D N; Pollard, R B; Suzuki, F

2001-04-07

Mice 6 days after thermal injury (TI-mice) did not respond to lipopolysaccharide (LPS) stimulation for production of serum interleukin 12 (IL-12; 2 h after LPS stimulation, <20 pg/ml in TI-mice; 1091+/-162 pg/ml in normal mice). However, 2 h after LPS stimulation, 1456+/-118 pg/ml of IL-12 were demonstrated in sera of TI-mice previously treated with a 10 mg/kg i.p. dose of glycyrrhizin (GR). IL-12 was not induced by LPS in sera of normal mice inoculated with burn-associated type 2 T cells (IL-4/IL-10-producing CD8+CD11b+TCRgamma/delta+T cells isolated from spleens of TI-mice). However, IL-12 production was induced by LPS in sera of these mice previously treated with GR or a mixture of monoclonal antibodies (mAbs) for type 2 cytokines. Also, IL-12 production was induced by LPS in TI-mice inoculated with CD4+T cells from spleens of GR-treated normal mice (GR-CD4+T cells, 5x10(6)cells/mouse). Since GR-CD4+T cells have been shown to be antagonistic cells against production of type 2 cytokines by burn-associated type 2 T cells, these results indicate that IL-12 unresponsiveness shown in TI-mice is recovered by GR through the regulation of burn-associated type 2 T cell responses. Copyright 2001 Academic Press.

[Therapeutic efficacy of compound Xuanju capsule on autoimmune prostatitis in rats: an experimental study].

PubMed

Li, Tian-Fu; Wu, Qiu-Yue; Li, Wei-Wei; Zhang, Cui; Li, Na; Shang, Xue-Jun; Xia, Xin-Yi; Xu, Hao-Qin; Huang, Yu-Feng

2014-05-01

To evaluate the therapeutic effect of Compound Xuanju Capsule (CXC) on autoimmune prostatitis in rat models. Sixty healthy male Wistar rats were randomly divided into five groups of equal number: blank control, low-concentration purified prostate protein (low-conc PPP), low-conc PPP + CXC treatment, high-concentration PPP (hi-con PPP), and hi-conc PPP + CXC treatment. Autoimmune prostatitis models were established by intragastric administration of PPP solution at 15 mg/ml (low concentration) and 80 mg/ml, respectively. At 30 days after modeling, the rats in the blank control and low-conc and hi-conc PPP model groups were treated with normal saline, and those in the other two groups with CXC at a daily dose of 0.068 g/ml. At 30, 45, and 60 days, all the animals were sacrificed for observation of pathological changes in the prostate tissue and determination of the levels of IL-8, IL-10, and TNF-alpha in the serum. Compared with the PPP models, the hi-conc PPP + CXC group showed significantly reduced levels of IL-8 and TNF-alpha in the serum at 45 days ([148.54 +/- 17.23] and [62.14 +/- 5.59] pg/ml vs [100.77 +/- 11.08] and [32.63 +/- 2.91] pg/ml, P < 0.05) and at 60 days ([143.69 +/- 17.28] and [59.38 +/- 5.50] pg/mlvs [95.77 +/-10.53] and [29.63 +/- 2.66] pg/ml, P < 0.05), and so did the low-cone PPP + CXC group at 45 days ([128.47 +/- 12.21] and [40.43 +/- 3.64] pg/ml vs [111.76 +/- 10.07] and [35.44 +/- 3.17] pg/ml, P < 0.05) and at 60 days ([131.07 +/- 10.93] and [43.34 +/- 3.91] pg/ml vs [97.46 +/- 8.75] and [30.44 +/- 2.75] pg/ml, P < 0.05). The serum level of IL-10 was remarkably elevated in the hi-cone PPP + CXC group as compared with that of the PPP models at 45 and 60 days ([189.14 +/- 16.78] and [184.14 +/- 15.89] pg/ml vs [230.48 +/- 29.96] and [248.48 +/- 31.03] pg/ml, P < 0.05), and so was it in low-cone PPP + CXC group ([223.14 +/- 17.87] and [224.14 +/- 17.93] pg/ml vs [231.42 +/- 23.18] and [249.42 +/- 24.97] pg/ml, P < 0.05). Pathological examination revealed morphological damages to the prostate tissue and infiltration of inflammatory cells in the model rats, but no obvious changes in the normal controls. At 15 days of treatment, the rats in the PPP + CXC group showed enlarged prostate glandular cavity, mild proliferation of epithelial cells, no obvious infiltration of inflammatory cells in the interstitial tissue, and a few visible fibrous tissues under the light microscope. Compound Xuanju Capsule is efficacious on autoimmune prostatis in rats by reducing inflammatory changes in the prostate tissue and improving the expression of inflammatory factors.

Implantation of In Vitro Tissue Engineered Muscle Repair Constructs and Bladder Acellular Matrices Partially Restore In Vivo Skeletal Muscle Function in a Rat Model of Volumetric Muscle Loss Injury

DTIC Science & Technology

2014-01-01

thickness abdominal wall defects. Tissue Eng 12, 1929, 2006. 7. Gamba, P.G., Conconi, M.T., Lo Piccolo, R., Zara , G., Spi nazzi, R., and Parnigotto... Zara , G., Sabatti, M., Marzaro, M., et al. Homologous muscle acellular matrix seeded with autologous myoblasts as a tissue engineering approach to

Mutations in cell elongation genes mreB, mrdA and mrdB suppress the shape defect of RodZ-deficient cells.

PubMed

Shiomi, Daisuke; Toyoda, Atsushi; Aizu, Tomoyuki; Ejima, Fumio; Fujiyama, Asao; Shini, Tadasu; Kohara, Yuji; Niki, Hironori

2013-03-01

RodZ interacts with MreB and both factors are required to maintain the rod shape of Escherichia coli. The assembly of MreB into filaments regulates the subcellular arrangement of a group of enzymes that synthesizes the peptidoglycan (PG) layer. However, it is still unknown how polymerization of MreB determines the rod shape of bacterial cells. Regulatory factor(s) are likely to be involved in controlling the function and dynamics of MreB. We isolated suppressor mutations to partially recover the rod shape in rodZ deletion mutants and found that some of the suppressor mutations occurred in mreB. All of the mreB mutations were in or in the vicinity of domain IA of MreB. Those mreB mutations changed the property of MreB filaments in vivo. In addition, suppressor mutations were found in the periplasmic regions in PBP2 and RodA, encoded by mrdA and mrdB genes. Similar to MreB and RodZ, PBP2 and RodA are pivotal to the cell wall elongation process. Thus, we found that mutations in domain IA of MreB and in the periplasmic domain of PBP2 and RodA can restore growth and rod shape to ΔrodZ cells, possibly by changing the requirements of MreB in the process. © 2013 Blackwell Publishing Ltd.

Mutations in cell elongation genes mreB, mrdA and mrdB suppress the shape defect of RodZ-deficient cells

PubMed Central

Shiomi, Daisuke; Toyoda, Atsushi; Aizu, Tomoyuki; Ejima, Fumio; Fujiyama, Asao; Shini, Tadasu; Kohara, Yuji; Niki, Hironori

2013-01-01

RodZ interacts with MreB and both factors are required to maintain the rod shape of Escherichia coli. The assembly of MreB into filaments regulates the subcellular arrangement of a group of enzymes that synthesizes the peptidoglycan (PG) layer. However, it is still unknown how polymerization of MreB determines the rod shape of bacterial cells. Regulatory factor(s) are likely to be involved in controlling the function and dynamics of MreB. We isolated suppressor mutations to partially recover the rod shape in rodZ deletion mutants and found that some of the suppressor mutations occurred in mreB. All of the mreB mutations were in or in the vicinity of domain IA of MreB. Those mreB mutations changed the property of MreB filaments in vivo. In addition, suppressor mutations were found in the periplasmic regions in PBP2 and RodA, encoded by mrdA and mrdB genes. Similar to MreB and RodZ, PBP2 and RodA are pivotal to the cell wall elongation process. Thus, we found that mutations in domain IA of MreB and in the periplasmic domain of PBP2 and RodA can restore growth and rod shape to ΔrodZ cells, possibly by changing the requirements of MreB in the process. PMID:23301723

The polygalacturonase gene BcMF2 from Brassica campestris is associated with intine development

PubMed Central

Huang, Li; Cao, Jiashu; Zhang, Aihong; Ye, Yiqun; Zhang, Yuchao; Liu, Tingting

2009-01-01

Brassica campestris Male Fertility 2 (BcMF2) is a putative polygalacturonase (PG) gene previously isolated from the flower bud of Chinese cabbage (Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis). This gene was found to be expressed specifically in tapetum and pollen after the tetrad stage of anther development. Antisense RNA technology was used to study the function of BcMF2 in Chinese cabbage. Scanning and transmission electron microscopy revealed that there were deformities in the transgenic mature pollen grains such as abnormal location of germinal furrows. In addition, the homogeneous pectic exintine layer facing the exterior seemed to be overdeveloped and predominantly occupied the intine, thus reversing the normal proportional distribution of the internal endintine layer and the external exintine layer. Since it is a continuation of the intine layer, the pollen tube wall could not grow normally. This resulted in the formation of a balloon-like swelling structure in the pollen tube tip in nearly 80% of the transgenic pollen grains. Premature degradation of tapetum was also found in these transgenic plants, which displayed decreased expression of the BcMF2 gene. BcMF2 might therefore encode a new PG with an important role in pollen wall development, possibly via regulation of pectin's dynamic metabolism. PMID:19039102

Biochemical evaluation of the anticancer potential of the polyamine-based nanocarrier Nano11047

PubMed Central

Ferrari, Elena; Xie, Ying; Yu, Fei; Marton, Laurence J.; Oupicky, David; Casero, Robert A.

2017-01-01

Synthesizing polycationic polymers directly from existing drugs overcomes the drug-loading limitations often associated with pharmacologically inert nanocarriers. We recently described nanocarriers formed from a first-generation polyamine analogue, bis(ethyl)norspermine (BENSpm), that could simultaneously target polyamine metabolism while delivering therapeutic nucleic acids. In the current study, we describe the synthesis and evaluation of self-immolative nanocarriers derived from the second-generation polyamine analogue PG-11047. Polyamines are absolutely essential for proliferation and their metabolism is frequently dysregulated in cancer. Through its effects on polyamine metabolism, PG-11047 effectively inhibits tumor growth in cancer cell lines of multiple origins as well as in human tumor mouse xenografts. Promising clinical trials have been completed verifying the safety and tolerance of this rotationally restricted polyamine analogue. We therefore used PG-11047 as the basis for Nano11047, a biodegradable, prodrug nanocarrier capable of targeting polyamine metabolism. Following exposure of lung cancer cell lines to Nano11047, uptake and intracellular degradation into the parent compound PG-11047 was observed. The release of PG-11047 highly induced the polyamine catabolic enzyme activities of spermidine/spermine N1-acetyltransferase (SSAT) and spermine oxidase (SMOX). By contrast, the activity of ornithine decarboxylase (ODC), a rate-limiting enzyme in polyamine biosynthesis and a putative oncogene, was decreased. Consequently, intracellular levels of the natural polyamines were depleted concurrent with tumor cell growth inhibition. This availability of Nano11047 as a novel drug form and potential nucleic acid delivery vector will potentially benefit and encourage future clinical studies. PMID:28423064

Architecture of dermatophyte cell Walls: Electron microscopic and biochemical analysis

NASA Technical Reports Server (NTRS)

Nozawa, Y.; Kitajima, Y.

1984-01-01

A review with 83 references on the cell wall structure of dermatophytes is presented. Topics discussed include separation and preparation of cell walls; microstructure of cell walls by electron microscopy; chemical composition of cell walls; structural model of cell walls; and morphological structure of cell walls.

Effects of OK-432 (picibanil) on the estrogen receptors of MCF-7 cells and potentiation of antiproliferative effects of tamoxifen in combination with OK-432.

PubMed

Aoyagi, H; Iino, Y; Takeo, T; Horii, Y; Morishita, Y; Horiuchi, R

1997-01-01

OK-432 (picibanil), a streptococcal preparation, has a strong biological response modifier (BRM) function and is expected to produce clinical improvement and prolongation of survival in treated cancer patients in Japan. We were interested in whether OK-432 augments estrogen receptor (ER) levels in breast cancer. To investigate the effect of the BRMs on cellular growth and the characteristics of ER and progesterone receptors (PgR) in the human breast cancer cell line MCF-7, we used OK-432, Krestin (PSK), a protein-bound polysaccharide extracted from Coriolus versicolor, and lentinan, a fungal branched (1...3)-beta-D-glycan. OK432 and PSK dose dependently inhibited DNA synthesis of MCF-7 cells, and the 50% inhibitory concentrations of OK-432 and PSK were 1.2 KE (klinische Einheit, clinical unit)/ml and 200 micrograms/ml, respectively. Lentinan showed no direct anticancer effect in vitro. We found that OK-432 induced a 2-fold increase in ER levels in MCF-7 cells at 0.005 KE/ml, but not in PgR. Lentinan and low-dose PSK did not change ER or PgR levels, but high-dose PSK decreased ER and PgR. We also studied the combined effect of OK-432 and antiestrogens, tamoxifen (TAM) and DP-TAT-59. The combined treatment with OK-432 and TAM showed an additive inhibitory effect on MCF-7 cells. These results suggest that OK-432 may augment the therapeutic effect of TAM in breast cancer.

Arabidopsis Regenerating Protoplast: A Powerful Model System for Combining the Proteomics of Cell Wall Proteins and the Visualization of Cell Wall Dynamics

PubMed Central

Yokoyama, Ryusuke; Kuki, Hiroaki; Kuroha, Takeshi; Nishitani, Kazuhiko

2016-01-01

The development of a range of sub-proteomic approaches to the plant cell wall has identified many of the cell wall proteins. However, it remains difficult to elucidate the precise biological role of each protein and the cell wall dynamics driven by their actions. The plant protoplast provides an excellent means not only for characterizing cell wall proteins, but also for visualizing the dynamics of cell wall regeneration, during which cell wall proteins are secreted. It therefore offers a unique opportunity to investigate the de novo construction process of the cell wall. This review deals with sub-proteomic approaches to the plant cell wall through the use of protoplasts, a methodology that will provide the basis for further exploration of cell wall proteins and cell wall dynamics. PMID:28248244

Comparison of Ganglion Cell and Retinal Nerve Fiber Layer Thickness in Pigment Dispersion Syndrome, Pigmentary Glaucoma, and Healthy Subjects with Spectral-domain OCT.

PubMed

Arifoglu, Hasan Basri; Simavli, Huseyin; Midillioglu, Inci; Berk Ergun, Sule; Simsek, Saban

2017-01-01

To evaluate the ganglion cell complex (GCC) and retinal nerve fiber layer (RNFL) thickness in pigment dispersion syndrome (PDS) and pigmentary glaucoma (PG) with RTVue spectral domain optical coherence tomography (SD-OCT). A total of 102 subjects were enrolled: 29 with PDS, 18 with PG, and 55 normal subjects. Full ophthalmic examination including visual field analysis was performed. SD-OCT was used to analyze GCC superior, GCC inferior, and average RNFL thickness. To compare the discrimination capabilities, the areas under the receiver operating characteristic curves were assessed. Superior GCC, inferior GCC, and RNFL thickness values of patients with PG were statistically signicantly lower than those of patients with PDS (p < 0.001) and healthy individuals (p < 0.001 for all). No statistically significant difference was found between PDS and normal subjects in same parameters (p > 0.05). The SD-OCT-derived GCC and RNFL thickness parameters can be useful to discriminate PG from both PDS and normal subjects.

Withaferin A protects against spinal cord injury by inhibiting apoptosis and inflammation in mice.

PubMed

Yan, Xianlei; Huang, Guangxiang; Liu, Quan; Zheng, Jiemin; Chen, Hongmou; Huang, Qidan; Chen, Jiakang; Huang, Heqing

2017-12-01

Withaferin A (WFA) exhibits diverse pharmaceutical applications on human diseases, including rheumatoid arthritis, cancers and microbial infection. We evaluated the neuroprotective role of WFA using a mouse model of spinal cord injury (SCI). BALB/c mice were administrated 10 mg/kg of WFA. Gene expression was measured by real-time PCR, western blot and immunohistochemistry. Cell morphology and apoptosis were determined by H&E staining and TUNEL assay. Motor function was evaluated by the BBB functional scale for continuous 7 weeks. WFA significantly improved neurobehavioural function and alleviated histological alteration of spinal cord tissues in traumatized mice. Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) significantly increased in WFA-treated mice. Meanwhile, the expression of Nogo-A and RhoA remarkably decreased in the presence of WFA. Furthermore, the apoptotic cell death was attenuated in mice treated with WFA (31.48 ± 2.50% vs. 50.08 ± 2.08%) accompanied by decreased bax and increased bcl-2. In addition, WFA decreased the expression of pro-inflammatory mediators such as IL-1β (11.20 ± 1.96 ng/mL vs. 17.59 ± 1.42 ng/mL) and TNF-α (57.38 ± 3.57 pg/mL vs. 95.06 ± 9.13 pg/mL). The anti-inflammatory cytokines including TGF-β1 (14.32 ± 1.04 pg/mL vs. 9.37 ± 1.17 pg/mL) and IL-10 (116.80 ± 6.91 pg/mL vs. 72.33 ± 9.35 pg/mL) were elevated after WFA administration. This study demonstrated that WFA has a neuroprotective role by inhibition of apoptosis and inflammation after SCI in mice.

Co-Administration of TiO2 Nanowired Mesenchymal Stem Cells with Cerebrolysin Potentiates Neprilysin Level and Reduces Brain Pathology in Alzheimer's Disease.

PubMed

Sharma, Hari Shanker; Muresanu, Dafin Fior; Lafuente, José Vicente; Patnaik, Ranjana; Tian, Z Ryan; Ozkizilcik, Asya; Castellani, Rudy J; Mössler, Herbert; Sharma, Aruna

2018-01-01

Neprilysin (NPL), the rate-limiting enzyme for amyloid beta peptide (AβP), appears to play a crucial role in the pathogenesis of Alzheimer's disease (AD). Since mesenchymal stem cells (MSCs) and/or cerebrolysin (CBL, a combination of neurotrophic factors and active peptide fragments) have neuroprotective effects in various CNS disorders, we examined nanowired delivery of MSCs and CBL on NPL content and brain pathology in AD using a rat model. AD-like symptoms were produced by intraventricular (i.c.v.) administration of AβP (1-40) in the left lateral ventricle (250 ng/10 μl, once daily) for 4 weeks. After 30 days, the rats were examined for NPL and AβP concentrations in the brain and related pathology. Co-administration of TiO2-nanowired MSCs (10 6 cells) with 2.5 ml/kg CBL (i.v.) once daily for 1 week after 2 weeks of AβP infusion significantly increased the NPL in the hippocampus (400 pg/g) from the untreated control group (120 pg/g; control 420 ± 8 pg/g brain) along with a significant decrease in the AβP deposition (45 pg/g from untreated control 75 pg/g; saline control 40 ± 4 pg/g). Interestingly, these changes were much less evident when the MSCs or CBL treatment was given alone. Neuronal damages, gliosis, and myelin vesiculation were also markedly reduced by the combined treatment of TiO2, MSCs, and CBL in AD. These observations are the first to show that co-administration of TiO2-nanowired CBL and MSCs has superior neuroprotective effects in AD probably due to increasing the brain NPL level effectively, not reported earlier.

Characterization of the Sclerotinia sclerotiorum cell wall proteome.

PubMed

Liu, Longzhou; Free, Stephen J

2016-08-01

We used a proteomic analysis to identify cell wall proteins released from Sclerotinia sclerotiorum hyphal and sclerotial cell walls via a trifluoromethanesulfonic acid (TFMS) digestion. Cell walls from hyphae grown in Vogel's glucose medium (a synthetic medium lacking plant materials), from hyphae grown in potato dextrose broth and from sclerotia produced on potato dextrose agar were used in the analysis. Under the conditions used, TFMS digests the glycosidic linkages in the cell walls to release intact cell wall proteins. The analysis identified 24 glycosylphosphatidylinositol (GPI)-anchored cell wall proteins and 30 non-GPI-anchored cell wall proteins. We found that the cell walls contained an array of cell wall biosynthetic enzymes similar to those found in the cell walls of other fungi. When comparing the proteins in hyphal cell walls grown in potato dextrose broth with those in hyphal cell walls grown in the absence of plant material, it was found that a core group of cell wall biosynthetic proteins and some proteins associated with pathogenicity (secreted cellulases, pectin lyases, glucosidases and proteases) were expressed in both types of hyphae. The hyphae grown in potato dextrose broth contained a number of additional proteins (laccases, oxalate decarboxylase, peroxidase, polysaccharide deacetylase and several proteins unique to Sclerotinia and Botrytis) that might facilitate growth on a plant host. A comparison of the proteins in the sclerotial cell wall with the proteins in the hyphal cell wall demonstrated that sclerotia formation is not marked by a major shift in the composition of cell wall protein. We found that the S. sclerotiorum cell walls contained 11 cell wall proteins that were encoded only in Sclerotinia and Botrytis genomes. © 2015 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.

Aloe vera attenuated gastric injury on indomethacin-induced gastropathy in rats.

PubMed

Werawatganon, Duangporn; Rakananurak, Narisorn; Sallapant, Sasipim; Prueksapanich, Piyapan; Somanawat, Kanjana; Klaikeaw, Naruemon; Rerknimitr, Rungsun

2014-12-28

To evaluate the protective effects of Aloe vera on gastric injury in rats with indomethacin (IMN)-induced gastropathy. Male Sprague-Dawley rats were randomly divided into three groups. Group 1 (control, n = 6) was given distilled water (DW) orally. Group 2 (IMN, n = 6) was given oral IMN (150 mg/kg) dissolved in 5% sodium bicarbonate (NaHCO3 (-)) at time 0 and 4 h. Group 3 (Aloe vera-treated, n = 6) was given oral Aloe vera (150 mg/kg) dissolved in DW and IMN at time 0 and 4 h. Eight hours later, the stomach was removed to determine gastric malondialdehyde (MDA), the number of interleukin (IL)-18 positive stained cells (%) by immunohistochemistry, and for histopathological examination. Then, the serum was collected to determine tumor necrosis factor (TNF)-α and cytokine-induced neutrophil chemoattractant (CINC)-1 by sandwich enzyme linked immunosorbent assay method. In the IMN group, serum TNF-α, CINC-1 and gastric MDA were significantly increased when compared to the control group (27.78 ± 1.52 pg/mL vs 85.07 ± 49.11 pg/mL, P = 0.009; 104.55 ± 45.80 pg/mL vs 1054.70 ± 20.38 pg/mL, and 1.74 ± 0.21 nmol/mg vs 9.36 ± 1.07 nmol/mg protein, P = 0.000, respectively). The mean level of TNF-α, CINC-1 and gastric MDA in the Aloe vera-treated group were improved as compared with the IMN group (85.07 ± 49.11 pg/mL vs 35.19 ± 1.61 pg/mL, P = 0.021; 1054.70 ± 20.38 pg/mL vs 813.56 ± 239.04 pg/mL, P = 0.025; and 9.36 ± 1.07 nmol/mg vs 2.67 ± 0.64 nmol/mg protein, P = 0.000, respectively). The number of IL-18 positive stained cells (%) in the gastric epithelial cells of the IMN group was significantly higher than the control group (5.01% ± 3.73% vs 30.67% ± 2.03%, P = 0.000, respectively). In contrast, Aloe vera treatment decreased the number of IL-18 positive stained cells (%) significantly when compared with the IMN group (30.67% ± 2.03% vs 13.21% ± 1.10%, P = 0.000, respectively). Most rats in the IMN group developed moderate to severe gastric inflammation and erosions. The gastric erosions and neutrophil infiltration scores were significantly reduced in the Aloe vera-treated group. Aloe vera attenuated IMN-induced gastropathy in rats by the reduction of oxidative stress, inflammation, and improvement of gastric histopathology.

Aloe vera attenuated gastric injury on indomethacin-induced gastropathy in rats

PubMed Central

Werawatganon, Duangporn; Rakananurak, Narisorn; Sallapant, Sasipim; Prueksapanich, Piyapan; Somanawat, Kanjana; Klaikeaw, Naruemon; Rerknimitr, Rungsun

2014-01-01

AIM: To evaluate the protective effects of Aloe vera on gastric injury in rats with indomethacin (IMN)-induced gastropathy. METHODS: Male Sprague-Dawley rats were randomly divided into three groups. Group 1 (control, n = 6) was given distilled water (DW) orally. Group 2 (IMN, n = 6) was given oral IMN (150 mg/kg) dissolved in 5% sodium bicarbonate (NaHCO3-) at time 0 and 4 h. Group 3 (Aloe vera-treated, n = 6) was given oral Aloe vera (150 mg/kg) dissolved in DW and IMN at time 0 and 4 h. Eight hours later, the stomach was removed to determine gastric malondialdehyde (MDA), the number of interleukin (IL)-18 positive stained cells (%) by immunohistochemistry, and for histopathological examination. Then, the serum was collected to determine tumor necrosis factor (TNF)-α and cytokine-induced neutrophil chemoattractant (CINC)-1 by sandwich enzyme linked immunosorbent assay method. RESULTS: In the IMN group, serum TNF-α, CINC-1 and gastric MDA were significantly increased when compared to the control group (27.78 ± 1.52 pg/mL vs 85.07 ± 49.11 pg/mL, P = 0.009; 104.55 ± 45.80 pg/mL vs 1054.70 ± 20.38 pg/mL, and 1.74 ± 0.21 nmol/mg vs 9.36 ± 1.07 nmol/mg protein, P = 0.000, respectively). The mean level of TNF-α, CINC-1 and gastric MDA in the Aloe vera-treated group were improved as compared with the IMN group (85.07 ± 49.11 pg/mL vs 35.19 ± 1.61 pg/mL, P = 0.021; 1054.70 ± 20.38 pg/mL vs 813.56 ± 239.04 pg/mL, P = 0.025; and 9.36 ± 1.07 nmol/mg vs 2.67 ± 0.64 nmol/mg protein, P = 0.000, respectively). The number of IL-18 positive stained cells (%) in the gastric epithelial cells of the IMN group was significantly higher than the control group (5.01% ± 3.73% vs 30.67% ± 2.03%, P = 0.000, respectively). In contrast, Aloe vera treatment decreased the number of IL-18 positive stained cells (%) significantly when compared with the IMN group (30.67% ± 2.03% vs 13.21% ± 1.10%, P = 0.000, respectively). Most rats in the IMN group developed moderate to severe gastric inflammation and erosions. The gastric erosions and neutrophil infiltration scores were significantly reduced in the Aloe vera-treated group. CONCLUSION: Aloe vera attenuated IMN-induced gastropathy in rats by the reduction of oxidative stress, inflammation, and improvement of gastric histopathology. PMID:25561799

[Pyoderma gangrenosum following hematopoietic stem cell transplantation].

PubMed

Eddou, H; Ennouhi, A; Sina, M; Zinebi, A; El Benaye, J; Moudden, M K; Doghmi, K; Malfuson, J-V; Mikdame, M; El Baaj, M

2018-05-07

Pyoderma gangrenosum (PG) is a rare form of neutrophilic dermatosis and is a potential complication in a number of systemic diseases. These include blood diseases, which represent 3.5% of cases, with the main forms being monoclonal gammopathy and acute myeloid leukemia. Herein we report a case of pyoderma gangrenosum in a female patient who had undergone haematopoietic stem cell allograft six months earlier as part of her treatment for acute T-cell leukemia. This condition forms one of the general disorders potentially associated with PG and is a dermatological disorder that can occur in marrow graft patients. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Hyperandrogenism from an ovarian interstitial-cell tumor in an alpaca.

PubMed

Gilbert, Rosanne; Kutzler, Michelle; Valentine, Beth A; Semevolos, Stacy

2006-11-01

An 8-year-old intact female Huacaya alpaca (Lama pacos) was presented for recent development of male behavior. Serum testosterone concentration was determined to be 969.1 pg/ml by using radioimmunoassay, while the range in 33 healthy female adult intact alpacas was 11.7-62.1 pg/ml. An ovarian mass was suspected, and an exploratory laparotomy was performed. A tan mass was present on the left ovary. Histologically, the mass was composed of closely packed, plump, polygonal cells with central round nuclei with granular chromatin and abundant eosinophilic finely granular to vesiculate cytoplasm. An ovarian benign interstitial (Leydig) cell tumor was diagnosed.

Seeds as natural matrices for immobilization of Aspergillus niger mycelium producing pectinases.

PubMed

Fiedurek, J; Szczodrak, J; Rogalski, J

1995-04-01

A simple method for the immobilization of Aspergillus niger mycelium producing polygalacturonase (PG) and pectinesterase (PE) is described. Fungal conidia were immobilized on wheat, rye, barley, peas, buckwheat and mustards seeds. Spongy mycelia overgrowing the seed surfaces on mineral medium with pectin produced extracellular PG and PE; the highest production was reached on the wheat carrier. Some of the variables influencing the enzymatic activity have been optimized. After every 24 h, a culture liquid with 6.8-7.8 U of PG ml-1 and 7.0-10.1 U of PE ml-1 was obtained. This procedure also made possible repeated batch enzyme production and, as many as eight subsequent 24-h batches could be fermented by using the same carrier without any loss of PG activity. The addition of sodium orthovanadate (1 mmol) into the medium with pectin caused a significant increase in PG and PE activity produced by free cells of A. niger (by 1.59-fold and 1.67-fold respectively), and only 0.47-fold of PG activity in case of the immobilized mycelium.

The Role of Auxin in Cell Wall Expansion

PubMed Central

2018-01-01

Plant cells are surrounded by cell walls, which are dynamic structures displaying a strictly regulated balance between rigidity and flexibility. Walls are fairly rigid to provide support and protection, but also extensible, to allow cell growth, which is triggered by a high intracellular turgor pressure. Wall properties regulate the differential growth of the cell, resulting in a diversity of cell sizes and shapes. The plant hormone auxin is well known to stimulate cell elongation via increasing wall extensibility. Auxin participates in the regulation of cell wall properties by inducing wall loosening. Here, we review what is known on cell wall property regulation by auxin. We focus particularly on the auxin role during cell expansion linked directly to cell wall modifications. We also analyze downstream targets of transcriptional auxin signaling, which are related to the cell wall and could be linked to acid growth and the action of wall-loosening proteins. All together, this update elucidates the connection between hormonal signaling and cell wall synthesis and deposition. PMID:29565829

The Role of Auxin in Cell Wall Expansion.

PubMed

Majda, Mateusz; Robert, Stéphanie

2018-03-22

Plant cells are surrounded by cell walls, which are dynamic structures displaying a strictly regulated balance between rigidity and flexibility. Walls are fairly rigid to provide support and protection, but also extensible, to allow cell growth, which is triggered by a high intracellular turgor pressure. Wall properties regulate the differential growth of the cell, resulting in a diversity of cell sizes and shapes. The plant hormone auxin is well known to stimulate cell elongation via increasing wall extensibility. Auxin participates in the regulation of cell wall properties by inducing wall loosening. Here, we review what is known on cell wall property regulation by auxin. We focus particularly on the auxin role during cell expansion linked directly to cell wall modifications. We also analyze downstream targets of transcriptional auxin signaling, which are related to the cell wall and could be linked to acid growth and the action of wall-loosening proteins. All together, this update elucidates the connection between hormonal signaling and cell wall synthesis and deposition.

Cell Wall Remodeling Enzymes Modulate Fungal Cell Wall Elasticity and Osmotic Stress Resistance

PubMed Central

Ene, Iuliana V.; Walker, Louise A.; Schiavone, Marion; Lee, Keunsook K.; Martin-Yken, Hélène; Dague, Etienne; Gow, Neil A. R.; Munro, Carol A.

2015-01-01

ABSTRACT The fungal cell wall confers cell morphology and protection against environmental insults. For fungal pathogens, the cell wall is a key immunological modulator and an ideal therapeutic target. Yeast cell walls possess an inner matrix of interlinked β-glucan and chitin that is thought to provide tensile strength and rigidity. Yeast cells remodel their walls over time in response to environmental change, a process controlled by evolutionarily conserved stress (Hog1) and cell integrity (Mkc1, Cek1) signaling pathways. These mitogen-activated protein kinase (MAPK) pathways modulate cell wall gene expression, leading to the construction of a new, modified cell wall. We show that the cell wall is not rigid but elastic, displaying rapid structural realignments that impact survival following osmotic shock. Lactate-grown Candida albicans cells are more resistant to hyperosmotic shock than glucose-grown cells. We show that this elevated resistance is not dependent on Hog1 or Mkc1 signaling and that most cell death occurs within 10 min of osmotic shock. Sudden decreases in cell volume drive rapid increases in cell wall thickness. The elevated stress resistance of lactate-grown cells correlates with reduced cell wall elasticity, reflected in slower changes in cell volume following hyperosmotic shock. The cell wall elasticity of lactate-grown cells is increased by a triple mutation that inactivates the Crh family of cell wall cross-linking enzymes, leading to increased sensitivity to hyperosmotic shock. Overexpressing Crh family members in glucose-grown cells reduces cell wall elasticity, providing partial protection against hyperosmotic shock. These changes correlate with structural realignment of the cell wall and with the ability of cells to withstand osmotic shock. PMID:26220968

Engineering streptokinase for generation of active site-labeled plasminogen analogs*

PubMed Central

Laha, Malabika; Panizzi, Peter; Nahrendorf, Matthias; Bock, Paul E.

2011-01-01

We previously demonstrated that streptokinase (SK) can be used to generate active site-labeled fluorescent analogs of plasminogen (Pg) by virtue of its non-proteolytic activation of the zymogen. The method is versatile and allows for stoichiometric and active site-specific incorporation of any one of many molecular probes. The limitation of the labeling approach is that it is both time-consuming and low yield. Here we demonstrate an improved method for the preparation of labeled Pg analogs by the use of an engineered SK mutant fusion protein with both COOH- and NH2-terminal His6-tags. The NH2-terminal tag is followed by a tobacco etch virus proteinase cleavage site to ensure that the SK Ile1 residue, essential for conformational activation of Pg, is preserved. The SK COOH-terminal Lys414 residue and residues Arg253-Leu260 in the SK β-domain were deleted to prevent cleavage by plasmin (Pm), and to disable Pg substrate binding to the SK·Pg*/Pm catalytic complexes, respectively. Near-elimination of Pm generation with the SKΔ(R253-L260)ΔK414-His6 mutant increased the yield of labeled Pg 2.6-fold and reduced the time required >2-fold. The versatility of the labeling method was extended to the application of Pg labeled with a near-infrared probe to quantitate Pg receptors on immune cells by flow cytometry. PMID:21570944

Molecular cloning and expression profile of an abiotic stress and hormone responsive MYB transcription factor gene from Panax ginseng.

PubMed

Afrin, Sadia; Zhu, Jie; Cao, Hongzhe; Huang, Jingjia; Xiu, Hao; Luo, Tiao; Luo, Zhiyong

2015-04-01

The v-myb avian myeloblastosis viral oncogene homolog (MYB) family constitutes one of the most abundant groups of transcription factors and plays vital roles in developmental processes and defense responses in plants. A ginseng (Panax ginseng C.A. Meyer) MYB gene was cloned and designated as PgMYB1. The cDNA of PgMYB1 is 762 base pairs long and encodes the R2R3-type protein consisting 238 amino acids. Subcellular localization showed that PgMYB1-mGFP5 fusion protein was specifically localized in the nucleus. To understand the functional roles of PgMYB1, we investigated the expression patterns of PgMYB1 in different tissues and under various conditions. Quantitative real-time polymerase chain reaction and western blot analysis showed that PgMYB1 was expressed at higher level in roots, leaves, and lateral roots than in stems and seeds. The expression of PgMYB1 was up-regulated by abscisic acid, salicylic acid, NaCl, and cold (chilling), and down-regulated by methyl jasmonate. These results suggest that PgMYB1 might be involved in responding to environmental stresses and hormones. © The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

In Vivo and in Vitro Synthesis of Phosphatidylglycerol by an Escherichia coli Cardiolipin Synthase.

PubMed

Li, Chijun; Tan, Brandon K; Zhao, Jinshi; Guan, Ziqiang

2016-11-25

Phosphatidylglycerol (PG) makes up 5-20% of the phospholipids of Escherichia coli and is essential for growth in wild-type cells. PG is synthesized from the dephosphorylation of its immediate precursor, phosphatidylglycerol phosphate (PGP) whose synthase in E. coli is PgsA. Using genetic, biochemical, and highly sensitive mass spectrometric approaches, we identified an alternative mechanism for PG synthesis in E. coli that is PgsA independent. The reaction of synthesis involves the conversion of phosphatidylethanolamine and glycerol into PG and is catalyzed by ClsB, a phospholipase D-type cardiolipin synthase. This enzymatic reaction is demonstrated herein both in vivo and in vitro as well as by using the purified ClsB protein. When the growth medium was supplemented with glycerol, the expression of E. coli ClsB significantly increased PG and cardiolipin levels, with the growth deficiency of pgsA null strain also being complemented under such conditions. Identification of this alternative mechanism for PG synthesis not only expands our knowledge of bacterial anionic phospholipid biosynthesis, but also sheds light on the biochemical functions of the cls gene redundancy in E. coli and other bacteria. Finally, the PGP-independent PG synthesis in E. coli may also have important implications for the understanding of PG biosynthesis in eukaryotes that remains incomplete. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

CCL3 and CXCL12 production in vitro by dental pulp fibroblasts from permanent and deciduous teeth stimulated by Porphyromonas gingivalis LPS

PubMed Central

SIPERT, Carla Renata; MORANDINI, Ana Carolina de Faria; MODENA, Karin Cristina da Silva; DIONÍSIO, Thiago José; MACHADO, Maria Aparecida Andrade Moreira; de OLIVEIRA, Sandra Helena Penha; CAMPANELLI, Ana Paula; SANTOS, Carlos Ferreira

2013-01-01

Objective: The aim of this study was to compare the production of the chemokines CCL3 and CXCL12 by cultured dental pulp fibroblasts from permanent (PDPF) and deciduous (DDPF) teeth under stimulation by Porphyromonas gingivalis LPS (PgLPS). Material and Methods: Primary culture of fibroblasts from permanent (n=3) and deciduous (n=2) teeth were established using an explant technique. After the fourth passage, fibroblasts were stimulated by increasing concentrations of PgLPS (0 - 10 µg/mL) at 1, 6 and 24 h. The cells were tested for viability through MTT assay, and production of the chemokines CCL3 and CXCL12 was determined through ELISA. Comparisons among samples were performed using One-way ANOVA for MTT assay and Two-way ANOVA for ELISA results. Results: Cell viability was not affected by the antigen after 24 h of stimulation. PgLPS induced the production of CCL3 by dental pulp fibroblasts at similar levels for both permanent and deciduous pulp fibroblasts. Production of CXCL12, however, was significantly higher for PDPF than DDPF at 1 and 6 h. PgLPS, in turn, downregulated the production of CXCL12 by PDPF but not by DDPF. Conclusion: These data suggest that dental pulp fibroblasts from permanent and deciduous teeth may present a differential behavior under PgLPS stimulation. PMID:23739851

Regulatory effect and mechanism of gastrin and its antagonists on colorectal carcinoma

PubMed Central

He, Shuang-Wu; Shen, Kang-Qiang; He, Yu-Jun; Xie, Bin; Zhao, Yan-Ming

1999-01-01

AIM: To explore the effect and mechanism of gastrin and its an tagonists proglumide and somatostatin on colorectal carcinoma and their clinical significance. METHODS: A model of transplanted human colonic carcinoma was established from SW480 cell line in gymnomouse body. The volume and weight of transplanted carcinoma was observed under the effect of pentagatrin (PG), proglumide (PGL) and octapeptide somotostatin (SMS201-995, SMS). The cAMP content of carcinoma cell was determined by radioimmunoassay and the DNA, protein content and cell cycle were determined by flow-cytometry. The amount of viable cells was determined by MTT colorimetric analysis, IP3 content was determined by radioimmuno assay, Ca2+ concentration in cell by fluorometry and PKC activity by isotopic enzymolysis. The expression of gastrin, c-myc, c-fos and rasP21 in 48 case s of colorectal carcinoma tissue was detected by the immuno-cytochemistry SP method. Argyrophilia nucleolar organizer regions was determined with argyrophilia stain. RESULTS: The volume, weight, cAMP, DNA and protein content in carcinoma cell, cell amount and proliferation index of S and G2M phase in PG group were all significantly higher than those of control group. When PG was at the concentration of 25 mg/L, the amount of viable cells, IP3 content and Ca2+ concentration in cell and membrane PKC activity in PG group were significantly higher than those in control group; when PGL was at a concentration of 32 mg/L, they dropped to the lowest level in PG (25 mg/L) + PGL group, but without significant difference from the control group. The positive expression rate of gastrin, c-myc, c-fos and rasP21 in carcinoma tissue was 39.6%, 54.2%, 47.9% and 54.2% respectively and significantly higher than that in mucosa 3 cm and 6 cm adjacent to carcinoma tissue and normal colorectal mucosa. The positive expression rate of gastrin of highly-differentiated adenocarcinoma group was significantly higher than that of poorly-differentiated and mucinous adenoc arcinoma groups. The AgNORs count of carcinoma tissue was significantly higher than that in mucosa 3 cm and 6 cm adjacent to carcinoma tissue and norm al colorectal mucosa; and the positive expression of c-myc and c-fos and the A gNORs count in gastrin-positive group was significantly higher than those in gastrin-negative group. CONCLUSION: Pentagastrin has a promoting effect on the growth of transplanted human colonic carcinoma from SW480 cell line. PGL has no obvious effect on the growth of human colonic carcinoma SW480 cell line, but could inhibit the growth promoting effect of PG on transplanted carcinoma. Somatostatin can not only inhibit the growth of transplanted human colonic carcinoma from SW480 cell line directly but also depress the growth-promoting effect of gastrin on the transplanted carcinoma. Some colorectal carcinoma cells can produce and secrete gastrin through autocrine, highly-differentiated adenocarcinoma express the highest level gastrin. Endogenous gastrin can stimulate the cell division and proliferation of carcinoma cell and promote the growth of colorectal carcinoma regulating the expression of oncogene c-myc, c-fos. Our study has provided experimental basis for the adjuvant treatment using gastrin antagonist such as PGL, so matostatin of patients with colorectal carcinoma. PMID:11819478

Cell Wall Remodeling Enzymes Modulate Fungal Cell Wall Elasticity and Osmotic Stress Resistance.

PubMed

Ene, Iuliana V; Walker, Louise A; Schiavone, Marion; Lee, Keunsook K; Martin-Yken, Hélène; Dague, Etienne; Gow, Neil A R; Munro, Carol A; Brown, Alistair J P

2015-07-28

The fungal cell wall confers cell morphology and protection against environmental insults. For fungal pathogens, the cell wall is a key immunological modulator and an ideal therapeutic target. Yeast cell walls possess an inner matrix of interlinked β-glucan and chitin that is thought to provide tensile strength and rigidity. Yeast cells remodel their walls over time in response to environmental change, a process controlled by evolutionarily conserved stress (Hog1) and cell integrity (Mkc1, Cek1) signaling pathways. These mitogen-activated protein kinase (MAPK) pathways modulate cell wall gene expression, leading to the construction of a new, modified cell wall. We show that the cell wall is not rigid but elastic, displaying rapid structural realignments that impact survival following osmotic shock. Lactate-grown Candida albicans cells are more resistant to hyperosmotic shock than glucose-grown cells. We show that this elevated resistance is not dependent on Hog1 or Mkc1 signaling and that most cell death occurs within 10 min of osmotic shock. Sudden decreases in cell volume drive rapid increases in cell wall thickness. The elevated stress resistance of lactate-grown cells correlates with reduced cell wall elasticity, reflected in slower changes in cell volume following hyperosmotic shock. The cell wall elasticity of lactate-grown cells is increased by a triple mutation that inactivates the Crh family of cell wall cross-linking enzymes, leading to increased sensitivity to hyperosmotic shock. Overexpressing Crh family members in glucose-grown cells reduces cell wall elasticity, providing partial protection against hyperosmotic shock. These changes correlate with structural realignment of the cell wall and with the ability of cells to withstand osmotic shock. The C. albicans cell wall is the first line of defense against external insults, the site of immune recognition by the host, and an attractive target for antifungal therapy. Its tensile strength is conferred by a network of cell wall polysaccharides, which are remodeled in response to growth conditions and environmental stress. However, little is known about how cell wall elasticity is regulated and how it affects adaptation to stresses such as sudden changes in osmolarity. We show that elasticity is critical for survival under conditions of osmotic shock, before stress signaling pathways have time to induce gene expression and drive glycerol accumulation. Critical cell wall remodeling enzymes control cell wall flexibility, and its regulation is strongly dependent on host nutritional inputs. We also demonstrate an entirely new level of cell wall dynamism, where significant architectural changes and structural realignment occur within seconds of an osmotic shock. Copyright © 2015 Ene et al.

Occurrence and gas/particle partitioning of short- and medium-chain chlorinated paraffins in the atmosphere of Fildes Peninsula of Antarctica

NASA Astrophysics Data System (ADS)

Ma, Xindong; Zhang, Haijun; Zhou, Hongqiang; Na, Guangshui; Wang, Zhen; Chen, Chen; Chen, Jingwen; Chen, Jiping

2014-06-01

Chlorinated paraffins (CPs) were measured in air samples at a remote air monitoring site established in Georgia King Island, Fildes Peninsula of Antarctica (Great Wall Station, China) to study the long-range atmospheric transport of these anthropogenic pollutants to the Antarctic. Gas- and particle-phase CPs were collected using polyurethane foam plugs (PUF) and glass fiber filters (GFF) respectively during summertime of 2012. The total atmospheric levels of SCCPs and MCCPs ranged from 9.6 to 20.8 pg m-3 (average: 14.9 pg m-3) and 3.7-5.2 pg m-3 (average: 4.5 pg m-3), respectively. C10 and C11 carbon chain homologues with Cl5 and Cl6 chlorine atoms predominated in SCCP formula groups both in gas- and particle-phase. Significant linear correlation was found between gas/particle partition coefficients (KP) and sub-cooled liquid vapor pressures (pL°) (R2 = 0.437, p < 0.01), as well as KP and octanol-air partition coefficients (KOA) (R2 = 0.442, p < 0.01). Absolute slope values of two regression models (0.31 and 0.39) were less than 0.6 indicating that the way of absorption into organic matter of aerosol played a more important role on atmospheric partitioning and transferring of CPs in remote Antarctic area. Both the Junge-Pankow model and the KOA-based model tended to underestimate the sorption of lower chlorinated CPs and overestimate the sorption of highly chlorinated CPs.

Potentiation of Sodium Metabisulfite Toxicity by Propylene Glycol in Both in Vitro and in Vivo Systems.

PubMed

Yoo, Jean; Lim, Yeon-Mi; Kim, Haewon; Kim, Eun-Ji; Lee, Doo-Hee; Lee, Byeongwoo; Kim, Pilje; Yu, Seung Do; Kim, Hyun-Mi; Yoon, Byung-Il; Shim, Ilseob

2018-01-01

Many consumer products used in our daily lives result in inhalation exposure to a variety of chemicals, although the toxicities of the active ingredients are not well known; furthermore, simultaneous exposure to chemical mixtures occurs. Sodium metabisulfite (SM) and propylene glycol (PG) are used in a variety of products. Both the cytotoxicity and the sub-acute inhalation toxicity of each chemical and their mixtures were evaluated. Assays for cell viability, membrane damage, and lysosome damage demonstrated that SM over 100 μg/ml induced significant cytotoxicity; moreover, when PG, which was not cytotoxic, was mixed with SM, the cytotoxicity of the mixture was enhanced. Solutions of 1, 5, and 20% SM, each with 1% PG solution, were prepared, and the whole body of rats was exposed to aerosols of the mixture for 6 h/day, 5 days/week for 2 weeks. The rats were sacrificed 1 (exposure group) or 7 days (recovery group) after termination of the exposure. The actual concentration of SM in the low-, medium-, and high-exposure groups was 3.91 ± 1.26, 35.73 ± 6.01, and 80.98 ± 5.47 mg/m 3 , respectively, and the actual concentration of PG in each group was 6.47 ± 1.25, 8.68 ± 0.6, and 8.84 ± 1.77 mg/m 3 . The repeated exposure to SM and PG caused specific clinical signs including nasal sound, sneeze, and eye irritation which were not found in SM single exposure. In addition, the body weight of treatment group rats decreased compared to that of the control group rats in a time-dependent manner. The total protein concentration and lactate dehydrogenase activity in the bronchoalveolar lavage fluid (BALF) increased. Histopathological analysis of the lungs, liver, and nasal cavity was performed. Adverse effects were observed in the nasal cavity, with squamous cell metaplasia identified in the front of the nasal cavity in all high-exposure groups, which completely recovered 7 days after exposure was terminated. Whereas inhalation of SM for 2 weeks only reduced body weight in the high-dose group, inhalation of SM and PG mixtures for 2 weeks significantly decreased body weight and induced metaplasia of the respiratory epithelium into squamous cells in the medium- and high-dose groups. In conclusion, PG potentiated the toxicity of SM in human lung epithelial cells and the inhalation toxicity in rats.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burch, S.W.; Goven, A.J.; Fitzpatrick, L.C.

An in vitro assay has been developed for rapid (48 h) evaluation of cytotoxic effects of exposure (24 h) of earthworm coelomocytes. The assay, inhibition of phagocytosis (24 h) of stained yeast cells and cell viability, links a traditional soil bioassay organism (Lumbricus terrestris) with a laboratory protocol for use in evaluating physical/chemical fractions resulting from terrestrial TIE manipulations. The assay was developed using copper sulfate as a reference toxicant. Copper exposures as low as 2--4 pg/ml. resulted in 20--60% inhibition of phagocytosis without significant decrease in cell viability. Exposures above 10 pg/ml resulted in reduced cell viability and inhibitionmore » of phagocytosis. The assay was successfully applied to terrestrial TIE fractions derived from extractions of soil from a PCP contaminated wood treatment site.« less

RF generator interlock by plasma grid bias current - An alternate to Hα interlock

NASA Astrophysics Data System (ADS)

Bandyopadhyay, M.; Gahlaut, A.; Yadav, R. K.; Pandya, K.; Tyagi, H.; Vupugalla, M.; Bhuyan, M.; Bhagora, J.; Chakraborty, A.

2017-08-01

ROBIN is inductively coupled plasma (ICP) based negative hydrogen ion source, operated with a 100kW, 1MHz Tetrode based RF generator (RFG). Inductive plasma ignition by the RFG in ROBIN is associated with electron seeding by a hot filament and a gas puff. RFG is triggered by the control system to deliver power just at the peak pressure of the gas puff. Once plasma is ignited due to proper impedance matching, a bright light, dominated by Hα (˜656nm wavelength) radiation is available inside RF driver which is used as a feedback signal to the RFG to continue its operation. If impedance matching is not correct, plasma is not produced due to lack of power coupling and bright light is not available. During such condition, reflected RF power may damage the RFG. Therefore, to protect the RFG, it needs to be switched off automatically within 200ms by the control system in such cases. This plasma light based RFG interlock is adopted from BATMAN ion source. However, in case of vacuum immersed RF ion source in reactor grade NBI system, such plasma light based interlock may not be feasible due to lack of adequate optical fiber interfaces. In reactor grade NBI system, neutron and gamma radiations have impact on materials which may lead to frequent maintenance and machine down time. The present demonstration of RFG interlock by Bias Current (BC) in ROBIN testbed gives an alternate option in this regard. In ROBIN, a bias plate (BP) is placed in the plasma chamber near the plasma grid (PG). BP is electrically connected to the plasma chamber wall of the ion source and PG is isolated from the wall. A high current ˜85 A direct current (DC) power supply of voltage in the range of 0 - 33V is connected between the PG and the BP in such a way that PG can be biased positively with respect to the BP or plasma chamber. This arrangement is actually made to absorb electrons and correspondingly reduce co-extracted electron current during beam extraction. However, in case of normal plasma operation, BC rises due to the presence of plasma electrons, almost in the same timescale as plasma light detection system and so, BC signal can also be used as RFG interlock. The BC signal transmission is through optical isolation to reduce noise interference with the signal. The response of the current monitoring signal available from the PG power supply of ROBIN is quite slow (in the order of few tens of milliseconds). Therefore, a fast response current detection electronic circuit having the ability to generate a PG current detection pulse with adjustable threshold set point has been developed and integrated with ROBIN, and the above concept has been demonstrated in ROBIN recently. The present paper will discuss this experimental activity and its results.

Sequence intrinsic somatic mutation mechanisms contribute to affinity maturation of VRC01-class HIV-1 broadly neutralizing antibodies

PubMed Central

Hwang, Joyce K.; Wang, Chong; Du, Zhou; Meyers, Robin M.; Kepler, Thomas B.; Neuberg, Donna; Kwong, Peter D.; Mascola, John R.; Joyce, M. Gordon; Bonsignori, Mattia; Haynes, Barton F.; Yeap, Leng-Siew; Alt, Frederick W.

2017-01-01

Variable regions of Ig chains provide the antigen recognition portion of B-cell receptors and derivative antibodies. Ig heavy-chain variable region exons are assembled developmentally from V, D, J gene segments. Each variable region contains three antigen-contacting complementarity-determining regions (CDRs), with CDR1 and CDR2 encoded by the V segment and CDR3 encoded by the V(D)J junction region. Antigen-stimulated germinal center (GC) B cells undergo somatic hypermutation (SHM) of V(D)J exons followed by selection for SHMs that increase antigen-binding affinity. Some HIV-1–infected human subjects develop broadly neutralizing antibodies (bnAbs), such as the potent VRC01-class bnAbs, that neutralize diverse HIV-1 strains. Mature VRC01-class bnAbs, including VRC-PG04, accumulate very high SHM levels, a property that hinders development of vaccine strategies to elicit them. Because many VRC01-class bnAb SHMs are not required for broad neutralization, high overall SHM may be required to achieve certain functional SHMs. To elucidate such requirements, we used a V(D)J passenger allele system to assay, in mouse GC B cells, sequence-intrinsic SHM-targeting rates of nucleotides across substrates representing maturation stages of human VRC-PG04. We identify rate-limiting SHM positions for VRC-PG04 maturation, as well as SHM hotspots and intrinsically frequent deletions associated with SHM. We find that mature VRC-PG04 has low SHM capability due to hotspot saturation but also demonstrate that generation of new SHM hotspots and saturation of existing hotspot regions (e.g., CDR3) does not majorly influence intrinsic SHM in unmutated portions of VRC-PG04 progenitor sequences. We discuss implications of our findings for bnAb affinity maturation mechanisms. PMID:28747530

Experimental basis for a role for sulfhydryls and dopamine in ulcerogenesis: a primer for cytoprotection--organoprotection.

PubMed

Szabo, S

1986-01-01

This brief review presents the evolution of the concept of cytoprotection which was originally described by Robert (1979) to represent prevention of chemically induced hemorrhagic gastric erosions without inhibiting acid secretion. Prostaglandins (PG) and sulfhydryls (SH) protect only against deep hemorrhagic necrosis in the mucosa without altering the initial damage to surface epithelial cells. Organ integrity and function are thus maintained (i.e., organoprotection) despite the loss of several layers of mucosal cells. While both PG and SH are natural products it must be stressed that only SH compounds can enter directly into protective reactions (e.g., free radical scavenging, modification of receptor SH groups, oxidation of certain structural and enzyme proteins). In addition, SH compounds also stimulate PG synthesis. A major target of gastroprotection by either PG or SH is the preservation of mucosal microvasculature to maintain blood flow for rapid restitution and cell proliferation. Dopamine-related compounds are reviewed because of their possible role in duodenal ulceration. Dopamine and dopamine agonists are antiulcerogens in duodenal ulcer models. Dopamine antagonists are proulcerogens and the dopamine neurotoxin MPTP causes duodenal ulcer in experimental animals. The mechanism of duodenal antiulcerogenic effect involves inhibition of gastric acid and pepsin secretion, stimulation of duodenal bicarbonate secretion, correction of duodenal dysmotility, and maybe increased blood flow. Because of their multiple beneficial effects, SH compounds and dopamine drugs are good models for gastroenteroprotection.

Targeted Deletions of COX-2 and Atherogenesis in Mice

PubMed Central

Hui, Yiqun; Ricciotti, Emanuela; Crichton, Irene; Yu, Zhou; Wang, Dairong; Stubbe, Jane; Wang, Miao; Puré, Ellen; FitzGerald, Garret A.

2010-01-01

Background While the dominant product of vascular cyclooxygenase (COX)-2, prostacyclin (PGI2), restrains atherogenesis, inhibition and deletion of COX-2 have yielded conflicting results in mouse models of atherosclerosis. Floxed mice were used to parse distinct cellular contributions of COX-2 in macrophages (Mac) and T cells (TC) to atherogenesis. Methods and Results Deletion of Mac COX-2 (MacKO) was attained using LysMCre mice and suppressed completely lipopolysaccharide (LPS) stimulated Mac prostaglandin (PG) formation and LPS evoked systemic PG biosynthesis by ∼ 30%. LPS stimulated COX-2 expression was suppressed in polymorphonuclear leucocytes (PMN) isolated from MacKOs, but PG formation was not even detected in PMN supernatants from control mice. Atherogenesis was attenuated when MacKOs were crossed into hyperlipidemic LdlR KOs. Deletion of Mac COX-2 appeared to remove a restraint on COX-2 expression in lesional non-leukocyte (CD45 and CD11b negative) vascular cells that express vascular cell adhesion molecule and variably, α-smooth muscle actin and vimentin, portending a shift in PG profile and consequent atheroprotection. Basal expression of COX-2 was minimal in TCs, but use of CD4Cre to generate TC knockouts (TCKOs) depressed its modest upregulation by anti-CD3ε. However, biosynthesis of PGs, TC composition in lymphatic organs and atherogenesis in LDLR KOs were unaltered in TCKOs. Conclusions Mac COX-2, primarily a source of thromboxane A2 and PGE2, promotes atherogenesis and exerts a restraint on enzyme expression by lesional cells suggestive of vascular smooth muscle cells, a prominent source of atheroprotective PGI2. TC COX-2 does not influence detectably TC development or function nor atherogenesis in mice. PMID:20530000

Light-induced translocation of Pyronine G from mitochondria to nucleoli in monkey kidney CV-1 cells

NASA Astrophysics Data System (ADS)

Geze, Marc; Dellinger, M.; Bazin, M.; Santus, Rene C.

1996-12-01

Pyronine G (3,6-bis-N,N-dimethylaminoxanthylium chloride; PG) is a cationic dye that concentrates in mitochondria of living cells due to the high membrane potential of these organelles, similarly to rhodamine 123 and many other cationic dyes. Pyronine G also shows a preferential affinity for RNA. Upon light irradiation PG has been shown to induce cell death, but the photosensitizing properties of this molecule and the mechanism of cell death are not well understood. Microfluorometry and most particularly microspectrofluorometry are now powerful non-invasive techniques for quantitative studies of single living cells in real time which allow, for example, knowing how living cells are affected by photosensitization. To demonstrate the usefulness of image acquisition with high resolution and high sensitive camera, we present data on photosensitizer relocalization during illumination leading to functional and structural damage in the cells.

A Transcriptomic Analysis of Xylan Mutants Does Not Support the Existence of a Secondary Cell Wall Integrity System in Arabidopsis

PubMed Central

Faria-Blanc, Nuno; Mortimer, Jenny C.; Dupree, Paul

2018-01-01

Yeast have long been known to possess a cell wall integrity (CWI) system, and recently an analogous system has been described for the primary walls of plants (PCWI) that leads to changes in plant growth and cell wall composition. A similar system has been proposed to exist for secondary cell walls (SCWI). However, there is little data to support this. Here, we analyzed the stem transcriptome of a set of cell wall biosynthetic mutants in order to investigate whether cell wall damage, in this case caused by aberrant xylan synthesis, activates a signaling cascade or changes in cell wall synthesis gene expression. Our data revealed remarkably few changes to the transcriptome. We hypothesize that this is because cells undergoing secondary cell wall thickening have entered a committed programme leading to cell death, and therefore a SCWI system would have limited impact. The absence of transcriptomic responses to secondary cell wall alterations may facilitate engineering of the secondary cell wall of plants. PMID:29636762

A Transcriptomic Analysis of Xylan Mutants Does Not Support the Existence of a Secondary Cell Wall Integrity System in Arabidopsis.

PubMed

Faria-Blanc, Nuno; Mortimer, Jenny C; Dupree, Paul

2018-01-01

Yeast have long been known to possess a cell wall integrity (CWI) system, and recently an analogous system has been described for the primary walls of plants (PCWI) that leads to changes in plant growth and cell wall composition. A similar system has been proposed to exist for secondary cell walls (SCWI). However, there is little data to support this. Here, we analyzed the stem transcriptome of a set of cell wall biosynthetic mutants in order to investigate whether cell wall damage, in this case caused by aberrant xylan synthesis, activates a signaling cascade or changes in cell wall synthesis gene expression. Our data revealed remarkably few changes to the transcriptome. We hypothesize that this is because cells undergoing secondary cell wall thickening have entered a committed programme leading to cell death, and therefore a SCWI system would have limited impact. The absence of transcriptomic responses to secondary cell wall alterations may facilitate engineering of the secondary cell wall of plants.

Mechanisms of gastroprotection.

PubMed

Konturek, S J

1990-01-01

Gastric mucosa is constantly exposed to various irritants, but it usually maintains its integrity owing to several lines of defense, including mucus-alkaline secretion, mucosal hydrophobicity, rich mucosal blood flow, stabilization of tissue lysosomes, maintenance of mucosal sulfhydryls, and rapid proliferation and renewal of mucosal cells. Prostaglandins (PG) inhibit experimental gastric mucosal damage and ulcerations induced by a wide variety of agents, hence PG have been proposed to contribute to the overall protective process by activation of various mucosal defence lines--particularly by prevention of vasocongestion, ischemia, and deep hemorrhagic necrosis. The relation between tissue PG generation and mucosal protection does not appear to be closely related, and probably only minute amounts of PG are required to maintain mucosal integrity. In contrast to PG, other products of arachidonate metabolism, such as TxA2, LTC4 or LTD4, and the related lipid, platelet-activating factor, appear to mediate mucosal damage mainly by the disturbance in mucosal microcirculation and tissue ischemia. Gastroprotection can be achieved by stimulation of mucosal biosynthesis of protective PG or by the inhibition of the release or action of the proulcerogenic arachidonate metabolites. Certain natural substances, such as sulfhydryls, epidermal growth factor, or polyamines, protect the mucosa via a PG-independent mechanism, probably by enhancing the tissue repair processes.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential histone modification and protein expression associated with cell wall removal and regeneration in rice (Oryza sativa).

PubMed

Tan, Feng; Zhang, Kangling; Mujahid, Hana; Verma, Desh Pal S; Peng, Zhaohua

2011-02-04

The cell wall is a critical extracellular structure that provides protection and structural support in plant cells. To study the biological function of the cell wall and the regulation of cell wall resynthesis, we examined cellular responses to enzymatic removal of the cell wall in rice (Oryza sativa) suspension cells using proteomic approaches. We find that removal of cell wall stimulates cell wall synthesis from multiple sites in protoplasts instead of from a single site as in cytokinesis. Nucleus DAPI stain and MNase digestion further show that removal of the cell wall is concomitant with substantial chromatin reorganization. Histone post-translational modification studies using both Western blots and isotope labeling assisted quantitative mass spectrometry analyses reveal that substantial histone modification changes, particularly H3K18(AC) and H3K23(AC), are associated with the removal and regeneration of the cell wall. Label-free quantitative proteome analyses further reveal that chromatin associated proteins undergo dramatic changes upon removal of the cell wall, along with cytoskeleton, cell wall metabolism, and stress-response proteins. This study demonstrates that cell wall removal is associated with substantial chromatin change and may lead to stimulation of cell wall synthesis using a novel mechanism.

Cyclic 2,3-diphosphoglycerate as a component of a new branch in gluconeogenesis in Methanobacterium thermoautotrophicum delta H.

PubMed

Gorkovenko, A; Roberts, M F

1993-07-01

A unique compound, cyclic 2,3-diphosphoglycerate (cDPG), is the major soluble carbon and phosphorus solute in Methanobacterium thermoautotrophicum delta H under optimal conditions of cell growth. It is a component of an unusual branch in gluconeogenesis in these bacteria. [U-13C]acetate pulse-[12C]acetate chase methodology was used to observe the relationship between cDPG and other metabolites (2-phosphoglycerate and 2,3-diphosphoglycerate [2-PG and 2,3-DPG, respectively]) of this branch. It was demonstrated that cells could grow exponentially under conditions in which 2-PG and 2,3-DPG, rather than cDPG, were the major solutes. While the total concentration of these three phosphorylated molecules was maintained, rapid interconversion of 13C label among them was observed. Label flow from 2-PG to 2,3-DPG to cDPG to polymer is the usual direction in this pathway in exponentially growing cells, while the reverse reactions sometimes predominate in the stationary phase. Evidence of the presence of a polymeric compound in this pathway was provided by 13C nuclear magnetic resonance (one-dimensional and two-dimensional INADEQUATE) studies of solubilized cell debris.

Cyclic 2,3-diphosphoglycerate as a component of a new branch in gluconeogenesis in Methanobacterium thermoautotrophicum delta H.

PubMed Central

Gorkovenko, A; Roberts, M F

1993-01-01

A unique compound, cyclic 2,3-diphosphoglycerate (cDPG), is the major soluble carbon and phosphorus solute in Methanobacterium thermoautotrophicum delta H under optimal conditions of cell growth. It is a component of an unusual branch in gluconeogenesis in these bacteria. [U-13C]acetate pulse-[12C]acetate chase methodology was used to observe the relationship between cDPG and other metabolites (2-phosphoglycerate and 2,3-diphosphoglycerate [2-PG and 2,3-DPG, respectively]) of this branch. It was demonstrated that cells could grow exponentially under conditions in which 2-PG and 2,3-DPG, rather than cDPG, were the major solutes. While the total concentration of these three phosphorylated molecules was maintained, rapid interconversion of 13C label among them was observed. Label flow from 2-PG to 2,3-DPG to cDPG to polymer is the usual direction in this pathway in exponentially growing cells, while the reverse reactions sometimes predominate in the stationary phase. Evidence of the presence of a polymeric compound in this pathway was provided by 13C nuclear magnetic resonance (one-dimensional and two-dimensional INADEQUATE) studies of solubilized cell debris. Images PMID:8320225

Biosynthesis of the unique trans-delta 3-hexadecenoic acid component of chloroplast phosphatidylglycerol: evidence concerning its site and mechanism of formation.

PubMed

Ohnishi, M; Thompson, G A

1991-08-01

As in most higher plants, chloroplast membranes of the green alga Dunaliella salina contain phosphatidylglycerol (PG) that is rich in trans-delta 3-hexadecenoic acid (16:1t), a fatty acid found nowhere else in the cell. After labeling D. salina with exogenous [3H]myristic acid [( 3H]14:0), the cis-unsaturated fatty acids of monogalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol as well as PG had higher specific radioactivities in chloroplast envelopes than in thylakoids. In contrast, 16:1t was very slow to become radioactive, and its specific radioactivity was several times higher in isolated thylakoids than in envelopes after brief (3-20 min) labeling with [3H]14:0. Analysis of individual PG molecular species revealed that the fatty acid paired with 16:1t was also labeled slowly. Thus linoleate (18:2) released from a 16:1t-containing PG had a 350-fold (at 3 min) to 20-fold (at 60 min) lower specific radioactivity than did 18:2 from a palmitate (16:0)-containing PG. The findings suggest that the substrates for trans-desaturation are 16:0-containing PG molecular species which are readily labeled from [3H]14:0 in the envelope but are diluted by the large pool of thylakoid PG before penetrating to the desaturation site. By examining the labeling patterns of individual PG molecular species classes, it was concluded that D. salina 16:1t is formed from 16:0 linked to 18:2/16:0 PG and 18:3/16:0 PG by a trans-desaturase located within the inner recesses of the thylakoid compartment.

Generation of TNFalpha and interleukin-6 by peritoneal macrophages after overnight dwells with bicarbonate- or lactate-buffered dialysis fluid.

PubMed

Liberek, Tomasz; Lichodziejewska-Niemierko, Monika; Knopinska-Posluszny, Wanda; Schaub, Thomas P; Kirchgessner, Judith; Passlick-Deetjen, Jutta; Rutkowski, Boleslaw

2002-01-01

In order to evaluate the biocompatibility profile of a newly designed peritoneal dialysis fluid (PDF), we evaluated peritoneal leukocyte (PMphi) cytokine release following overnight in vivo dwells using standard, lactate-buffered, single-chamber bag PDF (Lac-PDF) and purely bicarbonate-buffered, double-chamber bag PDF containing 34 (Bic-PDF) or 39 (Bic Hi-PDF) mmol/L bicarbonate. A randomized, open, crossover clinical trial with single weekly test dwells was performed in stable, long-term continuous ambulatory PD patients (n = 8). During 8-hour overnight dwells, PMphi were exposed to different PDF containing 1.5% glucose. After drainage, peritoneal cells were isolated and incubated with RPMI 1640 medium for 2 or 3 hours, with and without stimulation by lipopolysaccharide (LPS). Ex vivo release of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 was measured by specific ELISA technique. After pre-exposure to Lac-PDF, PMphi generated 242 +/- 279 pg TNFalpha/10(6) cells and 157 +/- 105 pg IL-6/10(6) cells. When pre-exposed to Bic-PDF and Bic Hi-PDF, TNFa and IL-6 production of PMphi was not significantly different from Lac-PDF. After LPS stimulation (100 ng/mL), PMD secretion of TNFalpha and IL-6 pre-exposed to three PDF revealed no significant differences between groups: TNFalpha was 2,864 +/- 1,216, 2,910 +/- 1,202, and 3,291 +/- 558 pg/10(6) cells after overnight dwells with Lac-PDF, Bic-PDF, and Bic Hi-PDF, respectively. Comparably, LPS-stimulated (100 pg/ mL) PMphi showed IL-6 secretion of 891 +/- 335, 1,380 +/- 1,149, and 1,442 +/- 966 pg/10(6) cells for Lac-PDF, Bic-PDF, and Bic Hi-PDF. After long-term overnight dwells, initial pH, the different buffers, and varying glucose degradation product levels of PDF do not strongly affect PMphi function with respect to cytokine release. The lack of significant differences between fluids may result from the complete dialysate equilibration achieved during the overnight intraperitoneal dwell.

Plant cell wall proteomics: the leadership of Arabidopsis thaliana

PubMed Central

Albenne, Cécile; Canut, Hervé; Jamet, Elisabeth

2013-01-01

Plant cell wall proteins (CWPs) progressively emerged as crucial components of cell walls although present in minor amounts. Cell wall polysaccharides such as pectins, hemicelluloses, and cellulose represent more than 90% of primary cell wall mass, whereas hemicelluloses, cellulose, and lignins are the main components of lignified secondary walls. All these polymers provide mechanical properties to cell walls, participate in cell shape and prevent water loss in aerial organs. However, cell walls need to be modified and customized during plant development and in response to environmental cues, thus contributing to plant adaptation. CWPs play essential roles in all these physiological processes and particularly in the dynamics of cell walls, which requires organization and rearrangements of polysaccharides as well as cell-to-cell communication. In the last 10 years, plant cell wall proteomics has greatly contributed to a wider knowledge of CWPs. This update will deal with (i) a survey of plant cell wall proteomics studies with a focus on Arabidopsis thaliana; (ii) the main protein families identified and the still missing peptides; (iii) the persistent issue of the non-canonical CWPs; (iv) the present challenges to overcome technological bottlenecks; and (v) the perspectives beyond cell wall proteomics to understand CWP functions. PMID:23641247

Production of polygalacturonases by Aspergillus oryzae in stirred tank and internal- and external-loop airlift reactors.

PubMed

Fontana, Roselei Claudete; da Silveira, Maurício Moura

2012-11-01

The production of endo- and exo-polygalacturonase (PG) by Aspergillus oryzae was assessed in stirred tank reactors (STRs), internal-loop airlift reactors (ILARs) and external-loop airlift reactors (ELARs). For STR production, we compared culture media formulated with either pectin (WBE) or partially hydrolyzed pectin. The highest enzyme activities were obtained in medium that contained 50% pectin in hydrolyzed form (WBE5). PG production in the three reactor types was compared for WBE5 and low salt WBE medium, with additional salts added at 48, 60 and 72h (WBES). The ELARs performed better than the ILARs in WBES medium where the exo-PG was the same concentration as for STRs and the endo-PG was 20% lower. These results indicate that PG production is higher under experimental conditions that result in higher cell growth with minimum pH values less than 3.0. Copyright © 2012 Elsevier Ltd. All rights reserved.

Detection of Cyclooxygenase-2-Derived Oxygenation Products of the Endogenous Cannabinoid 2-Arachidonoylglycerol in Mouse Brain.

PubMed

Morgan, Amanda J; Kingsley, Philip J; Mitchener, Michelle M; Altemus, Megan; Patrick, Toni A; Gaulden, Andrew D; Marnett, Lawrence J; Patel, Sachin

2018-05-09

Cyclooxygenase-2 (COX-2) catalyzes the formation of prostaglandins, which are involved in immune regulation, vascular function, and synaptic signaling. COX-2 also inactivates the endogenous cannabinoid (eCB) 2-arachidonoylglycerol (2-AG) via oxygenation of its arachidonic acid backbone to form a variety of prostaglandin glyceryl esters (PG-Gs). Although this oxygenation reaction is readily observed in vitro and in intact cells, detection of COX-2-derived 2-AG oxygenation products has not been previously reported in neuronal tissue. Here we show that 2-AG is metabolized in the brain of transgenic COX-2-overexpressing mice and mice treated with lipopolysaccharide to form multiple species of PG-Gs that are detectable only when monoacylglycerol lipase is concomitantly blocked. Formation of these PG-Gs is prevented by acute pharmacological inhibition of COX-2. These data provide evidence that neuronal COX-2 is capable of oxygenating 2-AG to form a variety PG-Gs in vivo and support further investigation of the physiological functions of PG-Gs.

Cell Wall Structure of Coccoid Green Algae as an Important Trade-Off Between Biotic Interference Mechanisms and Multidimensional Cell Growth.

PubMed

Dunker, Susanne; Wilhelm, Christian

2018-01-01

Coccoid green algae can be divided in two groups based on their cell wall structure. One group has a highly chemical resistant cell wall (HR-cell wall) containing algaenan. The other group is more susceptible to chemicals (LR-cell wall - Low resistant cell wall). Algaenan is considered as important molecule to explain cell wall resistance. Interestingly, cell wall types (LR- and HR-cell wall) are not in accordance with the taxonomic classes Chlorophyceae and Trebouxiophyceae, which makes it even more interesting to consider the ecological function. It was already shown that algaenan helps to protect against virus, bacterial and fungal attack, but in this study we show for the first time that green algae with different cell wall properties show different sensitivity against interference competition with the cyanobacterium Microcystis aeruginosa . Based on previous work with co-cultures of M. aeruginosa and two green algae ( Acutodesmus obliquus and Oocystis marssonii ) differing in their cell wall structure, it was shown that M. aeruginosa could impair only the growth of the green algae if they belong to the LR-cell wall type. In this study it was shown that the sensitivity to biotic interference mechanism shows a more general pattern within coccoid green algae species depending on cell wall structure.

Seasonal changes in pancreatic B-cell function in euthermic yellow-bellied marmots.

PubMed

Florant, G L; Lawrence, A K; Williams, K; Bauman, W A

1985-08-01

Fasting plasma insulin (PI) and glucose (PG) concentrations were measured throughout the body weight cycle of marmots. Animals gained weight during summer, and in late fall body weight peaked, after which they ceased feeding. Each month euthermic animals were injected intra-arterially with either dextrose (500 mg/kg) or porcine insulin (0.1 U/kg), and blood samples were collected over the subsequent 2 h. During weight gain fasting PI concentration and pancreatic B-cell response to injected dextrose increased markedly. Maximal insulin release to a dextrose challenge was measured during peak body weight or when body weight initially began to decline. The PG concentration after exogenous insulin administration was slight (less than 10%) in the fall but increased approximately 25% in the spring after marmots lost weight. Basal PG levels were not significantly different throughout the year. Basal fasting PI concentrations were significantly higher during the fall (P less than 0.01). It is suggested that in the fall, when marmots are obese, hyperinsulinemia and peripheral insulin resistance appear. Furthermore, in two animals with an increase in body weight of approximately 30% or less over the summer, peripheral resistance was demonstrable, albeit not as marked as in animals that appropriately doubled their body weights when given food ad libitum. Thus we hypothesize that factors other than adiposity, i.e., food intake, central nervous system input to the pancreatic B-cell, and/or changes in B-cell sensitivity to PG, may contribute to the observed peripheral insulin resistance and may be involved in body weight regulation.

The N-Linked Outer Chain Mannans and the Dfg5p and Dcw1p Endo-α-1,6-Mannanases Are Needed for Incorporation of Candida albicans Glycoproteins into the Cell Wall

PubMed Central

Ao, Jie; Chinnici, Jennifer L.; Maddi, Abhiram

2015-01-01

A biochemical pathway for the incorporation of cell wall protein into the cell wall of Neurospora crassa was recently proposed. In this pathway, the DFG-5 and DCW-1 endo-α-1,6-mannanases function to covalently cross-link cell wall protein-associated N-linked galactomannans, which are structurally related to the yeast outer chain mannans, into the cell wall glucan-chitin matrix. In this report, we demonstrate that the mannosyltransferase enzyme Och1p, which is needed for the synthesis of the N-linked outer chain mannan, is essential for the incorporation of cell wall glycoproteins into the Candida albicans cell wall. Using endoglycosidases, we show that C. albicans cell wall proteins are cross-linked into the cell wall via their N-linked outer chain mannans. We further demonstrate that the Dfg5p and Dcw1p α-1,6-mannanases are needed for the incorporation of cell wall glycoproteins into the C. albicans cell wall. Our results support the hypothesis that the Dfg5p and Dcw1p α-1,6-mannanases incorporate cell wall glycoproteins into the C. albicans cell wall by cross-linking outer chain mannans into the cell wall glucan-chitin matrix. PMID:26048011

DNA amount of X and B chromosomes in the grasshoppers Eyprepocnemis plorans and Locusta migratoria.

PubMed

Ruiz-Ruano, F J; Ruiz-Estévez, M; Rodríguez-Pérez, J; López-Pino, J L; Cabrero, J; Camacho, J P M

2011-01-01

We analyzed the DNA amount in X and B chromosomes of 2 XX/X0 grasshopper species (Eyprepocnemis plorans and Locusta migratoria), by means of Feulgen image analysis densitometry (FIAD), using previous estimates in L. migratoria as standard (5.89 pg). We first analyzed spermatids of 0B males and found a bimodal distribution of integrated optical densities (IODs), suggesting that one peak corresponded to +X and the other to -X spermatids. The difference between the 2 peaks corresponded to the X chromosome DNA amount, which was 1.28 pg in E. plorans and 0.80 pg in L. migratoria. In addition, the +X peak in E. plorans gave an estimate of the C-value in this species (10.39 pg). We next analyzed diplotene cells from 1B males in E. plorans and +B males in L. migratoria (a species where Bs are mitotically unstable and no integer B number can be defined for an individual) and measured B chromosome IOD relative to X chromosome IOD, within the same cell, taking advantage of the similar degree of condensation for both positively heteropycnotic chromosomes at this meiotic stage. From this proportion, we estimated the DNA amount for 3 different B chromosome variants found in individuals from 3 E. plorans Spanish populations (0.54 pg for B1 from Saladares, 0.51 pg for B2 from Salobreña and 0.64 for B24 from Torrox). Likewise, we estimated the DNA amount of the B chromosome in L. migratoria to be 0.15 pg. To automate measurements, we wrote a GPL3 licensed Python program (pyFIA). We discuss the utility of the present approach for estimating X and B chromosome DNA amount in a variety of situations, and the meaning of the DNA amount estimates for X and B chromosomes in these 2 species. Copyright © 2011 S. Karger AG, Basel.

Regulation of Cell Wall Biogenesis in Saccharomyces cerevisiae: The Cell Wall Integrity Signaling Pathway

PubMed Central

Levin, David E.

2011-01-01

The yeast cell wall is a strong, but elastic, structure that is essential not only for the maintenance of cell shape and integrity, but also for progression through the cell cycle. During growth and morphogenesis, and in response to environmental challenges, the cell wall is remodeled in a highly regulated and polarized manner, a process that is principally under the control of the cell wall integrity (CWI) signaling pathway. This pathway transmits wall stress signals from the cell surface to the Rho1 GTPase, which mobilizes a physiologic response through a variety of effectors. Activation of CWI signaling regulates the production of various carbohydrate polymers of the cell wall, as well as their polarized delivery to the site of cell wall remodeling. This review article centers on CWI signaling in Saccharomyces cerevisiae through the cell cycle and in response to cell wall stress. The interface of this signaling pathway with other pathways that contribute to the maintenance of cell wall integrity is also discussed. PMID:22174182

Insulin-like receptors and carbohydrate metabolism in gills of the euryhaline crab Neohelice granulata: Effects of osmotic stress.

PubMed

Trapp, Márcia; Valle, Sandra Costa; Pöppl, Alan Gomes; Chittó, Ana Lúcia Fernandes; Kucharski, Luiz Carlos; Da Silva, Roselis Silveira Martins

2018-06-01

The present study determined the effect of osmotic stress on the insulin-like receptor binding characteristics and on glucose metabolism in the anterior (AG) and posterior (PG) gills of the crab Neohelice granulata. Bovine insulin increased the capacity of the PG cell membrane to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) and the glucose uptake in the control crab group. The crabs were submitted to three periods of hyperosmotic (HR) and hyposmotic (HO) stress, for 24, 72 and 144 h, to investigate the insulin-like receptor phosphorylation capacity of gills. Acclimation to HO for 24 h or HR for 144 h of stress inhibited the effects of insulin in the PG, decreasing the capacity of insulin to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) and decreasing the glucose uptake. Hyperosmotic stress for the same period of 144 h significantly affected 125 I-insulin binding in the AG and PG. However, HO stress for 24 h significantly reduced 125 I-insulin-specific uptake only in the PG. Therefore, osmotic stress induces alterations in the gill insulin-like receptors that decrease insulin binding in the PG. These findings indicate that osmotic stress induced a pattern of insulin resistance in the PG. The free-glucose concentration in the PG decreased during acclimation to 144 h of HR stress and 24 h of HO stress. This decrease in the cell free-glucose concentration was not accompanied by a significant change in hemolymph glucose levels. In AG from the control group, neither the capacity of bovine insulin to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) nor the glucose uptake changed; however, genistein decreased tyrosine-kinase activity, confirming that this receptor belongs to the tyrosine-kinase family. Acclimation to HO (24 h) or HR (144 h) stress decreased tyrosine-kinase activity in the AG. This study provided new information on the mechanisms involved in the osmoregulation process in crustaceans, demonstrating for the first time in an estuarine crab that osmotic challenge inhibited insulin-like signaling and the effect of insulin on glucose uptake in the PG. Copyright © 2018 Elsevier Inc. All rights reserved.

Development of cost-effective plasmonic biosensor using partially embedded gold nanoparticles for detection of immunoglobulin proteins

NASA Astrophysics Data System (ADS)

Kumari, Sudha; Moirangthem, Rakesh S.

2018-02-01

This work illustrates a label-free sensing of biomolecules using a simple capillary sensor. Here, capillary biosensor was prepared by decorating inner walls of a glass capillary with gold nanoparticles that was employed to investigate the biomolecular interactions. As a demonstration, rabbit immunoglobulin G (IgG) and anti-rabbit IgG (anti-IgG) proteins were chosen as a model system to monitor the receptor-analyte interactions. A surface binding sensitivity of 409 pg mm-2 was able to achieve towards the detection of 10 nM concentration of anti-rabbit IgG. The presented plasmonic sensor provides multiple advantages over conventional LSPR sensor by lifting requirement of the flow cell, prolonged sample preparation, complicated measurement setup etc that may enable its usage in rapid diagnostic testing. We believed that our proposed plasmonic capillary sensor could represent a potential candidate for developing cost-effective, label-free and high sensitivity sensing device for detection of biological molecules at low concentration.

Polyphenols from Lonicera caerulea L. Berry Inhibit LPS-Induced Inflammation through Dual Modulation of Inflammatory and Antioxidant Mediators.

PubMed

Wu, Shusong; Yano, Satoshi; Chen, Jihua; Hisanaga, Ayami; Sakao, Kozue; He, Xi; He, Jianhua; Hou, De-Xing

2017-06-28

Lonicera caerulea L. berry polyphenols (LCBP) are considered as major components for bioactivity. This study aimed to clarify the molecular mechanisms by monitoring inflammatory and antioxidant mediator actions in lipopolysaccharide (LPS)-induced mouse paw edema and macrophage cell model. LCBP significantly attenuated LPS-induced paw edema (3.0 ± 0.1 to 2.8 ± 0.1 mm, P < 0.05) and reduced (P < 0.05) serum levels of monocyte chemotactic protein-1 (MCP-1, 100.9 ± 2.3 to 58.3 ± 14.5 ng/mL), interleukin (IL)-10 (1596.1 ± 424.3 to 709.7 ± 65.7 pg/mL), macrophage inflammatory protein (MIP)-1α (1761.9 ± 208.3 to 1369.1 ± 56.4 pg/mL), IL-6 (1262.8 ± 71.7 to 499.0 ± 67.1 pg/mL), IL-4 (93.3 ± 25.7 to 50.7 ± 12.5 pg/mL), IL-12(p-70) (580.4 ± 132.0 to 315.2 ± 35.1 pg/mL), and tumor necrosis factor-α (TNF-α, 2045.5 ± 264.9 to 1270.7 ± 158.6 pg/mL). Cell signaling analysis revealed that LCBP inhibited transforming growth factor β activated kinase-1 (TAK1)-mediated mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways, and enhanced the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and manganese-dependent superoxide dismutase (MnSOD) in earlier response. Moreover, cyanidin 3-glucoside (C3G) and (-)-epicatechin (EC), two major components of LCBP, directly bound to TAK1. These data demonstrated that LCBP might inhibit LPS-induced inflammation by modulating both inflammatory and antioxidant mediators.

Intracellular levels of hepatitis B virus DNA and pregenomic RNA in peripheral blood mononuclear cells of chronically infected patients.

PubMed

Lu, L; Zhang, H-Y; Yueng, Y-H; Cheung, K-F; Luk, J M; Wang, F-S; Lau, G K K

2009-02-01

It remains uncertain whether hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and pregenomic RNA (pgRNA) can be detected in the serum or peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis B (CHB) infection. We examined HBV cccDNA and pgRNA in the serum and PBMC, and investigated the effect of lamivudine therapy on the viral loads in the PBMC of CHB patients. Paired serum and PBMC samples from 50 treatment-naïve CHB patients [25 hepatitis B e antigen (HBeAg) positive and 25 HBeAg negative] were quantified for total HBV DNA, cccDNA and pgRNA by real time polymerase chain reaction. HBV cccDNA and pgRNA were below the lower detection limit in all serum samples, and in 84% of PBMC. HBV DNA (r = 0.889, P < 0.001) and pgRNA (r = 0.696, P < 0.001) in PBMC correlated with the HBV DNA in serum. In the longitudinal study, 30 patients treated with lamivudine therapy for a median duration of 34 weeks (range 12-48 weeks) were examined. The median HBV DNA reduction in PBMC before and after treatment was 1.318 (range -0.471 to 3.846) log units, which was significantly lower than serum HBV DNA reduction [3.371 (range -0.883 to 9.454) log units, P < 0.05]. HBV cccDNA and pgRNA were undetectable in the serum of CHB patients. HBV viral loads in PBMC correlated with serum HBV DNA. Lamivudine therapy had less effect on the HBV viral loads in PBMC compared with the serum viral loads.

Two DD-carboxypeptidases from Mycobacterium smegmatis affect cell surface properties through regulation of peptidoglycan cross-linking and glycopeptidolipids.

PubMed

Pandey, Satya Deo; Pal, Shilpa; Kumar N, Ganesh; Bansal, Ankita; Mallick, Sathi; Ghosh, Anindya S

2018-05-07

During the peptidoglycan (PG) maturation of mycobacteria, the glycan strands are interlinked by both 3-3 (between two meso-DAP) and 4-3 cross-links (between D-ala and meso-DAP), though there is a predominance (60-80%) of 3-3 cross-links. The DD-CPases act on pentapeptides to generate tetrapeptides that are used by LD-transpeptidases as substrates to form 3-3 cross-links. Therefore, DD-CPases play a crucial role in mycobacterial PG cross-link formation. However, the physiology of DD-CPases in mycobacteria is relatively unexplored. Here, we deleted two DD-CPase genes, msmeg_2433 , and msmeg_2432 , both individually and in combination, from Mycobacterium smegmatis mc 2 155. Though the single DD-CPase deletions had no significant impact on the mycobacterial physiology, many interesting functional alterations were observed in the double deletion mutant, viz. , a predominance in PG cross-link formation was shifted from 3-3 cross-links to 4-3, cell surface glycopeptidolipid (GPL) expression was reduced and susceptibility towards β-lactams and anti-tubercular agents was enhanced. Moreover, the existence of the double mutant within murine macrophages was better as compared to the parent. Interestingly, the complementation with any one of the DD-CPase genes could restore the wild-type phenotype. In a nutshell, we infer that the altered ratio of 4-3: 3-3 PG cross-links might have influenced the expression of surface GPLs, colony morphology, biofilm formation,, drug susceptibility and subsistence of the cells within macrophages. Importance The glycan strands in mycobacterial peptidoglycan (PG) are interlinked by both 3-3 and 4-3 cross-links. The DD-CPases generate tetrapeptides by acting on the pentapeptides, and LD-transpeptidases use tetrapeptides as substrates to form 3-3 cross-links. Here, we showed that simultaneous deletions of two DD-CPases alter the nature of PG cross-linking from 3-3 cross-links to 4-3 cross-links. The deletions subsequently decrease the expression of Glycopeptidolipids (a significant surface lipid present in many non-tuberculous mycobacteria including Mycobacterium smegmatis ) and affect other physiological parameters like cell morphology, growth rate, biofilm formation, antibiotic susceptibility and existence within murine macrophages. Thus, unraveling the physiology of DD-CPases might help us design anti-mycobacterial therapeutics in future. Copyright © 2018 American Society for Microbiology.

A model for cell wall dissolution in mating yeast cells: polarized secretion and restricted diffusion of cell wall remodeling enzymes induces local dissolution.

PubMed

Huberman, Lori B; Murray, Andrew W

2014-01-01

Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells.

A Model for Cell Wall Dissolution in Mating Yeast Cells: Polarized Secretion and Restricted Diffusion of Cell Wall Remodeling Enzymes Induces Local Dissolution

PubMed Central

Huberman, Lori B.; Murray, Andrew W.

2014-01-01

Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells. PMID:25329559

Patient-Generated Subjective Global Assessment as a prognosis tool in patients with multiple myeloma.

PubMed

Kim, Hae Su; Lee, Ji Yun; Lim, Sung Hee; Cho, Jaewon; Kim, Seok Jin; Jang, Jun Ho; Kim, Won Seog; Jung, Chul Won; Kim, Kihyun

2017-04-01

Disease-related weight loss is relatively common in patients with newly diagnosed multiple myeloma (MM), but limited data exist regarding the effects of nutritional status on survival. The aim of this study was to assess the relationship between malnutrition (as measured by Patient-Generated Subjective Global Assessment [PG-SGA]) and clinical characteristics of patients with MM, and to investigate the association between the PG-SGA score before chemotherapy and overall survival in MM patients. Using the PG-SGA score, we retrospectively explored the effect of malnutrition on the survival of Asian patients with MM. We divided 216 patients with MM into three groups based on their PG-SGA scores. Of these patients 23% (n = 50) had PG-SGA scores ≥9, indicating severe malnutrition requiring specialist nutrition intervention. Body mass index and serum hemoglobin were independently associated with PG-SGA scores (P < 0.05). The median survival time was not reached in nourished patients with PG-SGA scores of 0 to 3, 58.7 mo in moderately malnourished patients with PG-SGA scores of 4 to 8, and 35 mo in severely malnourished patients with PG-SGA scores ≥9 (P = 0.001). Multivariate analysis revealed that PG-SGA scores ≥9 compared with PG-SGA scores of 0 to 3 (hazard ratio [HR], 2.347; 95% confidence interval [CI], 1.271-4.334; P = 0.006), International Staging System (ISS) stage III compared with ISS stage I (HR, 2.360; 95% CI, 1.271-4.379; P = 0.007), and autologous stem cell transplantation (HR, 0.388; 95% CI, 0.248-0.606; P < 0.001) were associated with overall survival. A higher PG-SGA score before chemotherapy was associated with reduced survival among patients with MM. Nutritional evaluation should be an integral part of the clinical assessment of MM patients, and the PG-SGA score would be an appropriate tool to evaluate nutritional status. Copyright © 2016 Elsevier Inc. All rights reserved.

Membrane-anchored Plakoglobins Have Multiple Mechanisms of Action in Wnt Signaling

PubMed Central

Klymkowsky, Michael W.; Williams, Bart O.; Barish, Grant D.; Varmus, Harold E.; Vourgourakis, Yanni E.

1999-01-01

In Wnt signaling, β-catenin and plakoglobin transduce signals to the nucleus through interactions with TCF-type transcription factors. However, when plakoglobin is artificially engineered to restrict it to the cytoplasm by fusion with the transmembrane domain of connexin (cnxPg), it efficiently induces a Wnt-like axis duplication phenotype in Xenopus. In Xenopus embryos, maternal XTCF3 normally represses ventral expression of the dorsalizing gene Siamois. Two models have been proposed to explain the Wnt-like activity of cnxPg: 1) that cnxPg inhibits the machinery involved in the turnover of cytosolic β-catenin, which then accumulates and inhibits maternal XTCF3, and 2) that cnxPg directly acts to inhibit XTCF3 activity. To distinguish between these models, we created a series of N-terminal deletion mutations of cnxPg and examined their ability to induce an ectopic axis in Xenopus, activate a TCF-responsive reporter (OT), stabilize β-catenin, and colocalize with components of the Wnt signaling pathway. cnxPg does not colocalize with the Wnt pathway component Dishevelled, but it does lead to the redistribution of APC and Axin, two proteins involved in the regulation of β-catenin turnover. Expression of cnxPg increases levels of cytosolic β-catenin; however, this effect does not completely explain its signaling activity. Although cnxPg and Wnt-1 stabilize β-catenin to similar extents, cnxPg activates OT to 10- to 20-fold higher levels than Wnt-1. Moreover, although LEF1 and TCF4 synergize with β-catenin and plakoglobin to activate OT, both suppress the signaling activity of cnxPg. In contrast, XTCF3 suppresses the signaling activity of both β-catenin and cnxPg. Both exogenous XLEF1 and XTCF3 are sequestered in the cytoplasm of Xenopus cells by cnxPg. Based on these data, we conclude that, in addition to its effects on β-catenin, cnxPg interacts with other components of the Wnt pathway, perhaps TCFs, and that these interactions contribute to its signaling activity. PMID:10512857

Membrane-anchored plakoglobins have multiple mechanisms of action in Wnt signaling.

PubMed

Klymkowsky, M W; Williams, B O; Barish, G D; Varmus, H E; Vourgourakis, Y E

1999-10-01

In Wnt signaling, beta-catenin and plakoglobin transduce signals to the nucleus through interactions with TCF-type transcription factors. However, when plakoglobin is artificially engineered to restrict it to the cytoplasm by fusion with the transmembrane domain of connexin (cnxPg), it efficiently induces a Wnt-like axis duplication phenotype in Xenopus. In Xenopus embryos, maternal XTCF3 normally represses ventral expression of the dorsalizing gene Siamois. Two models have been proposed to explain the Wnt-like activity of cnxPg: 1) that cnxPg inhibits the machinery involved in the turnover of cytosolic beta-catenin, which then accumulates and inhibits maternal XTCF3, and 2) that cnxPg directly acts to inhibit XTCF3 activity. To distinguish between these models, we created a series of N-terminal deletion mutations of cnxPg and examined their ability to induce an ectopic axis in Xenopus, activate a TCF-responsive reporter (OT), stabilize beta-catenin, and colocalize with components of the Wnt signaling pathway. cnxPg does not colocalize with the Wnt pathway component Dishevelled, but it does lead to the redistribution of APC and Axin, two proteins involved in the regulation of beta-catenin turnover. Expression of cnxPg increases levels of cytosolic beta-catenin; however, this effect does not completely explain its signaling activity. Although cnxPg and Wnt-1 stabilize beta-catenin to similar extents, cnxPg activates OT to 10- to 20-fold higher levels than Wnt-1. Moreover, although LEF1 and TCF4 synergize with beta-catenin and plakoglobin to activate OT, both suppress the signaling activity of cnxPg. In contrast, XTCF3 suppresses the signaling activity of both beta-catenin and cnxPg. Both exogenous XLEF1 and XTCF3 are sequestered in the cytoplasm of Xenopus cells by cnxPg. Based on these data, we conclude that, in addition to its effects on beta-catenin, cnxPg interacts with other components of the Wnt pathway, perhaps TCFs, and that these interactions contribute to its signaling activity.

A study of the native cell wall structures of the marine alga Ventricaria ventricosa (Siphonocladales, Chlorophyceae) using atomic force microscopy.

PubMed

Eslick, Enid M; Beilby, Mary J; Moon, Anthony R

2014-04-01

A substantial proportion of the architecture of the plant cell wall remains unknown with a few cell wall models being proposed. Moreover, even less is known about the green algal cell wall. Techniques that allow direct visualization of the cell wall in as near to its native state are of importance in unravelling the spatial arrangement of cell wall structures and hence in the development of cell wall models. Atomic force microscopy (AFM) was used to image the native cell wall of living cells of Ventricaria ventricosa (V. ventricosa) at high resolution under physiological conditions. The cell wall polymers were identified mainly qualitatively via their structural appearance. The cellulose microfibrils (CMFs) were easily recognizable and the imaging results indicate that the V. ventricosa cell wall has a cross-fibrillar structure throughout. We found the native wall to be abundant in matrix polysaccharides existing in different curing states. The soft phase matrix polysaccharides susceptible by the AFM scanning tip existed as a glutinous fibrillar meshwork, possibly incorporating both the pectic- and hemicellulosic-type substances. The hard phase matrix producing clearer images, revealed coiled fibrillar structures associated with CMFs, sometimes being resolved as globular structures by the AFM tip. The coiling fibrillar structures were also seen in the images of isolated cell wall fragments. The mucilaginous component of the wall was discernible from the gelatinous cell wall matrix as it formed microstructural domains over the surface. AFM has been successful in imaging the native cell wall and revealing novel findings such as the 'coiling fibrillar structures' and cell wall components which have previously not been seen, that is, the gelatinous matrix phase.

The enzyme-sensitive release of prodigiosin grafted β-cyclodextrin and chitosan magnetic nanoparticles as an anticancer drug delivery system: Synthesis, characterization and cytotoxicity studies.

PubMed

Rastegari, Banafsheh; Karbalaei-Heidari, Hamid Reza; Zeinali, Sedigheh; Sheardown, Heather

2017-10-01

In present investigation, two glucose based smart tumor-targeted drug delivery systems coupled with enzyme-sensitive release strategy are introduced. Magnetic nanoparticles (Fe 3 O 4 ) were grafted with carboxymethyl chitosan (CS) and β-cyclodextrin (β-CD) as carriers. Prodigiosin (PG) was used as the model anti-tumor drug, targeting aggressive tumor cells. The morphology, properties and composition and grafting process were characterized by transmission electron microscope (TEM), Fourier transform infrared spectroscopy (FT-IR), vibration sample magnetometer (VSM), X-ray diffraction (XRD) analysis. The results revealed that the core crystal size of the nanoparticles synthesized were 14.2±2.1 and 9.8±1.4nm for β-CD and CS-MNPs respectively when measured using TEM; while dynamic light scattering (DLS) gave diameters of 121.1 and 38.2nm. The saturation magnetization (Ms) of bare magnetic nanoparticles is 50.10emucm -3 , while modification with β-CD and CS gave values of 37.48 and 65.01emucm -3 , respectively. The anticancer compound, prodigiosin (PG) was loaded into the NPs with an encapsulation efficiency of approximately 81% for the β-CD-MNPs, and 92% for the CS-MNPs. This translates to a drug loading capacity of 56.17 and 59.17mg/100mg MNPs, respectively. Measurement of in vitro release of prodigiosin from the loaded nanocarriers in the presence of the hydrolytic enzymes, alpha-amylase and chitosanase showed that 58.1 and 44.6% of the drug was released after one-hour of incubation. Cytotoxicity studies of PG-loaded nanocarriers on two cancer cell lines, MCF-7 and HepG2, and on a non-cancerous control, NIH/3T3 cells, revealed that the drug loaded nanoparticles had greater efficacy on the cancer cell lines. The selective index (SI) for free PG on MCF-7 and HepG2 cells was 1.54 and 4.42 respectively. This parameter was reduced for PG-loaded β-CD-MNPs to 1.27 and 1.85, while the SI for CS-MNPs improved considerably to 7.03 on MCF-7 cells. Complementary studies by fluorescence and confocal microscopy and flow cytometry confirm specific targeting of the nanocarriers to the cancer cells. The results suggest that CS-MNPs have higher potency and are better able to target the prodigiosin toxicity effect on cancerous cells than β-CD-MNPs. Copyright © 2017 Elsevier B.V. All rights reserved.

Efficiency of pH-Sensitive Fusogenic Polymer-Modified Liposomes as a Vaccine Carrier

PubMed Central

Watarai, Shinobu; Iwase, Tana; Tajima, Tomoko; Yuba, Eiji; Kono, Kenji

2013-01-01

The usefulness of pH-sensitive fusogenic polymer-(succinylated poly(glycidol)-(SucPG-) modified liposomes as a vaccine carrier in the induction of immune responses was evaluated. Mice were intraperitoneally immunized with ovalbumin- (OVA-) containing SucPG-modified liposomes. After immunization, significant OVA-specific antibodies were detected in the serum. When sera were analyzed for isotype distribution, OVA-specific IgG1 antibody responses were noted in mice immunized with OVA-containing polymer-unmodified liposomes, whereas immunization with OVA-containing SucPG-modified liposomes resulted in the induction of OVA-specific IgG1, IgG2a, and IgG3 Ab responses. In spleen lymphocytes from mice immunized with OVA-containing SucPG-modified liposomes, both IFN-γ-(Th1-type-) and IL-4-(Th2 type-) specific mRNA were detected. Moreover, substantial production of IFN-γ and IL-4 was demonstrated in spleen cells from OVA-containing SucPG-modified liposomes in vitro. These results suggest that the pH-sensitive fusogenic polymer-(SucPG-) modified liposomes would serve effectively as an antigen delivery vehicle for inducing Th1 and Th2 immune responses. PMID:23431260

Efficiency of pH-sensitive fusogenic polymer-modified liposomes as a vaccine carrier.

PubMed

Watarai, Shinobu; Iwase, Tana; Tajima, Tomoko; Yuba, Eiji; Kono, Kenji

2013-01-01

The usefulness of pH-sensitive fusogenic polymer-(succinylated poly(glycidol)-(SucPG-) modified liposomes as a vaccine carrier in the induction of immune responses was evaluated. Mice were intraperitoneally immunized with ovalbumin- (OVA-) containing SucPG-modified liposomes. After immunization, significant OVA-specific antibodies were detected in the serum. When sera were analyzed for isotype distribution, OVA-specific IgG1 antibody responses were noted in mice immunized with OVA-containing polymer-unmodified liposomes, whereas immunization with OVA-containing SucPG-modified liposomes resulted in the induction of OVA-specific IgG1, IgG2a, and IgG3 Ab responses. In spleen lymphocytes from mice immunized with OVA-containing SucPG-modified liposomes, both IFN-γ-(Th1-type-) and IL-4-(Th2 type-) specific mRNA were detected. Moreover, substantial production of IFN-γ and IL-4 was demonstrated in spleen cells from OVA-containing SucPG-modified liposomes in vitro. These results suggest that the pH-sensitive fusogenic polymer-(SucPG-) modified liposomes would serve effectively as an antigen delivery vehicle for inducing Th1 and Th2 immune responses.

Structural studies and molecular dynamics simulations suggest a processive mechanism of exolytic lytic transglycosylase from Campylobacter jejuni.

PubMed

Vijayaraghavan, Jagamya; Kumar, Vijay; Krishnan, Nikhil P; Kaufhold, Ross T; Zeng, Ximin; Lin, Jun; van den Akker, Focco

2018-01-01

The bacterial soluble lytic transglycosylase (LT) breaks down the peptidoglycan (PG) layer during processes such as cell division. We present here crystal structures of the soluble LT Cj0843 from Campylobacter jejuni with and without bulgecin A inhibitor in the active site. Cj0843 has a doughnut shape similar but not identical to that of E. coli SLT70. The C-terminal catalytic domain is preceded by an L-domain, a large helical U-domain, a flexible linker, and a small N-terminal NU-domain. The flexible linker allows the NU-domain to reach over and complete the circular shape, using residues conserved in the Epsilonproteobacteria LT family. The inner surface of the Cj0843 doughnut is mostly positively charged including a pocket that has 8 Arg/Lys residues. Molecular dynamics simulations with PG strands revealed a potential functional role for this pocket in anchoring the negatively charged terminal tetrapeptide of the PG during several steps in the reaction including homing and aligning the PG strand for exolytic cleavage, and subsequent ratcheting of the PG strand to enhance processivity in degrading PG strands.

Altered Cell Wall Plasticity Can Restrict Plant Growth under Ammonium Nutrition.

PubMed

Podgórska, Anna; Burian, Maria; Gieczewska, Katarzyna; Ostaszewska-Bugajska, Monika; Zebrowski, Jacek; Solecka, Danuta; Szal, Bożena

2017-01-01

Plants mainly utilize inorganic forms of nitrogen (N), such as nitrate (NO 3 - ) and ammonium (NH 4 + ). However, the composition of the N source is important, because excess of NH 4 + promotes morphological disorders. Plants cultured on NH 4 + as the sole N source exhibit serious growth inhibition, commonly referred to as "ammonium toxicity syndrome." NH 4 + -mediated suppression of growth may be attributable to both repression of cell elongation and reduction of cell division. The precondition for cell enlargement is the expansion of the cell wall, which requires the loosening of the cell wall polymers. Therefore, to understand how NH 4 + nutrition may trigger growth retardation in plants, properties of their cell walls were analyzed. We found that Arabidopsis thaliana using NH 4 + as the sole N source has smaller cells with relatively thicker cell walls. Moreover, cellulose, which is the main load-bearing polysaccharide revealed a denser assembly of microfibrils. Consequently, the leaf blade tissue showed elevated tensile strength and indicated higher cell wall stiffness. These changes might be related to changes in polysaccharide and ion content of cell walls. Further, NH 4 + toxicity was associated with altered activities of cell wall modifying proteins. The lower activity and/or expression of pectin hydrolyzing enzymes and expansins might limit cell wall expansion. Additionally, the higher activity of cell wall peroxidases can lead to higher cross-linking of cell wall polymers. Overall, the NH 4 + -mediated inhibition of growth is related to a more rigid cell wall structure, which limits expansion of cells. The changes in cell wall composition were also indicated by decreased expression of Feronia , a receptor-like kinase involved in the control of cell wall extension.

Altered Cell Wall Plasticity Can Restrict Plant Growth under Ammonium Nutrition

PubMed Central

Podgórska, Anna; Burian, Maria; Gieczewska, Katarzyna; Ostaszewska-Bugajska, Monika; Zebrowski, Jacek; Solecka, Danuta; Szal, Bożena

2017-01-01

Plants mainly utilize inorganic forms of nitrogen (N), such as nitrate (NO3–) and ammonium (NH4+). However, the composition of the N source is important, because excess of NH4+ promotes morphological disorders. Plants cultured on NH4+ as the sole N source exhibit serious growth inhibition, commonly referred to as “ammonium toxicity syndrome.” NH4+-mediated suppression of growth may be attributable to both repression of cell elongation and reduction of cell division. The precondition for cell enlargement is the expansion of the cell wall, which requires the loosening of the cell wall polymers. Therefore, to understand how NH4+ nutrition may trigger growth retardation in plants, properties of their cell walls were analyzed. We found that Arabidopsis thaliana using NH4+ as the sole N source has smaller cells with relatively thicker cell walls. Moreover, cellulose, which is the main load-bearing polysaccharide revealed a denser assembly of microfibrils. Consequently, the leaf blade tissue showed elevated tensile strength and indicated higher cell wall stiffness. These changes might be related to changes in polysaccharide and ion content of cell walls. Further, NH4+ toxicity was associated with altered activities of cell wall modifying proteins. The lower activity and/or expression of pectin hydrolyzing enzymes and expansins might limit cell wall expansion. Additionally, the higher activity of cell wall peroxidases can lead to higher cross-linking of cell wall polymers. Overall, the NH4+-mediated inhibition of growth is related to a more rigid cell wall structure, which limits expansion of cells. The changes in cell wall composition were also indicated by decreased expression of Feronia, a receptor-like kinase involved in the control of cell wall extension. PMID:28848567

[Production and mechanism of CCL5 by macrophages in U14 cervical cancer-bearing mice during infection].

PubMed

Ren, Hong; Ren, Guoli; Sun, Limin; Fan, Xiuhua; Wang, Yuran; Li, Xiaoxi

2015-05-01

To investigate the production and mechanism of chemokine (C-C motif) ligand 5 (CCL5) by macrophages in U14 cervical cancer-bearing mice during infection. The U14 cervical cancer cells were injected in C57BL/6 mice to induce tumor-bearing condition. Lipopolysaccharide (LPS) was injected into C57BL/6 mice to induce infection. The protein expression of CCL5 in the serum and the CCL5 mRNA expression in inflammatory cells were measured by ELISA and fluorescence quantitative-PCR in four groups. Macrophages were induced in the tumor conditioned medium (TCM) which extracted from mice serum. The protein expression levels of CCL5, prostaglandin E2 (PGE2) and cyclic adenosine monophosphate (cAMP) in the medium and CCL5, PGE2 and cAMP mRNA expression in the macrophages were detected in different groups. In order to determine whether the inhibition was related to PGE2, selective cyclooxygenase 2(COX-2) inhibitor NS398 was used to reverse this phenomenon and protein kinase A (PKA) inhibitor H89 demonstrated the mechanism through blocking cAMP/PKA signaling pathway. (1) The protein and mRNA level of CCL5 in tumor-bearing mice were respectively (151 ± 35) pg/ml and 1.0, which were lower than those in the tumor-free mice (691 ± 85) pg/ml and 4.5 ± 0.8, there were significant difference between them (all P < 0.05). The protein and mRNA level of PGE2 in tumor-bearing mice were (1 198 ± 83) pg/ml and 5.8 ± 0.8, which were higher than those in the tumor-free mice (187 ± 25) pg/ml and 1.0, the difference were significant (all P < 0.05). The protein and mRNA level of CCL5 in tumor-free + LPS mice were (4 049 ± 141) pg/ml and 31.5 ± 2.0, which were higher than those in the tumor-bearing + LPS mice (1 951 ± 71) pg/ml and 12.1 ± 2.8, the difference were also significant (P < 0.05). The protein and mRNA level of PGE2 in tumor-free + LPS mice were (676 ± 70) pg/ml and 3.4 ± 0.4, which were lower than those in tumor-bearing + LPS mice (2 550 ± 382) pg/ml and 11.6 ± 0.9, the difference were also significant (all P < 0.05). (2) Macrophages were cultured in vitro using TCM derived from mice. The protein and mRNA level of CCL5 in tumor-bearing mice TCM were respectively (1 626 ± 177) pg/ml and 28.6 ± 1.2, which were higher than those in the tumor-free mice TCM [(27 ± 3) pg/ml and 1.0], there were significant difference (P < 0.05). The protein and mRNA level of PGE2 in tumor-bearing mice TCM were (790 ± 156) pg/ml and 1.7 ± 0.3, which were higher than those in the tumor-free mice TCM [(448 ± 115) pg/ml, 1.0], the difference were significant (all P < 0.05). The protein and mRNA level of cAMP in tumor-bearing mice TCM were (164 ± 30) pg/ml and 1.6 ± 0.3, which weres higher than those in the tumor-free mice TCM [(118 ± 25) pg/ml,1.0], the difference were significant (all P < 0.05). The protein and mRNA level of CCL5 in tumor-free + LPS mice TCM were (10 475 ± 742) pg/ml and 212.0 ± 5.7, which were higher than those in the tumor-bearing + LPS mice TCM [(6 375 ± 530) pg/ml, 142.3 ± 2.5], the difference were significant (all P < 0.05). The protein and mRNA level of PGE2 in tumor-free + LPS mice TCM were (2 438 ± 95) pg/ml and 4.3 ± 0.7, which weres lower than those in the tumor-bearing + LPS mice TCM [(3 441 ± 163) pg/ml, 5.9 ± 0.3], the difference were significant (all P < 0.05). The protein and mRNA level of cAMP in tumor-free + LPS mice TCM were (340 ± 13) pg/ml and 4.1 ± 0.4, which were lower than those in the tumor-bearing + LPS mice TCM [(542 ± 42) pg/ml, 5.4 ± 0.5], the difference were significant (all P < 0.05). (3) Using COX-2 inhibitor NS398 in the tumor-bearing + LPS mice, the protein and mRNA level of CCL5, PGE2 and cAMP were (7 691 ± 269) pg/ml and 159.0 ± 8.9, (2 820 ± 152) pg/ml and 4.9 ± 0.3, (465 ± 8) pg/ml and 4.3 ± 0.4, respectively, and there were significant difference (all P < 0.05), compared to before treatment. Using PKA inhibitor H89 in the tumor-bearing + LPS mice, the protein and mRNA level of CCL5, PGE2 and cAMP were (8 375 ± 520) pg/ml and 177.0 ± 8.8, (2 650 ± 35) pg/ml and 4.7 ± 0.4, (368 ± 13) pg/ml and 3.1 ± 0.7, respectively, and there were significant difference (all P < 0.05), compared to before treatment. TCM of U14 cells activated macrophages to release PGE2 could inhibit the expression of CCL5 levels by cAMP/PKA signaling pathway.

[Analysis of virulence factors of Porphyromonas endodontalis based on comparative proteomics technique].

PubMed

Li, H; Ji, H; Wu, S S; Hou, B X

2016-12-09

Objective: To analyze the protein expression profile and the potential virulence factors of Porphyromonas endodontalis (Pe) via comparison with that of two strains of Porphyromonas gingivalis (Pg) with high and low virulences, respectively. Methods: Whole cell comparative proteomics of Pe ATCC35406 was examined and compared with that of high virulent strain Pg W83 andlow virulent strain Pg ATCC33277, respectively. Isobaric tags for relative and absolute quantitation (iTRAQ) combined with nano liquid chromatography-tandem mass spectrometry (Nano-LC-MS/MS) were adopted to identify and quantitate the proteins of Pe and two strains of Pg with various virulences by using the methods of isotopically labeled peptides, mass spectrometric detection and bioinformatics analysis. The biological functions of similar proteins expressed by Pe ATCC35406 and two strains of Pg were quantified and analyzed. Results: Totally 1 210 proteins were identified while Pe compared with Pg W83. There were 130 proteins (10.74% of the total proteins) expressed similarly, including 89 known functional proteins and 41 proteins of unknown functions. Totally 1 223 proteins were identified when Pe compared with Pg ATCC33277. There were 110 proteins (8.99% of the total proteins) expressed similarly, including 72 known functional proteins and 38 proteins of unknown functions. The similarly expressed proteins in Pe and Pg strains with various virulences mainly focused on catalytic activity and binding function, including recombination activation gene (RagA), lipoprotein, chaperonin Dnak, Clp family proteins (ClpC and ClpX) and various iron-binding proteins. They were involved in metabolism and cellular processes. In addition, the type and number of similar virulence proteins between Pe and high virulence Pg were higher than those between Pe and low virulence Pg. Conclusions: Lipoprotein, oxygen resistance protein, iron binding protein were probably the potential virulence factors of Pe ATCC35406. It was speculated that pathogenicity of Pe was more similar to high virulence Pg than that to low virulence strain.

Dendritic polyglycerol sulfate as a novel platform for paclitaxel delivery: pitfalls of ester linkage

NASA Astrophysics Data System (ADS)

Sousa-Herves, Ana; Würfel, Patrick; Wegner, Nicole; Khandare, Jayant; Licha, Kai; Haag, Rainer; Welker, Pia; Calderón, Marcelo

2015-02-01

In this study, dendritic polyglycerol sulfate (dPGS) is evaluated as a delivery platform for the anticancer, tubulin-binding drug paclitaxel (PTX). The conjugation of PTX to dPGS is conducted via a labile ester linkage. A non-sulfated dendritic polyglycerol (dPG) is used as a control, and the labeling with an indocarbocyanine dye (ICC) renders multifunctional conjugates that can be monitored by fluorescence microscopy. The conjugates are characterized by 1H NMR, UV-vis measurements, and RP-HPLC. In vitro cytotoxicity of PTX and dendritic conjugates is evaluated using A549 and A431 cell lines, showing a reduced cytotoxic efficacy of the conjugates compared to PTX. The study of uptake kinetics reveals a linear, non saturable uptake in tumor cells for dPGS-PTX-ICC, while dPG-PTX-ICC is hardly taken up. Despite the marginal uptake of dPG-PTX-ICC, it prompts tubulin polymerization to a comparable extent as PTX. These observations suggest a fast ester hydrolysis and premature drug release, as confirmed by HPLC measurements in the presence of plasma enzymes.In this study, dendritic polyglycerol sulfate (dPGS) is evaluated as a delivery platform for the anticancer, tubulin-binding drug paclitaxel (PTX). The conjugation of PTX to dPGS is conducted via a labile ester linkage. A non-sulfated dendritic polyglycerol (dPG) is used as a control, and the labeling with an indocarbocyanine dye (ICC) renders multifunctional conjugates that can be monitored by fluorescence microscopy. The conjugates are characterized by 1H NMR, UV-vis measurements, and RP-HPLC. In vitro cytotoxicity of PTX and dendritic conjugates is evaluated using A549 and A431 cell lines, showing a reduced cytotoxic efficacy of the conjugates compared to PTX. The study of uptake kinetics reveals a linear, non saturable uptake in tumor cells for dPGS-PTX-ICC, while dPG-PTX-ICC is hardly taken up. Despite the marginal uptake of dPG-PTX-ICC, it prompts tubulin polymerization to a comparable extent as PTX. These observations suggest a fast ester hydrolysis and premature drug release, as confirmed by HPLC measurements in the presence of plasma enzymes. Electronic supplementary information (ESI) available: 1H NMR spectra of the conjugates, HPLC chromatograms, internalization images of dPGS-PTX-ICC (5), elimination kinetics of dPGS-PTX-ICC (5) and dPGS-ICC (7), comparison of IC50 values of PTX and dPGS-PTX (3) in A431 and A549 cell lines and cell viability of dPGS amine (1). See DOI: 10.1039/c4nr04428b

Virologic and immunologic outcome of HAART in Human Immunodeficiency Virus (HIV)-1 infected patients with and without tuberculosis (TB) and latent TB infection (LTBI) in Addis Ababa, Ethiopia.

PubMed

Kassa, Desta; Gebremichael, Gebremedhin; Alemayehu, Yodit; Wolday, Dawit; Messele, Tsehaynesh; van Baarle, Debbie

2013-01-01

HIV/TB coinfection remains a major challenge even after the initiation of HAART. Little is known about Mycobacterium tuberculosis (Mtb) specific immune restoration in relation to immunologic and virologic outcomes after long-term HAART during co-infections with latent and active TB. A total of 232 adults, including 59 HIV patients with clinical TB (HIV + TB+), 125 HIV patients without clinical TB (HIV + TB-), 13 HIV negative active TB patients (HIV-TB+), and 10 HIV negative Tuberculin Skin TST positive (HIV-TST+), and 25 HIV-TST- individuals were recruited. HAART was initiated in 113 HIV + patients (28 TB + and 85 TB-), and anti-TB treatment for all TB cases. CD4+ T-cell count, HIV RNA load, and IFN-γ responses to ESAT-6/CFP-10 were measured at baseline, 6 months (M6), 18 months (M18) and 24 months (M24) after HAART initiation. The majority of HIV + TB- (70%, 81%, 84%) as well as HIV + TB + patients (60%, 77%, 80%) had virologic success (HIV RNA < 50 copies/ml) by M6, M18 and M24, respectively. HAART also significantly increased CD4+ T-cell counts at 2 years in HIV + TB + (from 110.3 to 289.9 cells/μl), HIV + TB- patients (197.8 to 332.3 cells/μl), HIV + TST- (199 to 347 cells/μl) and HIV + TST + individuals (195 to 319 cells/μl). Overall, there was no significant difference in the percentage of patients that achieved virologic success and in total CD4+ counts increased between HIV patients with and without TB or LTBI. The Mtb specific IFN-γ response at baseline was significantly lower in HIV + TB + (3.6 pg/ml) compared to HIV-TB + patients (34.4 pg/ml) and HIV + TST + (46.3 pg/ml) individuals; and in HIV-TB + patients compared to HIV-TST + individuals (491.2 pg/ml). By M18 on HAART, the IFN-γ response remained impaired in HIV + TB + patients (18.1 pg/ml) while it normalized in HIV + TST + individuals (from 46.3 to 414.2 pg/ml). Our data show that clinical and latent TB infections do not influence virologic and immunologic outcomes of ART in HIV patients. Despite this, HAART was unable to restore optimal TB responsiveness as measured by Mtb specific IFN-γ response in HIV/TB patients. Improvement of Mtb-specific immune restoration should be the focus of future therapeutic strategies.

The Cell Wall of the Human Fungal Pathogen Aspergillus fumigatus: Biosynthesis, Organization, Immune Response, and Virulence.

PubMed

Latgé, Jean-Paul; Beauvais, Anne; Chamilos, Georgios

2017-09-08

More than 90% of the cell wall of the filamentous fungus Aspergillus fumigatus comprises polysaccharides. Biosynthesis of the cell wall polysaccharides is under the control of three types of enzymes: transmembrane synthases, which are anchored to the plasma membrane and use nucleotide sugars as substrates, and cell wall-associated transglycosidases and glycosyl hydrolases, which are responsible for remodeling the de novo synthesized polysaccharides and establishing the three-dimensional structure of the cell wall. For years, the cell wall was considered an inert exoskeleton of the fungal cell. The cell wall is now recognized as a living organelle, since the composition and cellular localization of the different constitutive cell wall components (especially of the outer layers) vary when the fungus senses changes in the external environment. The cell wall plays a major role during infection. The recognition of the fungal cell wall by the host is essential in the initiation of the immune response. The interactions between the different pattern-recognition receptors (PRRs) and cell wall pathogen-associated molecular patterns (PAMPs) orientate the host response toward either fungal death or growth, which would then lead to disease development. Understanding the molecular determinants of the interplay between the cell wall and host immunity is fundamental to combatting Aspergillus diseases.

Cell Wall Composition and Candidate Biosynthesis Gene Expression During Rice Development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Fan; Manisseri, Chithra; Fagerström, Alexandra

Cell walls of grasses, including cereal crops and biofuel grasses, comprise the majority of plant biomass and intimately influence plant growth, development and physiology. However, the functions of many cell wall synthesis genes, and the relationships among and the functions of cell wall components remain obscure. To better understand the patterns of cell wall accumulation and identify genes that act in grass cell wall biosynthesis, we characterized 30 samples from aerial organs of rice (Oryza sativa cv. Kitaake) at 10 developmental time points, 3-100 d post-germination. Within these samples, we measured 15 cell wall chemical components, enzymatic digestibility and 18more » cell wall polysaccharide epitopes/ligands. We also used quantitative reverse transcription-PCR to measure expression of 50 glycosyltransferases, 15 acyltransferases and eight phenylpropanoid genes, many of which had previously been identified as being highly expressed in rice. Most cell wall components vary significantly during development, and correlations among them support current understanding of cell walls. We identified 92 significant correlations between cell wall components and gene expression and establish nine strong hypotheses for genes that synthesize xylans, mixed linkage glucan and pectin components. This work provides an extensive analysis of cell wall composition throughout rice development, identifies genes likely to synthesize grass cell walls, and provides a framework for development of genetically improved grasses for use in lignocellulosic biofuel production and agriculture.« less

Fundoplication with 180-Degree Wrap During Esophagogastrostomy After Robotic Proximal Gastrectomy for Early Gastric Cancer.

PubMed

Ojima, Toshiyasu; Nakamori, Mikihito; Nakamura, Masaki; Hayata, Keiji; Maruoka, Shimpei; Yamaue, Hiroki

2018-04-20

Compared with total gastrectomy, proximal gastrectomy (PG) has potential advantages from a nutritional perspective, such as anemia and postoperative loss of body weight. However, PG is associated with some postoperative functional disorders, such as reflux esophagus (13-31%) and anastomotic stenosis (3-29%).1 We therefore developed a new procedure for fundoplication during esophago-gastrostomy after robotic PG (RPG). We performed RPG for early gastric cancer localized in the upper third of the stomach using the da Vinci Surgical System (Intuitive, Sunnyvale, CA). After RPG conclusion, intracorporeal esophago-gastrostomy was performed by side-to-side anastomosis using a linear 45 mm stapling device, Endo GIA purple cartridge.2 The post-excisional hole in the esophago-gastrostomy was closed with interrupted single-layered sutures by robotic suturing technique. Fundoplication was created by wrapping the remnant stomach around 180 degrees of the circumference of the esophagus; the remnant stomach was wrapped from the esophageal posterior wall towards the esophageal anterior wall. Four stitches were used for fixation. We did not add a bougie of esophago-gastrostomy when fashioning the wrap. In addition, we did not perform pyloroplasty. In our series with 15 patients, there were no postoperative complications. No patients had reflux symptoms. Our technique using the fundoplication with "clockwise" rotation attempts to prevent reflux by use of intragastric pressure to flatten the lower end of the esophagus into a valvate shape. Indeed, in fluoroscopic findings 4 days after surgery, there was no reflux to the esophagus of the contrast medium. In endoscopic findings 3 months after surgery, anastomotic stenosis was absent. We observed no endoscopic findings of reflux esophagitis. Formation of the pseudo-fornix was confirmed by wrapping the remnant stomach. RPG followed by fundoplication with 180-degree wrap may be a promising procedure for reflux esophagitis prevention.3,4 However, long-term follow-up is required to show benefits of this new procedure.4.

An Embryonic and Induced Pluripotent Stem Cell Model for Ovarian Granulosa Cell Development and Steroidogenesis.

PubMed

Lipskind, Shane; Lindsey, Jennifer S; Gerami-Naini, Behzad; Eaton, Jennifer L; O'Connell, Daniel; Kiezun, Adam; Ho, Joshua W K; Ng, Nicholas; Parasar, Parveen; Ng, Michelle; Nickerson, Michael; Demirci, Utkan; Maas, Richard; Anchan, Raymond M

2018-05-01

Embryoid bodies (EBs) can serve as a system for evaluating pluripotency, cellular differentiation, and tissue morphogenesis. In this study, we use EBs derived from mouse embryonic stem cells (mESCs) and human amniocyte-derived induced pluripotent stem cells (hAdiPSCs) as a model for ovarian granulosa cell (GC) development and steroidogenic cell commitment. We demonstrated that spontaneously differentiated murine EBs (mEBs) and human EBs (hEBs) displayed ovarian GC markers, such as aromatase (CYP19A1), FOXL2, AMHR2, FSHR, and GJA1. Comparative microarray analysis identified both shared and unique gene expression between mEBs and the maturing mouse ovary. Gene sets related to gonadogenesis, lipid metabolism, and ovarian development were significantly overrepresented in EBs. Of the 29 genes, 15 that were differentially regulated in steroidogenic mEBs displayed temporal expression changes between embryonic, postnatal, and mature ovarian tissues by polymerase chain reaction. Importantly, both mEBs and hEBs were capable of gonadotropin-responsive estradiol (E2) synthesis in vitro (217-759 pg/mL). Live fluorescence-activated cell sorting-sorted AMHR2 + granulosa-like cells from mEBs continued to produce E2 after purification (15.3 pg/mL) and secreted significantly more E2 than AMHR2 - cells (8.6 pg/mL, P < .05). We conclude that spontaneously differentiated EBs of both mESC and hAdiPSC origin can serve as a biologically relevant model for ovarian GC differentiation and steroidogenic cell commitment. These cells should be further investigated for therapeutic uses, such as stem cell-based hormone replacement therapy and in vitro maturation of oocytes.

The Impact of Microfibril Orientations on the Biomechanics of Plant Cell Walls and Tissues.

PubMed

Ptashnyk, Mariya; Seguin, Brian

2016-11-01

The microscopic structure and anisotropy of plant cell walls greatly influence the mechanical properties, morphogenesis, and growth of plant cells and tissues. The microscopic structure and properties of cell walls are determined by the orientation and mechanical properties of the cellulose microfibrils and the mechanical properties of the cell wall matrix. Viewing the shape of a plant cell as a square prism with the axis aligning with the primary direction of expansion and growth, the orientation of the microfibrils within the side walls, i.e. the parts of the cell walls on the sides of the cells, is known. However, not much is known about their orientation at the upper and lower ends of the cell. Here we investigate the impact of the orientation of cellulose microfibrils within the upper and lower parts of the plant cell walls by solving the equations of linear elasticity numerically. Three different scenarios for the orientation of the microfibrils are considered. We also distinguish between the microstructure in the side walls given by microfibrils perpendicular to the main direction of the expansion and the situation where the microfibrils are rotated through the wall thickness. The macroscopic elastic properties of the cell wall are obtained using homogenization theory from the microscopic description of the elastic properties of the cell wall microfibrils and wall matrix. It is found that the orientation of the microfibrils in the upper and lower parts of the cell walls affects the expansion of the cell in the lateral directions and is particularly important in the case of forces acting on plant cell walls and tissues.

High temperature induced disruption of the cell wall integrity and structure in Pleurotus ostreatus mycelia.

PubMed

Qiu, Zhiheng; Wu, Xiangli; Gao, Wei; Zhang, Jinxia; Huang, Chenyang

2018-05-30

Fungal cells are surrounded by a tight cell wall to protect them from harmful environmental conditions and to resist lysis. The synthesis and assembly determine the shape, structure, and integrity of the cell wall during the process of mycelial growth and development. High temperature is an important abiotic stress, which affects the synthesis and assembly of cell walls. In the present study, the chitin and β-1,3-glucan concentrations in the cell wall of Pleurotus ostreatus mycelia were changed after high-temperature treatment. Significantly higher chitin and β-1,3-glucan concentrations were detected at 36 °C than those incubated at 28 °C. With the increased temperature, many aberrant chitin deposition patches occurred, and the distribution of chitin in the cell wall was uneven. Moreover, high temperature disrupts the cell wall integrity, and P. ostreatus mycelia became hypersensitive to cell wall-perturbing agents at 36 °C. The cell wall structure tended to shrink or distorted after high temperature. The cell walls were observed to be thicker and looser by using transmission electron microscopy. High temperature can decrease the mannose content in the cell wall and increase the relative cell wall porosity. According to infrared absorption spectrum, high temperature broke or decreased the glycosidic linkages. Finally, P. ostreatus mycelial cell wall was easily degraded by lysing enzymes after high-temperature treatment. In other words, the cell wall destruction caused by high temperature may be a breakthrough for P. ostreatus to be easily infected by Trichoderma.

Co-culture of chondrons and mesenchymal stromal cells reduces the loss of collagen VI and improves extracellular matrix production.

PubMed

Owida, H A; De Las Heras Ruiz, T; Dhillon, A; Yang, Y; Kuiper, N J

2017-12-01

Adult articular chondrocytes are surrounded by a pericellular matrix (PCM) to form a chondron. The PCM is rich in hyaluronan, proteoglycans, and collagen II, and it is the exclusive location of collagen VI in articular cartilage. Collagen VI anchors the chondrocyte to the PCM. It has been suggested that co-culture of chondrons with mesenchymal stromal cells (MSCs) might enhance extracellular matrix (ECM) production. This co-culture study investigates whether MSCs help to preserve the PCM and increase ECM production. Primary bovine chondrons or chondrocytes or rat MSCs were cultured alone to establish a baseline level for ECM production. A xenogeneic co-culture monolayer model using rat MSCs (20, 50, and 80%) was established. PCM maintenance and ECM production were assessed by biochemical assays, immunofluorescence, and histological staining. Co-culture of MSCs with chondrons enhanced ECM matrix production, as compared to chondrocyte or chondron only cultures. The ratio 50:50 co-culture of MSCs and chondrons resulted in the highest increase in GAG production (18.5 ± 0.54 pg/cell at day 1 and 11 ± 0.38 pg/cell at day 7 in 50:50 co-culture versus 16.8 ± 0.61 pg/cell at day 1 and 10 ± 0.45 pg/cell at day 7 in chondron monoculture). The co-culture of MSCs with chondrons appeared to decelerate the loss of the PCM as determined by collagen VI expression, whilst the expression of high-temperature requirement serine protease A1 (HtrA1) demonstrated an inverse relationship to that of the collagen VI. Together, this implies that MSCs directly or indirectly inhibited HtrA1 activity and the co-culture of MSCs with chondrons enhanced ECM synthesis and the preservation of the PCM.

The impact of childhood obesity on inflammation, innate immune cell frequency, and metabolic microRNA expression.

PubMed

Carolan, Eirin; Hogan, Andrew E; Corrigan, Michelle; Gaotswe, Gadintshware; O'Connell, Jean; Foley, Niamh; O'Neill, Luke A; Cody, Declan; O'Shea, Donal

2014-03-01

Obesity is characterized by chronic inflammation, immune dysregulation, and alteration of gene expression, associated with type 2 diabetes mellitus and cardiovascular disease. The degree to which these changes occur in childhood obesity is not fully defined. The aim was to investigate the effect of childhood obesity on immune cell frequency, macrophage activation, cytokine production, and specific regulators of metabolic gene expression. Profiling was performed on peripheral blood from 29 obese and 20 nonobese children using real-time PCR, ELISA, and flow cytometry. Fasting glucose was similar in both groups, but there was a higher degree of insulin resistance in obese subjects (homeostasis model of assessment for insulin resistance, 4.8 vs 0.84; P < .001). Soluble CD163, a marker of macrophage polarization to a proinflammatory profile, was elevated in the obese compared to nonobese children (135 vs 105 ng/mL; P = .03). Invariant natural killer T cells were reduced in the obese children (CD3 T cells, 0.31 vs 0.53%; P = .001). Cytokine profiling revealed significantly elevated TNF-α (6.7 vs 5.1 pg/mL; P = .01) and leptin (1186 vs 432 pg/mL; P < .001) and reduced adiponectin (884 vs 1321 pg/mL; P = .001) in obese compared to nonobese children. Stimulation of peripheral blood mononuclear cells from obese children resulted in higher levels of IL-1β (2100 vs 1500 pg/mL; P = .018). There was a 4-fold increase in expression of microRNA33a (P = .001) and a 3-fold increase in microRNA33b (P = .017) in obese children. Childhood obesity is associated with changes in immune cell frequency, inflammatory environment, and regulation of metabolic gene expression. These changes have been causally linked to the onset of metabolic disease in adulthood and suggest the future trajectory of obese children to the development of type 2 diabetes mellitus and premature cardiovascular disease.

A case of cytomegalovirus colitis following immunosuppressive treatment for pyoderma gangrenosum.

PubMed

Kikuchi, Hidezumi; Nagamine, Hidehiro; Setoyama, Mitsuru

2005-04-01

We report a case of pyoderma gangrenosum (PG) complicated by cytomegalovirus (CMV)-induced colitis. A 79-year-old woman with PG was treated with corticosteroid and cyclosporin. She had blood in her stool and advancing anemia during the treatment. A colonoscopic biopsy specimen from the colon revealed typical CMV-infected cells with CMV inclusions confirmed by immunohistochemistry. Furthermore, there were many CMV-antigen-positive leukocytes, suggesting an active CMV infection, which is serious in compromised hosts. Although ulcerative colitis and Crohn's disease are well known as complications of PG, CMV enterocolitis should be considered in the differential diagnosis of enterocolitis in immunocompromised patients.

Endoplasmic reticulum-derived reactive oxygen species (ROS) is involved in toxicity of cell wall stress to Candida albicans.

PubMed

Yu, Qilin; Zhang, Bing; Li, Jianrong; Zhang, Biao; Wang, Honggang; Li, Mingchun

2016-10-01

The cell wall is an important cell structure in both fungi and bacteria, and hence becomes a common antimicrobial target. The cell wall-perturbing agents disrupt synthesis and function of cell wall components, leading to cell wall stress and consequent cell death. However, little is known about the detailed mechanisms by which cell wall stress renders fungal cell death. In this study, we found that ROS scavengers drastically attenuated the antifungal effect of cell wall-perturbing agents to the model fungal pathogen Candida albicans, and these agents caused remarkable ROS accumulation and activation of oxidative stress response (OSR) in this fungus. Interestingly, cell wall stress did not cause mitochondrial dysfunction and elevation of mitochondrial superoxide levels. Furthermore, the iron chelator 2,2'-bipyridyl (BIP) and the hydroxyl radical scavengers could not attenuate cell wall stress-caused growth inhibition and ROS accumulation. However, cell wall stress up-regulated expression of unfold protein response (UPR) genes, enhanced protein secretion and promoted protein folding-related oxidation of Ero1, an important source of ROS production. These results indicated that oxidation of Ero1 in the endoplasmic reticulum (ER), rather than mitochondrial electron transport and Fenton reaction, contributed to cell wall stress-related ROS accumulation and consequent growth inhibition. Our findings uncover a novel link between cell wall integrity (CWI), ER function and ROS production in fungal cells, and shed novel light on development of strategies promoting the antifungal efficacy of cell wall-perturbing agents against fungal infections. Copyright © 2016 Elsevier Inc. All rights reserved.

Regulation of cell wall biosynthesis.

PubMed

Zhong, Ruiqin; Ye, Zheng-Hua

2007-12-01

Plant cell walls differ in their amount and composition among various cell types and even in different microdomains of the wall of a given cell. Plants must have evolved regulatory mechanisms controlling biosynthesis, targeted secretion, and assembly of wall components to achieve the heterogeneity in cell walls. A number of factors, including hormones, the cytoskeleton, glycosylphosphatidylinositol-anchored proteins, phosphoinositides, and sugar nucleotide supply, have been implicated in the regulation of cell wall biosynthesis or deposition. In the past two years, there have been important discoveries in transcriptional regulation of secondary wall biosynthesis. Several transcription factors in the NAC and MYB families have been shown to be the key switches for activation of secondary wall biosynthesis. These studies suggest a transcriptional network comprised of a hierarchy of transcription factors is involved in regulating secondary wall biosynthesis. Further investigation and integration of the regulatory players participating in the making of cell walls will certainly lead to our understanding of how wall amounts and composition are controlled in a given cell type. This may eventually allow custom design of plant cell walls on the basis of our needs.

Vaccine to control the viral infection of fish

DOEpatents

Leong, Jo-Ann C.

1994-10-11

Subunit vaccines and their use for immunizing fish against infection by viruses are disclosed. In particular, plasmid pG8 is constructed by joining, with the plasmid pUC8, DNA which encodes the glycoprotein of infectious hematopoietic necrosis virus (IHNV). E. coli cells are transformed by pG8, whereby pure viral antigen is produced to provide a vaccine for the control of IHNV in fish.

Modulation of T-Cell Activation in an Experimental Model of Mammary Carcinoma.

DTIC Science & Technology

1998-07-01

K. R., A. D. Kirk, G. Pecher, F. W.W., and 0. J. Finn. 1997. A survivor of breast cancer with immunity to MUC-1 mucin, and lactational mastitis ...experiments, mice were injected with in DMEM:F12 (Biowhittaker) supplemented with 2% bovine anti-CD4 (GK 1.5,400 pg), anti-CD8 (2.43,600 pg), a combination

Relating Nanoscale Accessibility within Plant Cell Walls to Improved Enzyme Hydrolysis Yields in Corn Stover Subjected to Diverse Pretreatments.

PubMed

Crowe, Jacob D; Zarger, Rachael A; Hodge, David B

2017-10-04

Simultaneous chemical modification and physical reorganization of plant cell walls via alkaline hydrogen peroxide or liquid hot water pretreatment can alter cell wall structural properties impacting nanoscale porosity. Nanoscale porosity was characterized using solute exclusion to assess accessible pore volumes, water retention value as a proxy for accessible water-cell walls surface area, and solute-induced cell wall swelling to measure cell wall rigidity. Key findings concluded that delignification by alkaline hydrogen peroxide pretreatment decreased cell wall rigidity and that the subsequent cell wall swelling resulted increased nanoscale porosity and improved enzyme binding and hydrolysis compared to limited swelling and increased accessible surface areas observed in liquid hot water pretreated biomass. The volume accessible to a 90 Å dextran probe within the cell wall was found to be correlated to both enzyme binding and glucose hydrolysis yields, indicating cell wall porosity is a key contributor to effective hydrolysis yields.

Following the compositional changes of fresh grape skin cell walls during the fermentation process in the presence and absence of maceration enzymes.

PubMed

Zietsman, Anscha J J; Moore, John P; Fangel, Jonatan U; Willats, William G T; Trygg, Johan; Vivier, Melané A

2015-03-18

Cell wall profiling technologies were used to follow compositional changes that occurred in the skins of grape berries (from two different ripeness levels) during fermentation and enzyme maceration. Multivariate data analysis showed that the fermentation process yielded cell walls enriched in hemicellulose components because pectin was solubilized (and removed) with a reduction as well as exposure of cell wall proteins usually embedded within the cell wall structure. The addition of enzymes caused even more depectination, and the enzymes unravelled the cell walls enabling better access to, and extraction of, all cell wall polymers. Overripe grapes had cell walls that were extensively hydrolyzed and depolymerized, probably by natural grape-tissue-ripening enzymes, and this enhanced the impact that the maceration enzymes had on the cell wall monosaccharide profile. The combination of the techniques that were used is an effective direct measurement of the hydrolysis actions of maceration enzymes on the cell walls of grape berry skin.

Employing proteomic analysis to compare Paracoccidioides lutzii yeast and mycelium cell wall proteins.

PubMed

Araújo, Danielle Silva; de Sousa Lima, Patrícia; Baeza, Lilian Cristiane; Parente, Ana Flávia Alves; Melo Bailão, Alexandre; Borges, Clayton Luiz; de Almeida Soares, Célia Maria

2017-11-01

Paracoccidioidomycosis is an important systemic mycosis caused by thermodimorphic fungi of the Paracoccidioides genus. During the infective process, the cell wall acts at the interface between the fungus and the host. In this way, the cell wall has a key role in growth, environment sensing and interaction, as well as morphogenesis of the fungus. Since the cell wall is absent in mammals, it may present molecules that are described as target sites for new antifungal drugs. Despite its importance, up to now few studies have been conducted employing proteomics in for the identification of cell wall proteins in Paracoccidioides spp. Here, a detailed proteomic approach, including cell wall-fractionation coupled to NanoUPLC-MS E , was used to study and compare the cell wall fractions from Paracoccidioides lutzii mycelia and yeast cells. The analyzed samples consisted of cell wall proteins extracted by hot SDS followed by extraction by mild alkali. In summary, 512 proteins constituting different cell wall fractions were identified, including 7 predicted GPI-dependent cell wall proteins that are potentially involved in cell wall metabolism. Adhesins previously described in Paracoccidioides spp. such as enolase, glyceraldehyde-3-phosphate dehydrogenase were identified. Comparing the proteins in mycelium and yeast cells, we detected some that are common to both fungal phases, such as Ecm33, and some specific proteins, as glucanase Crf1. All of those proteins were described in the metabolism of cell wall. Our study provides an important elucidation of cell wall composition of fractions in Paracoccidioides, opening a way to understand the fungus cell wall architecture. Copyright © 2017 Elsevier B.V. All rights reserved.

Isolation of a potent antibiotic producer bacterium, especially against MRSA, from northern region of the Persian Gulf

PubMed Central

Darabpour, Esmaeil; Ardakani, Mohammad Roayaei; Motamedi, Hossein; Ronagh, Mohammad Taghi

2012-01-01

Nowadays, emergence and prevalence of MRSA (Methicillin Resistant Staphylococcus aureus) strain have become a great global concern in 21st century, so, it is necessary to discover new antibiotics against this pathogen. The aim of this study was isolation and evaluation marine bacteria from the Persian Gulf in order to finding antibiotic compounds against some pathogenic bacteria. For this purpose, water and sediment samples were collected from the Persian Gulf during March to October 2009. The antibacterial activity of the isolated bacteria was assessed using disc diffusion method. The Growth Curve Interference (GCI) parameter against MRSA was determined for the high potential antibiotic producing strain. The most important factors affecting fermentation conditions in antibiotic production were also optimized. Definite identification of intended isolate was confirmed by 16S rRNA sequencing. Altogether, 51 bacterial colony was isolated and among them only 3 bacterium showed antibacterial activity. Pseudoalteromonaspiscicida PG-01 isolated from a sediment sample was chosen as the best antibiotic producing strain. This strain was effective against all tested Gram-positive bacteria, had good anti-MRSA activity and also GCI value against MRSA was two times lower than MIC value. Among the optimized fermentation parameters, carbon and nitrogen sources play major role in efficacy of optimized antibiotic production. Ultrastructural study on the effect of intended antibiotic compounds on MRSA using TEM revealed that the target site for this compound is cell wall. Considering the antibacterial effect of PG-01 strain especially against MRSA, intended antibiotic compounds can gives hope for treatment of diseases caused by multi-drug resistant bacteria. PMID:22642595

Responses of human neutrophils to nicotine and/or Porphyromonas gingivalis.

PubMed

Al-Shibani, Nouf K; Labban, Nawaf Y; Kowolik, Michael J; Ruby, John D; Windsor, L Jack

2011-10-01

Tobacco smoking is considered a major modifiable risk factor for periodontal disease. Nicotine is the addictive ingredient in tobacco and has been shown to affect multiple cellular processes. Neutrophils are the first line of host defense and are critical cells in the maintenance of periodontal health through their role in the control of bacteria, but they can also contribute to the progression of periodontal disease by the production and release of reactive oxygen species (ROS). Virulence factors from periodontal pathogens, such as Porphyromonas gingivalis (Pg), stimulate the respiratory burst of neutrophils. The objective of this study is to explore the oxidative activity of neutrophils when stimulated with Pg, nicotine, or both. Neutrophils were separated from buffy coats by the double dextran gradient method. The generation of ROS by neutrophils was determined using luminol-dependent chemiluminescence assays. The reaction was followed for 90 minutes, and the neutrophil activation was recorded as the total integrated energy output. The Pg and Pg plus nicotine groups had a significantly higher active and peak chemiluminescence than the nicotine group (all with P <0.0001). The Pg and Pg with nicotine groups were not significantly different (P = 0.90). In the presence of Pg, the nicotine did not further enhance the ROS release by the neutrophils, suggesting that the bacteria induced the maximum ROS release in this model system.

Engineering cell wall synthesis mechanism for enhanced PHB accumulation in E. coli.

PubMed

Zhang, Xing-Chen; Guo, Yingying; Liu, Xu; Chen, Xin-Guang; Wu, Qiong; Chen, Guo-Qiang

2018-01-01

The rigidity of bacterial cell walls synthesized by a complicated pathway limit the cell shapes as coccus, bar or ellipse or even fibers. A less rigid bacterium could be beneficial for intracellular accumulation of poly-3-hydroxybutyrate (PHB) as granular inclusion bodies. To understand how cell rigidity affects PHB accumulation, E. coli cell wall synthesis pathway was reinforced and weakened, respectively. Cell rigidity was achieved by thickening the cell walls via insertion of a constitutive gltA (encoding citrate synthase) promoter in front of a series of cell wall synthesis genes on the chromosome of several E. coli derivatives, resulting in 1.32-1.60 folds increase of Young's modulus in mechanical strength for longer E. coli cells over-expressing fission ring FtsZ protein inhibiting gene sulA. Cell rigidity was weakened by down regulating expressions of ten genes in the cell wall synthesis pathway using CRISPRi, leading to elastic cells with more spaces for PHB accumulation. The regulation on cell wall synthesis changes the cell rigidity: E. coli with thickened cell walls accumulated only 25% PHB while cell wall weakened E. coli produced 93% PHB. Manipulation on cell wall synthesis mechanism adds another possibility to morphology engineering of microorganisms. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Salt stress causes cell wall damage in yeast cells lacking mitochondrial DNA.

PubMed

Gao, Qiuqiang; Liou, Liang-Chun; Ren, Qun; Bao, Xiaoming; Zhang, Zhaojie

2014-03-03

The yeast cell wall plays an important role in maintaining cell morphology, cell integrity and response to environmental stresses. Here, we report that salt stress causes cell wall damage in yeast cells lacking mitochondrial DNA (ρ 0 ). Upon salt treatment, the cell wall is thickened, broken and becomes more sensitive to the cell wall-perturbing agent sodium dodecyl sulfate (SDS). Also, SCW11 mRNA levels are elevated in ρ 0 cells. Deletion of SCW11 significantly decreases the sensitivity of ρ 0 cells to SDS after salt treatment, while overexpression of SCW11 results in higher sensitivity. In addition, salt stress in ρ 0 cells induces high levels of reactive oxygen species (ROS), which further damages the cell wall, causing cells to become more sensitive towards the cell wall-perturbing agent.

Cloning and functional characterization of a fatty acid transport protein (FATP) from the pheromone gland of a lichen moth, Eilema japonica, which secretes an alkenyl sex pheromone.

PubMed

Qian, Shuguang; Fujii, Takeshi; Ito, Katsuhiko; Nakano, Ryo; Ishikawa, Yukio

2011-01-01

Sex pheromones of moths are largely classified into two types based on the presence (Type I) or absence (Type II) of a terminal functional group. While Type-I sex pheromones are synthesized from common fatty acids in the pheromone gland (PG), Type-II sex pheromones are derived from hydrocarbons produced presumably in the oenocytes and transported to the PG via the hemolymph. Recently, a fatty acid transport protein (BmFATP) was identified from the PG of the silkworm Bombyx mori, which produces a Type-I sex pheromone (bombykol). BmFATP was shown to facilitate the uptake of extracellular fatty acids into PG cells for the synthesis of bombykol. To elucidate the presence and function of FATP in the PG of moths that produce Type-II sex pheromones, we explored fatp homologues expressed in the PG of a lichen moth, Eilema japonica, which secretes an alkenyl sex pheromone (Type II). A fatp homologue cloned from E. japonica (Ejfatp) was predominantly expressed in the PG, and its expression is upregulated shortly after eclosion. Functional expression of EjFATP in Escherichia coli enhanced the uptake of long chain fatty acids (C₁₈ and C₂₀), but not pheromone precursor hydrocarbons. To the best of our knowledge, this is the first report of the cloning and functional characterization of a FATP in the PG of a moth producing a Type-II sex pheromone. Although EjFATP is not likely to be involved in the uptake of pheromone precursors in E. japonica, the expression pattern of Ejfatp suggests a role for EjFATP in the PG not directly linked to pheromone biosynthesis. Copyright © 2010 Elsevier Ltd. All rights reserved.

Serological levels of apoptotic bodies, sFAS and TNF in lupus erythematosus.

PubMed

Alecu, M; Coman, G; Alecu, S

In our study we have investigated the presence of apoptotic bodies, soluble FAS receptor and TNF (tumor necrosis factor) in three clinical forms of lupus erythematosus. Determinations were performed in attack period of: systemic lupus erythematosus (SLE) for 20 patients, 20 patients with subacute cutaneous lupus erythematosus (SCLE), 20 patients with chronic discoid lupus erythematosus (DLE). Determinations were performed by ELISA (for apoptotic bodies, kit Boehringer, normal values 400-800 mU), (for sFAS, kit R&D Systems, normal values 4500-17000 pg/ml) (for TNF, ELISA kit R&D Systems, normal values 0.4-3.6 pg/ml). Results in SLE: apoptotic bodies were increased in 16 cases (980-1030); sFAS in 18 cases (17000-24000 pg/ml) TNF was increased in all 20 cases (40-140 pg/ml). In SCLE with multiple cutaneous lesions and without internal organs disturbance the apoptotic bodies were increased in 10 cases (960-1030 pg/ml), sFAS in 9 cases (17000-22000 pg/ml), and TNF alpha in 9 cases. In DLE, apoptotic bodies were increased in 2 patients (980-1010 pg/ml), sFAS in 3 patients (17000-20000 pg/ml) and TNF in 2 patients (20-40 pg/mil). Investigated values were slightly correlated with immune parameters (anti dsDNA antibodies), but they were correlated with the presence of renal disturbances or extension of cutaneous lesions. We consider that the presence of increased apoptotic bodies as a result of peripheral mononuclear cells apoptosis appear as a nauto-limiting mechanism in a pathological immune response. The increase of sFAS in lupus patients serum might be interpreted as an alteration of apoptosis respectively a deficit in apoptosis which has as a first consequence the persistence of B and T lymphocytes, activated, in the pathogen immune response.

Characterization of PgPepO, a bacterial homologue of endothelin-converting enzyme-1.

PubMed

Carson, Julie A; Ansai, Toshihiro; Awano, Shuji; Yu, Weixian; Takehara, Tadamichi; Turner, Anthony J

2002-08-01

PgPepO is a homologue of endothelin-converting enzyme-1 (ECE-1), with which it shares 31% identity. PgPepO was isolated from the periodontal pathogen Porphyromonas gingivalis. Recent studies have suggested a link between periodontal and cardiovascular disease, and several groups have suggested that bacterial and viral infections may contribute to the latter. P. gingivalis possesses the ability to invade, and multiply within, aortic endothelial cells and has been localized to atherosclerotic plaques. PgPepO was expressed and purified to homogeneity and we have begun detailed functional analysis, in terms of substrate preference and inhibitor specificity, in order to provide active-site comparisons with other members of the neprilysin (NEP)/ECE family. PgPepO possesses similar substrate specificity to ECE-1 and has been shown to cleave big endothelin-1 (big ET-1), big ET-2 and big ET-3, converting the substrates into their respective mature endothelin peptides. Substance P, angiotensin I, angiotensin II and neurotensin are all cleaved at multiple sites by PgPepO and the kinetics of these reactions have been compared. The potent vasoconstrictor urotensin II is not hydrolysed by PgPepO. Cleavage of bradykinin by PgPepO occurs at the Pro(7)-Phe(8) bond and is inhibited by the NEP and ECE-1 inhibitor phosphoramidon in a pH-dependent fashion (IC(50) =10 microM at pH 7.0) but not by thiorphan, an NEP-specific inhibitor. PgPepO activity is completely inhibited by EDTA. Characterization of this enzyme is important in elucidating possible links between periodontal pathogens and cardiovascular disorders such as atherosclerosis, and provides an opportunity to gain structural information on a bacterial protein with striking similarity to human ECE-1.

Effect of the phosphogypsum amendment of saline and agricultural soils on growth, productivity and antioxidant enzyme activities of tomato (Solanum lycopersicum L.).

PubMed

Smaoui-Jardak, Mariem; Kriaa, Walid; Maalej, Mohamed; Zouari, Mohamed; Kamoun, Lotfi; Trabelsi, Wassim; Ben Abdallah, Ferjani; Elloumi, Nada

2017-10-01

The objective of this study was to investigate the effects of phosphogypsum (PG) amendment on the physiochemical proprieties of saline and agricultural soils along with the growth, productivity and antioxidant enzyme activities of tomato plants ( Solanum lycopersicum L.) grown on the amended soils under controlled conditions. Obtained results showed that the amendment of saline soil (H) by PG induced a decrease in pH as well as in electrical conductivity. However, for the non saline soil (MC), there was a decrease in pH associated with an increase in electrical conductivity. For both soils, PG amendment led to an increase in Calcium (Ca) and sodium (Na), and a decrease in potassium (K) in plant tissues. Cadmium (Cd), Zinc (Zn) and Chromium (Cr) contents in different parts of plants increased in proportion with PG concentration in the soils. Apart from Cd, all the analyzed metals in tomato fruit were found to be below the recommended maximum allowable concentration (MAC). Our results showed that PG application, at doses not exceeding 20%, seems to be beneficial for growth, photosynthetic activity and productivity of tomato plants as well as in decreasing salinity of saline soils. In these conditions, the use of PG could be a promising project for the rehabilitation of marginalized and saline ecosystems with either ornamental or non-fruit species. For both soils, a significant accumulation of MDA in shoots was detected, reflecting cell membrane damage especially when the PG amendment reached 20%. Beyond 20 and 40% PG, tomato plants developed an enzymatic antioxidant defense system in response to salinity and heavy metal stress. However, at 80% PG, enzymes activities were significantly inhibited.

Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls.

PubMed

Marcus, Susan E; Verhertbruggen, Yves; Hervé, Cécile; Ordaz-Ortiz, José J; Farkas, Vladimir; Pedersen, Henriette L; Willats, William G T; Knox, J Paul

2008-05-22

Molecular probes are required to detect cell wall polymers in-situ to aid understanding of their cell biology and several studies have shown that cell wall epitopes have restricted occurrences across sections of plant organs indicating that cell wall structure is highly developmentally regulated. Xyloglucan is the major hemicellulose or cross-linking glycan of the primary cell walls of dicotyledons although little is known of its occurrence or functions in relation to cell development and cell wall microstructure. Using a neoglycoprotein approach, in which a XXXG heptasaccharide of tamarind seed xyloglucan was coupled to BSA to produce an immunogen, we have generated a rat monoclonal antibody (designated LM15) to the XXXG structural motif of xyloglucans. The specificity of LM15 has been confirmed by the analysis of LM15 binding using glycan microarrays and oligosaccharide hapten inhibition of binding studies. The use of LM15 for the analysis of xyloglucan in the cell walls of tamarind and nasturtium seeds, in which xyloglucan occurs as a storage polysaccharide, indicated that the LM15 xyloglucan epitope occurs throughout the thickened cell walls of the tamarind seed and in the outer regions, adjacent to middle lamellae, of the thickened cell walls of the nasturtium seed. Immunofluorescence analysis of LM15 binding to sections of tobacco and pea stem internodes indicated that the xyloglucan epitope was restricted to a few cell types in these organs. Enzymatic removal of pectic homogalacturonan from equivalent sections resulted in the abundant detection of distinct patterns of the LM15 xyloglucan epitope across these organs and a diversity of occurrences in relation to the cell wall microstructure of a range of cell types. These observations support ideas that xyloglucan is associated with pectin in plant cell walls. They also indicate that documented patterns of cell wall epitopes in relation to cell development and cell differentiation may need to be re-considered in relation to the potential masking of cell wall epitopes by other cell wall components.

The Interplay between Cell Wall Mechanical Properties and the Cell Cycle in Staphylococcus aureus

PubMed Central

Bailey, Richard G.; Turner, Robert D.; Mullin, Nic; Clarke, Nigel; Foster, Simon J.; Hobbs, Jamie K.

2014-01-01

The nanoscale mechanical properties of live Staphylococcus aureus cells during different phases of growth were studied by atomic force microscopy. Indentation to different depths provided access to both local cell wall mechanical properties and whole-cell properties, including a component related to cell turgor pressure. Local cell wall properties were found to change in a characteristic manner throughout the division cycle. Splitting of the cell into two daughter cells followed a local softening of the cell wall along the division circumference, with the cell wall on either side of the division circumference becoming stiffer. Once exposed, the newly formed septum was found to be stiffer than the surrounding, older cell wall. Deeper indentations, which were affected by cell turgor pressure, did not show a change in stiffness throughout the division cycle, implying that enzymatic cell wall remodeling and local variations in wall properties are responsible for the evolution of cell shape through division. PMID:25468333

KRE5 Suppression Induces Cell Wall Stress and Alternative ER Stress Response Required for Maintaining Cell Wall Integrity in Candida glabrata

PubMed Central

Sasaki, Masato; Ito, Fumie; Aoyama, Toshio; Sato-Okamoto, Michiyo; Takahashi-Nakaguchi, Azusa; Chibana, Hiroji; Shibata, Nobuyuki

2016-01-01

The maintenance of cell wall integrity in fungi is required for normal cell growth, division, hyphae formation, and antifungal tolerance. We observed that endoplasmic reticulum stress regulated cell wall integrity in Candida glabrata, which possesses uniquely evolved mechanisms for unfolded protein response mechanisms. Tetracycline-mediated suppression of KRE5, which encodes a predicted UDP-glucose:glycoprotein glucosyltransferase localized in the endoplasmic reticulum, significantly increased cell wall chitin content and decreased cell wall β-1,6-glucan content. KRE5 repression induced endoplasmic reticulum stress-related gene expression and MAP kinase pathway activation, including Slt2p and Hog1p phosphorylation, through the cell wall integrity signaling pathway. Moreover, the calcineurin pathway negatively regulated cell wall integrity, but not the reduction of β-1,6-glucan content. These results indicate that KRE5 is required for maintaining both endoplasmic reticulum homeostasis and cell wall integrity, and that the calcineurin pathway acts as a regulator of chitin-glucan balance in the cell wall and as an alternative mediator of endoplasmic reticulum stress in C. glabrata. PMID:27548283

Imaging the Dynamics of Cell Wall Polymer Deposition in the Unicellular Model Plant, Penium margaritaceum.

PubMed

Domozych, David; Lietz, Anna; Patten, Molly; Singer, Emily; Tinaz, Berke; Raimundo, Sandra C

2017-01-01

The unicellular green alga, Penium margaritaceum, represents a novel and valuable model organism for elucidating cell wall dynamics in plants. This organism's cell wall contains several polymers that are highly similar to those found in the primary cell walls of land plants. Penium is easily grown in laboratory culture and is effectively manipulated in various experimental protocols including microplate assays and correlative microscopy. Most importantly, Penium can be live labeled with cell wall-specific antibodies or other probes and returned to culture where specific cell wall developmental events can be monitored. Additionally, live cells can be rapidly cryo-fixed and cell wall surface microarchitecture can be observed with variable pressure scanning electron microscopy. Here, we describe the methodology for maintaining Penium for experimental cell wall enzyme studies.

Cell walls of the dimorphic fungal pathogens Sporothrix schenckii and Sporothrix brasiliensis exhibit bilaminate structures and sloughing of extensive and intact layers

PubMed Central

Walker, Louise A.; Niño-Vega, Gustavo; Mora-Montes, Héctor M.; Neves, Gabriela W. P.; Villalobos-Duno, Hector; Barreto, Laura; Garcia, Karina; Franco, Bernardo; Martínez-Álvarez, José A.; Munro, Carol A.; Gow, Neil A. R.

2018-01-01

Sporotrichosis is a subcutaneous mycosis caused by pathogenic species of the Sporothrix genus. A new emerging species, Sporothrix brasiliensis, is related to cat-transmitted sporotrichosis and has severe clinical manifestations. The cell wall of pathogenic fungi is a unique structure and impacts directly on the host immune response. We reveal and compare the cell wall structures of Sporothrix schenckii and S. brasiliensis using high-pressure freezing electron microscopy to study the cell wall organization of both species. To analyze the components of the cell wall, we also used infrared and 13C and 1H NMR spectroscopy and the sugar composition was determined by quantitative high-performance anion-exchange chromatography. Our ultrastructural data revealed a bi-layered cell wall structure for both species, including an external microfibrillar layer and an inner electron-dense layer. The inner and outer layers of the S. brasiliensis cell wall were thicker than those of S. schenckii, correlating with an increase in the chitin and rhamnose contents. Moreover, the outer microfibrillar layer of the S. brasiliensis cell wall had longer microfibrils interconnecting yeast cells. Distinct from those of other dimorphic fungi, the cell wall of Sporothrix spp. lacked α-glucan component. Interestingly, glycogen α-particles were identified in the cytoplasm close to the cell wall and the plasma membrane. The cell wall structure as well as the presence of glycogen α-particles varied over time during cell culture. The structural differences observed in the cell wall of these Sporothrix species seemed to impact its uptake by monocyte-derived human macrophages. The data presented here show a unique cell wall structure of S. brasiliensis and S. schenckii during the yeast parasitic phase. A new cell wall model for Sporothrix spp. is therefore proposed that suggests that these fungi molt sheets of intact cell wall layers. This observation may have significant effects on localized and disseminated immunopathology. PMID:29522522

The 1-hour post-load glucose level is more effective than HbA1c for screening dysglycemia.

PubMed

Jagannathan, Ram; Sevick, Mary Ann; Fink, Dorothy; Dankner, Rachel; Chetrit, Angela; Roth, Jesse; Buysschaert, Martin; Bergman, Michael

2016-08-01

To assess the performance of HbA1c and the 1-h plasma glucose (PG ≥ 155 mg/dl; 8.6 mmol/l) in identifying dysglycemia based on the oral glucose tolerance test (OGTT) from a real-world clinical care setting. This was a diagnostic test accuracy study. For this analysis, we tested the HbA1c diagnostic criteria advocated by the American Diabetes Association (ADA 5.7-6.4 %) and International Expert Committee (IEC 6.0-6.4 %) against conventional OGTT criteria. We also tested the utility of 1-h PG ≥ mg/dl; 8.6 mmol/l. Prediabetes was defined according to ADA-OGTT guidelines. Spearman correlation tests were used to determine the relationships between HbA1c, 1-h PG with fasting, 2-h PG and indices of insulin sensitivity and β-cell function. The levels of agreement between diagnostic methods were ascertained using Cohen's kappa coefficient (Κ). Receiver operating characteristic (ROC) curve was used to analyze the performance of the HbA1c and 1-h PG test in identifying prediabetes considering OGTT as reference diagnostic criteria. The diagnostic properties of different HbA1c thresholds were contrasted by determining sensitivity, specificity and likelihood ratios (LR). Of the 212 high-risk individuals, 70 (33 %) were identified with prediabetes, and 1-h PG showed a stronger association with 2-h PG, insulin sensitivity index, and β-cell function than HbA1c (P < 0.05). Furthermore, the level of agreement between 1-h PG ≥ 155 mg/dl (8.6 mmol/l) and the OGTT (Κ[95 % CI]: 0.40[0.28-0.53]) diagnostic test was stronger than that of ADA-HbA1c criteria 0.1[0.03-0.16] and IEC criteria (0.17[0.04-0.30]). The ROC (AUC[95 % CI]) for HbA1c and 1-h PG were 0.65[0.57-0.73] and 0.79[0.72-0.85], respectively. Importantly, 1-h PG ≥ 155 mg/dl (8.6 mmol/l) showed good sensitivity (74.3 % [62.4-84.0]) and specificity 69.7 % [61.5-77.1]) with a LR of 2.45. The ability of 1-h PG to discriminate prediabetes was better than that of HbA1c (∆AUC: -0.14; Z value: 2.5683; P = 0.01022). In a real-world clinical practice setting, the 1-h PG ≥ 155 mg/dl (8.6 mmol/l) is superior for detecting high-risk individuals compared with HbA1c. Furthermore, HbA1c is a less precise correlate of insulin sensitivity and β-cell function than the 1-h PG and correlates poorly with the 2-h PG during the OGTT.

The effect of additives on release and in vitro skin retention of flavonoids from emulsion and gel semisolid formulations.

PubMed

Dyja, R; Jankowski, A

2017-08-01

To assess the effect of two different additives (propylene glycol (PG) and polyethylene glycol 400 (PEG 400)) on release and in vitro skin retention of quercetin and chrysin from semisolid bases (amphiphilic creams and acidic carbomer gels). For obtaining semisolid formulations, flavonoids were pre-dissolved in the liquid (PG or PEG 400) or directly suspended in the semisolid base. Three chrysin formulations ('cream 0', 'PG-cream' and 'PEG 400-cream') and five quercetin formulations ('cream 0', 'PG cream', 'PEG 400 cream', 'gel 0' and 'PG gel') were prepared. The release studies were carried out in Franz diffusion cells by means of a cellulose membrane. The porcine ear skin was used in in vitro skin retention studies. The dissolution was a prerequisite to increase the release rates of tested flavonoids from obtained semisolid formulations. The cumulative amount of chrysin released after 6 h from 'PEG 400 cream' containing partly dissolved form of that flavonoid was higher than that from 'cream 0' or 'PG cream' containing its suspended form. The formulations containing quercetin dissolved in PG ('PG cream', 'PG gel') or PEG 400 ('PEG 400 cream') exhibited higher release rates of that flavonoid than corresponding semisolid suspensions ('cream 0' or 'gel 0'). The effects of both liquid additives (PG and PEG 400) on the cumulative amount of quercetin released after 6 h were comparable. However, there was no correlation between the release rate and the skin retention. The amounts of the flavonoids found in the skin were strongly affected by the type of the used solvent. While PG increased the skin retention of both flavonoids, PEG 400 had no effect on chrysin skin retention and delayed quercetin skin absorption. The proper choice of the solvent added to the semisolid base is crucial for enhanced skin delivery of the tested flavonoids. PG is more efficient absorption promoter than PEG 400 of both chrysin and quercetin. © 2017 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Effects of chondroitin sulfate and interleukin-1beta on human chondrocyte cultures exposed to pressurization: a biochemical and morphological study.

PubMed

Nerucci, F; Fioravanti, A; Cicero, M R; Collodel, G; Marcolongo, R

2000-07-01

Objective This study investigated the in vitro effects of chondroitin sulfate (CS) on human articular chondrocytes cultivated in the presence or in the absence of interleukin-1beta (IL-1beta) during 10 days of culture with and without pressurization cycles. Design The effects of CS (10 and 100 microg/ml) with and without IL-1beta were assessed in the culture medium of cells exposed to pressurization cycles in the form of synusoidal waves (minimum pressure 1 Mpa, maximum pressure 5 Mpa) and a frequency of 0.25 Hz for 3 h by immunoenzymatic method on microplates for the quantitative measurement of human proteoglycans (PG). On the 4th and 10th day of culture the cells were used for morphological analysis by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Results The presence of IL-1beta determines a significant decrease in PG concentration measured in the culture medium. When the cells are cultured in the presence of IL-1beta and CS, a statistically significant restoration of PG levels is observed. Under pressurization conditions, we observed that PG concentration in the medium of cells presents a significant increase at baseline conditions, in the presence of IL-1beta+CS10 and IL-1beta+CS100, but not with IL-1beta alone. The results concerning metabolic evaluation are confirmed by the morphologic findings obtained by TEM and SEM. Conclusions These in vitro studies confirm the protective role of CS, which counteracts the IL-1beta induced effects and they confirm the importance of pressure on chondrocyte metabolism and morphology.

The receptor-like kinase AtVRLK1 regulates secondary cell wall thickening.

PubMed

Huang, Cheng; Zhang, Rui; Gui, Jinshan; Zhong, Yu; Li, Laigeng

2018-04-20

During the growth and development of land plants, some specialized cells, such as tracheary elements, undergo secondary cell wall thickening. Secondary cell walls contain additional lignin, compared with primary cell walls, thus providing mechanical strength and potentially improving defenses against pathogens. However, the molecular mechanisms that initiate wall thickening are unknown. In this study, we identified an Arabidopsis thaliana leucine-rich repeat receptor-like kinase, encoded by AtVRLK1 (Vascular-Related RLK 1), that is specifically expressed in cells undergoing secondary cell wall thickening. Suppression of AtVRLK1expression resulted in a range of phenotypes that included retarded early elongation of the inflorescence stem, shorter fibers, slower root growth, and shorter flower filaments. In contrast, upregulation of AtVRLK1 led to longer fiber cells, reduced secondary cell wall thickening in fiber and vessel cells, and defects in anther dehiscence. Molecular and cellular analyses showed that downregulation of AtVRLK1 promoted secondary cell wall thickening and upregulation of AtVRLK1 enhanced cell elongation and inhibited secondary cell wall thickening. We propose that AtVRLK1 functions as a signaling component in coordinating cell elongation and cell wall thickening during growth and development. {copyright, serif} 2018 American Society of Plant Biologists. All rights reserved.

Spatio-temporal diversification of the cell wall matrix materials in the developing stomatal complexes of Zea mays.

PubMed

Giannoutsou, E; Apostolakos, P; Galatis, B

2016-11-01

The matrix cell wall materials, in developing Zea mays stomatal complexes are asymmetrically distributed, a phenomenon appearing related to the local cell wall expansion and deformation, the establishment of cell polarity, and determination of the cell division plane. In cells of developing Zea mays stomatal complexes, definite cell wall regions expand determinately and become locally deformed. This differential cell wall behavior is obvious in the guard cell mother cells (GMCs) and the subsidiary cell mother cells (SMCs) that locally protrude towards the adjacent GMCs. The latter, emitting a morphogenetic stimulus, induce polarization/asymmetrical division in SMCs. Examination of immunolabeled specimens revealed that homogalacturonans (HGAs) with a high degree of de-esterification (2F4- and JIM5-HGA epitopes) and arabinogalactan proteins are selectively distributed in the extending and deformed cell wall regions, while their margins are enriched with rhamnogalacturonans (RGAs) containing highly branched arabinans (LM6-RGA epitope). In SMCs, the local cell wall matrix differentiation constitutes the first structural event, indicating the establishment of cell polarity. Moreover, in the premitotic GMCs and SMCs, non-esterified HGAs (2F4-HGA epitope) are preferentially localized in the cell wall areas outlining the cytoplasm where the preprophase band is formed. In these areas, the forthcoming cell plate fuses with the parent cell walls. These data suggest that the described heterogeneity in matrix cell wall materials is probably involved in: (a) local cell wall expansion and deformation, (b) the transduction of the inductive GMC stimulus, and (c) the determination of the division plane in GMCs and SMCs.

Flow cytometric assay to assess short-term effects of personal care products on the marine microalga Tetraselmis suecica.

PubMed

Seoane, Marta; Esperanza, Marta; Rioboo, Carmen; Herrero, Concepción; Cid, Ángeles

2017-03-01

Large quantities of personal care products (PCPs) are used daily and many of their chemical ingredients are subsequently released into marine environments. Cultures of the marine microalga Tetraselmis suecica were exposed for 24 h to three emerging compounds included in the main classes of PCPs: the UV filter benzophenone-3 (BP-3), the disinfectant triclosan (TCS) and the fragrance tonalide (AHTN). Concentrations tested, expressed as cellular quota (pg cell -1 ), ranged from 5 to 40 for BP-3, from 2 to 16 for TCS and from 1.2 to 2.4 for AHTN. A small cytometric panel was carried out to evaluate key cytotoxicity biomarkers including inherent cell properties, growth and metabolic activity and cytoplasmic membrane properties. BP-3 caused a significant increase in growth rate, metabolic activity and chlorophyll a fluorescence from 10 pg cell -1 . However, growth and esterase activity decreased in cells exposed to all TCS and AHTN concentrations, except the lowest ones. Also these two compounds provoked a significant swelling of cells, more pronounced in the case of TCS-exposed cells. Although all treated cells remained viable, changes in membrane potential were observed. BP-3 and AHTN caused a significant depolarization of cells from 10 to 1.6 pg cell -1 , respectively; however all TCS concentrations assayed caused a noticeable hyperpolarization of cells. Metabolic activity and cytoplasmic membrane potential were the most sensitive parameters. It can be concluded that the toxicological model used and the toxicological parameters evaluated are suitable to assess the toxicity of these emerging contaminants. Copyright © 2016 Elsevier Ltd. All rights reserved.

Immunogold scanning electron microscopy can reveal the polysaccharide architecture of xylem cell walls

PubMed Central

Sun, Yuliang; Juzenas, Kevin

2017-01-01

Abstract Immunofluorescence microscopy (IFM) and immunogold transmission electron microscopy (TEM) are the two main techniques commonly used to detect polysaccharides in plant cell walls. Both are important in localizing cell wall polysaccharides, but both have major limitations, such as low resolution in IFM and restricted sample size for immunogold TEM. In this study, we have developed a robust technique that combines immunocytochemistry with scanning electron microscopy (SEM) to study cell wall polysaccharide architecture in xylem cells at high resolution over large areas of sample. Using multiple cell wall monoclonal antibodies (mAbs), this immunogold SEM technique reliably localized groups of hemicellulosic and pectic polysaccharides in the cell walls of five different xylem structures (vessel elements, fibers, axial and ray parenchyma cells, and tyloses). This demonstrates its important advantages over the other two methods for studying cell wall polysaccharide composition and distribution in these structures. In addition, it can show the three-dimensional distribution of a polysaccharide group in the vessel lateral wall and the polysaccharide components in the cell wall of developing tyloses. This technique, therefore, should be valuable for understanding the cell wall polysaccharide composition, architecture and functions of diverse cell types. PMID:28398585

Building a plant cell wall at a glance.

PubMed

Lampugnani, Edwin R; Khan, Ghazanfar Abbas; Somssich, Marc; Persson, Staffan

2018-01-29

Plant cells are surrounded by a strong polysaccharide-rich cell wall that aids in determining the overall form, growth and development of the plant body. Indeed, the unique shapes of the 40-odd cell types in plants are determined by their walls, as removal of the cell wall results in spherical protoplasts that are amorphic. Hence, assembly and remodeling of the wall is essential in plant development. Most plant cell walls are composed of a framework of cellulose microfibrils that are cross-linked to each other by heteropolysaccharides. The cell walls are highly dynamic and adapt to the changing requirements of the plant during growth. However, despite the importance of plant cell walls for plant growth and for applications that we use in our daily life such as food, feed and fuel, comparatively little is known about how they are synthesized and modified. In this Cell Science at a Glance article and accompanying poster, we aim to illustrate the underpinning cell biology of the synthesis of wall carbohydrates, and their incorporation into the wall, in the model plant Arabidopsis . © 2018. Published by The Company of Biologists Ltd.

Cell wall evolution and diversity

PubMed Central

Fangel, Jonatan U.; Ulvskov, Peter; Knox, J. P.; Mikkelsen, Maria D.; Harholt, Jesper; Popper, Zoë A.; Willats, William G.T.

2012-01-01

Plant cell walls display a considerable degree of diversity in their compositions and molecular architectures. In some cases the functional significance of a particular cell wall type appears to be easy to discern: secondary cells walls are often reinforced with lignin that provides durability; the thin cell walls of pollen tubes have particular compositions that enable their tip growth; lupin seed cell walls are characteristically thickened with galactan used as a storage polysaccharide. However, more frequently the evolutionary mechanisms and selection pressures that underpin cell wall diversity and evolution are unclear. For diverse green plants (chlorophytes and streptophytes) the rapidly increasing availability of transcriptome and genome data sets, the development of methods for cell wall analyses which require less material for analysis, and expansion of molecular probe sets, are providing new insights into the diversity and occurrence of cell wall polysaccharides and associated biosynthetic genes. Such research is important for refining our understanding of some of the fundamental processes that enabled plants to colonize land and to subsequently radiate so comprehensively. The study of cell wall structural diversity is also an important aspect of the industrial utilization of global polysaccharide bio-resources. PMID:22783271

Analysis of a novel autonomous marine hybrid power generation/energy storage system with a high-voltage direct current link

NASA Astrophysics Data System (ADS)

Wang, Li; Lee, Dong-Jing; Lee, Wei-Jen; Chen, Zhe

This paper presents both time-domain and frequency-domain simulated results of a novel marine hybrid renewable-energy power generation/energy storage system (PG/ESS) feeding isolated loads through an high-voltage direct current (HVDC) link. The studied marine PG subsystems comprise both offshore wind turbines and Wells turbines to respectively capture wind energy and wave energy from marine wind and ocean wave. In addition to wind-turbine generators (WTGs) and wave-energy turbine generators (WETGs) employed in the studied system, diesel-engine generators (DEGs) and an aqua electrolyzer (AE) absorbing a part of generated energy from WTGs and WETGs to generate available hydrogen for fuel cells (FCs) are also included in the PG subsystems. The ES subsystems consist of a flywheel energy storage system (FESS) and a compressed air energy storage (CAES) system to balance the required energy in the hybrid PG/ESS. It can be concluded from the simulation results that the proposed hybrid marine PG/ESS feeding isolated loads can stably operate to achieve system power-frequency balance condition.

DNA content of hepatocyte and erythrocyte nuclei of the spined loach (Cobitis taenia L.) and its polyploid forms.

PubMed

Juchno, Dorota; Lackowska, Bozena; Boron, Alicja; Kilarski, Wincenty

2010-09-01

We analyzed the DNA content of hepatocyte and erythrocyte nuclei of the spined loach Cobitis taenia (diploid) and its allopolyploid forms. Twenty triploid females and one tetraploid were used. At least 20,000 hepatocyte and erythrocyte nuclei were acquired and analyzed by flow cytometry. C. taenia erythrocyte nuclei contain 3.15 +/- 0.21 pg of DNA and the hepatocyte nuclei 4.45 +/- 0.46 pg of DNA. Triploid Cobitis have 5.08 +/- 0.41 pg of DNA in erythrocyte nuclei and 6.11 +/- 0.40 pg of DNA in hepatocyte nuclei, whereas the tetraploid erythrocyte and hepatocyte nuclei contained 6.60 and 7.40 pg of DNA, respectively. In general, the DNA contents correlate positively with the ploidy level of the fish investigated. The DNA content variation in the hepatocyte and erythrocyte nuclei may be due to differences in extent of chromatin condensation, which is more pronounced in the erythrocyte than hepatocyte nuclei, or to the several orders of ploidy that occur in the parenchymal liver cells.

Anorganic bovine bone and a silicate-based synthetic bone activate different microRNAs.

PubMed

Annalisa, Palmieri; Furio, Pezzetti; Ilaria, Zollino; Anna, Avantaggiato; Luca, Scapoli; Marcella, Martinelli; Marzia, Arlotti; Elena, Masiero; Carinci, Francesco

2008-09-01

Bio-Oss (BO), composed of anorganic bovine bone, is widely used in several bone regeneration procedures in oral surgery. PerioGlas (PG) is an alloplastic material that has been used for grafting of periodontal osseous defects since the 1990s. However, how these biomaterials alter osteoblast activity to promote bone formation is poorly understood. We attempted to address this question by using microRNA microarray techniques to investigate differences in translational regulation in osteoblasts exposed to BO and PG. By using miRNA microarrays containing 329 probes designed from human miRNA sequences, we investigated miRNAs whose expression was significantly modified in an osteoblast-like cell line (MG-63) cultured with BO vs PG. Three up-regulated miRNAs (mir-337, mir-200b, mir-377) and 4 down-regulated miRNAs (mir-130a, mir-214, mir-27a, mir-93) were identified. Our results indicated that BO and PG act on different miRNAs. Globally, PG causes activation of bone-forming signaling, whereas BO also activates cartilage-related pathways.

A simple, specific high-throughput enzyme-linked immunosorbent assay (ELISA) for quantitative determination of melatonin in cell culture medium.

PubMed

Li, Ye; Cassone, Vincent M

2015-09-01

A simple, specific, high-throughput enzyme-linked immunosorbent assay (ELISA) for quantitative determination of melatonin was developed for directly measuring melatonin in cell culture medium with 10% FBS. This assay adopts a commercial monoclonal melatonin antibody and melatonin-HRP conjugate, so it can be applied in multiple labs rapidly with low cost compared with commercial RIA and ELISA kits. In addition, the procedure is much simpler with only four steps: 1) sample/conjugate incubation, 2) plate washing, 3) TMB color reaction and 4) reading of results. The standards of the assay cover a wide working range from 100 pg/mL to 10 ng/mL. The sensitivity was 68 pg/mL in cell culture medium with 10% FBS and 26 pg/mL in PBS with as little as 25 μL sample volume. The recovery of melatonin from cell culture medium was 101.0%. The principal cross-reacting compound was 5-methoxytryptophol (0.1%). The variation coefficients of the assay, within and between runs, ranged between 6.68% and 15.76% in cell culture medium. The mean linearity of a series diluted cell culture medium sample was 105% (CV=5%), ranging between 98% and 111%, y=5.5263x+0.0646, R(2)=0.99. The assay enables small research and teaching labs to reliably measure this important neurohormone. Copyright © 2015 Elsevier B.V. All rights reserved.

Vaccine to Control the Viral Infection of Fish.

DOEpatents

Leong, JoAnn Ching

1994-10-11

Subunit vaccines and their use for immunizing fish against infection by viruses are disclosed. In particular, plasmid pG8 is constructed by joining, with the plasmid pUC8, DNA which encodes the glycoprotein of infectious hematopoietic necrosis virus (IHNV). E. coli cells are transformed by pG8, whereby pure viral antigen is produced to provide a vaccine for the control of IHNV in fish. 10 figs.

Distribution of alginate and cellulose and regulatory role of calcium in the cell wall of the brown alga Ectocarpus siliculosus (Ectocarpales, Phaeophyceae).

PubMed

Terauchi, Makoto; Nagasato, Chikako; Inoue, Akira; Ito, Toshiaki; Motomura, Taizo

2016-08-01

This work investigated a correlation between the three-dimensional architecture and compound-components of the brown algal cell wall. Calcium greatly contributes to the cell wall integrity. Brown algae have a unique cell wall consisting of alginate, cellulose, and sulfated polysaccharides. However, the relationship between the architecture and the composition of the cell wall is poorly understood. Here, we investigated the architecture of the cell wall and the effect of extracellular calcium in the sporophyte and gametophyte of the model brown alga, Ectocarpus siliculosus (Dillwyn) Lyngbye, using transmission electron microscopy, histochemical, and immunohistochemical studies. The lateral cell wall of vegetative cells of the sporophyte thalli had multilayered architecture containing electron-dense and negatively stained fibrils. Electron tomographic analysis showed that the amount of the electron-dense fibrils and the junctions was different between inner and outer layers, and between the perpendicular and tangential directions of the cell wall. By immersing the gametophyte thalli in the low-calcium (one-eighth of the normal concentration) artificial seawater medium, the fibrous layers of the lateral cell wall of vegetative cells became swollen. Destruction of cell wall integrity was also induced by the addition of sorbitol. The results demonstrated that electron-dense fibrils were composed of alginate-calcium fibrous gels, and electron negatively stained fibrils were crystalline cellulose microfibrils. It was concluded that the spatial arrangement of electron-dense fibrils was different between the layers and between the directions of the cell wall, and calcium was necessary for maintaining the fibrous layers in the cell wall. This study provides insights into the design principle of the brown algal cell wall.

Cell Wall Composition and Candidate Biosynthesis Gene Expression During Rice Development.

PubMed

Lin, Fan; Manisseri, Chithra; Fagerström, Alexandra; Peck, Matthew L; Vega-Sánchez, Miguel E; Williams, Brian; Chiniquy, Dawn M; Saha, Prasenjit; Pattathil, Sivakumar; Conlin, Brian; Zhu, Lan; Hahn, Michael G; Willats, William G T; Scheller, Henrik V; Ronald, Pamela C; Bartley, Laura E

2016-10-01

Cell walls of grasses, including cereal crops and biofuel grasses, comprise the majority of plant biomass and intimately influence plant growth, development and physiology. However, the functions of many cell wall synthesis genes, and the relationships among and the functions of cell wall components remain obscure. To better understand the patterns of cell wall accumulation and identify genes that act in grass cell wall biosynthesis, we characterized 30 samples from aerial organs of rice (Oryza sativa cv. Kitaake) at 10 developmental time points, 3-100 d post-germination. Within these samples, we measured 15 cell wall chemical components, enzymatic digestibility and 18 cell wall polysaccharide epitopes/ligands. We also used quantitative reverse transcription-PCR to measure expression of 50 glycosyltransferases, 15 acyltransferases and eight phenylpropanoid genes, many of which had previously been identified as being highly expressed in rice. Most cell wall components vary significantly during development, and correlations among them support current understanding of cell walls. We identified 92 significant correlations between cell wall components and gene expression and establish nine strong hypotheses for genes that synthesize xylans, mixed linkage glucan and pectin components. This work provides an extensive analysis of cell wall composition throughout rice development, identifies genes likely to synthesize grass cell walls, and provides a framework for development of genetically improved grasses for use in lignocellulosic biofuel production and agriculture. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Identification of an HIV-1 Clade A Envelope That Exhibits Broad Antigenicity and Neutralization Sensitivity and Elicits Antibodies Targeting Three Distinct Epitopes

PubMed Central

Hoffenberg, Simon; Powell, Rebecca; Carpov, Alexei; Wagner, Denise; Wilson, Aaron; Kosakovsky Pond, Sergei; Lindsay, Ross; Arendt, Heather; DeStefano, Joanne; Phogat, Sanjay; Poignard, Pascal; Fling, Steven P.; Simek, Melissa; LaBranche, Celia; Montefiori, David; Wrin, Terri; Phung, Pham; Burton, Dennis; Koff, Wayne; King, C. Richter; Parks, Christopher L.

2013-01-01

Broadly neutralizing antibodies (bNAbs) PG9 and PG16 were isolated from an International AIDS Vaccine Initiative (IAVI) Protocol G subject infected with human immunodeficiency virus type 1 (HIV-1) clade A. Both antibodies are highly potent and neutralize greater than 70% of viruses tested. We sought to begin immunogen design based on viral sequences from this patient; however, pseudoviruses prepared with 19 envelope sequences from this subject were resistant to neutralization by PG9 and PG16. Therefore, we used a bioinformatics approach to identify closely related viruses that were potentially sensitive to PG9 and PG16. A most-recent common ancestor (MRCA) sequence for the viral envelope (Env) was determined and aligned with 99 subtype A gp160 sequences from the Los Alamos HIV database. Virus BG505.W6M.ENV.C2 (BG505) was found to have the highest degree of homology (73%) to the MRCA sequence. Pseudoviruses prepared with this Env were sensitive to neutralization with a broad panel of bNAbs, including PG9 and PG16. When expressed by 293T cells as soluble gp120, the BG505 monomer bound well to both PG9 and PG16. We further showed that a point mutation (L111A) enabled more efficient production of a stable gp120 monomer that preserves the major neutralization epitopes. Finally, we showed that an adjuvanted formulation of this gp120 protein elicited neutralizing antibodies in rabbits (following a gp120 DNA vaccine prime) and that the antisera competed with bNAbs from 3 classes of nonoverlapping epitopes. Thus, the BG505 Env protein warrants further investigation as an HIV vaccine candidate, as a stand-alone protein, or as a component of a vaccine vector. PMID:23468492

Screening and characterization of plant cell walls using carbohydrate microarrays.

PubMed

Sørensen, Iben; Willats, William G T

2011-01-01

Plant cells are surrounded by cell walls built largely from complex carbohydrates. The primary walls of growing plant cells consist of interdependent networks of three polysaccharide classes: cellulose, cross-linking glycans (also known as hemicelluloses), and pectins. Cellulose microfibrils are tethered together by cross-linking glycans, and this assembly forms the major load-bearing component of primary walls, which is infiltrated with pectic polymers. In the secondary walls of woody tissues, pectins are much reduced and walls are reinforced with the phenolic polymer lignin. Plant cell walls are essential for plant life and also have numerous industrial applications, ranging from wood to nutraceuticals. Enhancing our knowledge of cell wall biology and the effective use of cell wall materials is dependent to a large extent on being able to analyse their fine structures. We have developed a suite of techniques based on microarrays probed with monoclonal antibodies with specificity for cell wall components, and here we present practical protocols for this type of analysis.

Characteristic thickened cell walls of the bracts of the 'eternal flower' Helichrysum bracteatum.

PubMed

Nishikawa, Kuniko; Ito, Hiroaki; Awano, Tatsuya; Hosokawa, Munetaka; Yazawa, Susumu

2008-07-01

Helichrysum bracteatum is called an 'eternal flower' and has large, coloured, scarious bracts. These maintain their aesthetic value without wilting or discoloration for many years. There have been no research studies of cell death or cell morphology of the scarious bract, and hence the aim of this work was to elucidate these characteristics for the bract of H. bracteatum. DAPI (4'6-diamidino-2-phenylindol dihydrochloride) staining and fluorescence microscopy were used for observation of cell nuclei. Light microscopy (LM), transmission electron microscopy (TEM) and polarized light microscopy were used for observation of cells, including cell wall morphology. Cell death occurred at the bract tip during the early stage of flower development. The cell wall was the most prominent characteristic of H. bracteatum bract cells. Characteristic thickened secondary cell walls on the inside of the primary cell walls were observed in both epidermal and inner cells. In addition, the walls of all cells exhibited birefringence. Characteristic thickened secondary cell walls have orientated cellulose microfibrils as well as general secondary cell walls of the tracheary elements. For comparison, these characters were not observed in the petal and bract tissues of Chrysanthemum morifolium. Bracts at anthesis are composed of dead cells. Helichrysum bracteatum bracts have characteristic thickened secondary cell walls that have not been observed in the parenchyma of any other flowers or leaves. The cells of the H. bracteatum bract differ from other tissues with secondary cell walls, suggesting that they may be a new cell type.

Peptidoglycan turnover and recycling in Gram-positive bacteria.

PubMed

Reith, Jan; Mayer, Christoph

2011-10-01

Bacterial cells are protected by an exoskeleton, the stabilizing and shape-maintaining cell wall, consisting of the complex macromolecule peptidoglycan. In view of its function, it could be assumed that the cell wall is a static structure. In truth, however, it is steadily broken down by peptidoglycan-cleaving enzymes during cell growth. In this process, named cell wall turnover, in one generation up to half of the preexisting peptidoglycan of a bacterial cell is released from the wall. This would result in a massive loss of cell material, if turnover products were not be taken up and recovered. Indeed, in the Gram-negative model organism Escherichia coli, peptidoglycan recovery has been recognized as a complex pathway, named cell wall recycling. It involves about a dozen dedicated recycling enzymes that convey cell wall turnover products to peptidoglycan synthesis or energy pathways. Whether Gram-positive bacteria also recover their cell wall is currently questioned. Given the much larger portion of peptidoglycan in the cell wall of Gram-positive bacteria, however, recovery of the wall material would provide an even greater benefit in these organisms compared to Gram-negatives. Consistently, in many Gram-positives, orthologs of recycling enzymes were identified, indicating that the cell wall may also be recycled in these organisms. This mini-review provides a compilation of information about cell wall turnover and recycling in Gram-positive bacteria during cell growth and division, including recent findings relating to muropeptide recovery in Bacillus subtilis and Clostridium acetobutylicum from our group. Furthermore, the impact of cell wall turnover and recycling on biotechnological processes is discussed.

RADIOAUTOGRAPHIC STUDY OF CELL WALL DEPOSITION IN GROWING PLANT CELLS

PubMed Central

Ray, Peter M.

1967-01-01

Segments cut from growing oat coleoptiles and pea stems were fed glucose-3H in presence and absence of the growth hormone indoleacetic acid (IAA). By means of electron microscope radioautography it was demonstrated that new cell wall material is deposited both at the wall surface (apposition) and within the preexisting wall structure (internally). Quantitative profiles for the distribution of incorporation with position through the thickness of the wall were obtained for the thick outer wall of epidermal cells. With both oat coleoptile and pea stem epidermal outer walls, it was found that a larger proportion of the newly synthesized wall material appeared to become incorporated within the wall in the presence of IAA. Extraction experiments on coleoptile tissue showed that activity that had been incorporated into the cell wall interior represented noncellulosic constituents, mainly hemicelluloses, whereas cellulose was deposited largely or entirely by apposition. It seems possible that internal incorporation of hemicelluloses plays a role in the cell wall expansion process that is involved in cell growth. PMID:6064369

Interactions of Condensed Tannins with Saccharomyces cerevisiae Yeast Cells and Cell Walls: Tannin Location by Microscopy.

PubMed

Mekoue Nguela, Julie; Vernhet, Aude; Sieczkowski, Nathalie; Brillouet, Jean-Marc

2015-09-02

Interactions between grape tannins/red wine polyphenols and yeast cells/cell walls was previously studied within the framework of red wine aging and the use of yeast-derived products as an alternative to aging on lees. Results evidenced a quite different behavior between whole cells (biomass grown to elaborate yeast-derived products, inactivated yeast, and yeast inactivated after autolysis) and yeast cell walls (obtained from mechanical disruption of the biomass). Briefly, whole cells exhibited a high capacity to irreversibly adsorb grape and wine tannins, whereas only weak interactions were observed for cell walls. This last point was quite unexpected considering the literature and called into question the real role of cell walls in yeasts' ability to fix tannins. In the present work, tannin location after interactions between grape and wine tannins and yeast cells and cell walls was studied by means of transmission electron microscopy, light epifluorescence, and confocal microscopy. Microscopy observations evidenced that if tannins interact with cell walls, and especially cell wall mannoproteins, they also diffuse freely through the walls of dead cells to interact with their plasma membrane and cytoplasmic components.

Synthetic Poly(L-Glutamic Acid)-conjugated CpG Exhibits Antitumor Efficacy With Increased Retention in Tumor and Draining Lymph Nodes After Intratumoral Injection in a Mouse Model of Melanoma.

PubMed

Ma, Qing; Zhou, Dapeng; DeLyria, Elizabeth S; Wen, Xiaoxia; Lu, Wei; Thapa, Prakash; Liu, Chengwen; Li, Dan; Bassett, Roland L; Overwijk, Willem W; Hwu, Patrick; Li, Chun

2017-01-01

There is an urgent need for new clinically applicable drug-delivery methods to enhance accumulation of immune-activating drugs in tumors. We synthesized a poly(L-glutamic acid)-CpG ODN2216 conjugate (PG-CpG) and injected it intratumorally into C57BL/6 mice bearing subcutaneous B16-ovalbumin melanoma. PG-CpG elicited the same potent antitumoral activity as CpG with respect to reducing tumor growth and triggering antigen-specific CD8 T-cell responses in this well-established solid tumor model. Moreover, PG-CpG was retained significantly longer in both tumor and draining lymph nodes than was free CpG after intratumoral injection. Specifically, 48 hours after injection, 26.5%±16.9% of the injected PG-CpG dose versus 4.72%±2.61% of free CpG remained at the tumor, and 1.53%±1.22% of the injected PG-CpG versus 0.37%±0.33% of free CpG was retained in the draining inguinal lymph nodes. These findings indicate that PG is an effective synthetic polymeric carrier for delivery of immunostimulatory agents to tumors and lymph nodes.

Impaired Photosynthesis in Phosphatidylglycerol-Deficient Mutant of Cyanobacterium Anabaena sp. PCC7120 with a Disrupted Gene Encoding a Putative Phosphatidylglycerophosphatase1

PubMed Central

Wu, Feng; Yang, Zhenle; Kuang, Tingyun

2006-01-01

Phosphatidylglycerol (PG) is a ubiquitous phospholipid in thylakoid membranes of cyanobacteria and chloroplasts and plays an important role in the structure and function of photosynthetic membranes. The last step of the PG biosynthesis is dephosphorylation of phosphatidylglycerophosphate (PGP) catalyzed by PGP phosphatase. However, the gene-encoding PGP phosphatase has not been identified and cloned from cyanobacteria or higher plants. In this study, we constructed a PG-deficient mutant from cyanobacterium Anabaena sp. PCC7120 with a disrupted gene (alr1715, a gene for Alr1715 protein, GenBank accession no. BAB78081) encoding a putative PGP phosphatase. The obtained mutant showed an approximately 30% reduction in the cellular content of PG. Following the reduction in the PG content, the photoautotrophical growth of the mutant was restrained, and the cellular content of chlorophyll was decreased. The decreases in net photosynthetic and photosystem II (PSII) activities on a cell basis also occurred in this mutant. Simultaneously, the photochemical efficiency of PSII was considerably declined, and less excitation energy was transferred toward PSII. These findings demonstrate that the alr1715 gene of Anabaena sp. PCC7120 is involved in the biosynthesis of PG and essential for photosynthesis. PMID:16815953

At the border: the plasma membrane-cell wall continuum.

PubMed

Liu, Zengyu; Persson, Staffan; Sánchez-Rodríguez, Clara

2015-03-01

Plant cells rely on their cell walls for directed growth and environmental adaptation. Synthesis and remodelling of the cell walls are membrane-related processes. During cell growth and exposure to external stimuli, there is a constant exchange of lipids, proteins, and other cell wall components between the cytosol and the plasma membrane/apoplast. This exchange of material and the localization of cell wall proteins at certain spots in the plasma membrane seem to rely on a particular membrane composition. In addition, sensors at the plasma membrane detect changes in the cell wall architecture, and activate cytoplasmic signalling schemes and ultimately cell wall remodelling. The apoplastic polysaccharide matrix is, on the other hand, crucial for preventing proteins diffusing uncontrollably in the membrane. Therefore, the cell wall-plasma membrane link is essential for plant development and responses to external stimuli. This review focuses on the relationship between the cell wall and plasma membrane, and its importance for plant tissue organization. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Plant and algal cell walls: diversity and functionality

PubMed Central

Popper, Zoë A.; Ralet, Marie-Christine; Domozych, David S.

2014-01-01

Background Although plants and many algae (e.g. the Phaeophyceae, brown, and Rhodophyceae, red) are only very distantly related they are united in their possession of carbohydrate-rich cell walls, which are of integral importance being involved in many physiological processes. Furthermore, wall components have applications within food, fuel, pharmaceuticals, fibres (e.g. for textiles and paper) and building materials and have long been an active topic of research. As shown in the 27 papers in this Special Issue, as the major deposit of photosynthetically fixed carbon, and therefore energy investment, cell walls are of undisputed importance to the organisms that possess them, the photosynthetic eukaryotes (plants and algae). The complexities of cell wall components along with their interactions with the biotic and abiotic environment are becoming increasingly revealed. Scope The importance of plant and algal cell walls and their individual components to the function and survival of the organism, and for a number of industrial applications, are illustrated by the breadth of topics covered in this issue, which includes papers concentrating on various plants and algae, developmental stages, organs, cell wall components, and techniques. Although we acknowledge that there are many alternative ways in which the papers could be categorized (and many would fit within several topics), we have organized them as follows: (1) cell wall biosynthesis and remodelling, (2) cell wall diversity, and (3) application of new technologies to cell walls. Finally, we will consider future directions within plant cell wall research. Expansion of the industrial uses of cell walls and potentially novel uses of cell wall components are both avenues likely to direct future research activities. Fundamentally, it is the continued progression from characterization (structure, metabolism, properties and localization) of individual cell wall components through to defining their roles in almost every aspect of plant and algal physiology that will present many of the major challenges in future cell wall research. PMID:25453142

Plant and algal cell walls: diversity and functionality.

PubMed

Popper, Zoë A; Ralet, Marie-Christine; Domozych, David S

2014-10-01

Although plants and many algae (e.g. the Phaeophyceae, brown, and Rhodophyceae, red) are only very distantly related they are united in their possession of carbohydrate-rich cell walls, which are of integral importance being involved in many physiological processes. Furthermore,wall components have applications within food, fuel, pharmaceuticals, fibres (e.g. for textiles and paper) and building materials and have long been an active topic of research. As shown in the 27 papers in this Special Issue, as the major deposit of photosynthetically fixed carbon, and therefore energy investment, cell walls are of undisputed importance to the organisms that possess them, the photosynthetic eukaryotes ( plants and algae). The complexities of cell wall components along with their interactions with the biotic and abiotic environment are becoming increasingly revealed. The importance of plant and algal cell walls and their individual components to the function and survival of the organism, and for a number of industrial applications, are illustrated by the breadth of topics covered in this issue, which includes papers concentrating on various plants and algae, developmental stages, organs, cell wall components, and techniques. Although we acknowledge that there are many alternative ways in which the papers could be categorized (and many would fit within several topics), we have organized them as follows: (1) cell wall biosynthesis and remodelling, (2) cell wall diversity, and (3) application of new technologies to cell walls. Finally, we will consider future directions within plant cell wall research. Expansion of the industrial uses of cell walls and potentially novel uses of cell wall components are both avenues likely to direct future research activities. Fundamentally, it is the continued progression from characterization (structure, metabolism, properties and localization) of individual cell wall components through to defining their roles in almost every aspect of plant and algal physiology that will present many of the major challenges in future cell wall research.

A molecular model of proteoglycan-associated electrostatic forces in cartilage mechanics.

PubMed

Buschmann, M D; Grodzinsky, A J

1995-05-01

Measured values of the swelling pressure of charged proteoglycans (PG) in solution (Williams RPW, and Comper WD; Biophysical Chemistry 36:223, 1990) and the ionic strength dependence of the equilibrium modulus of PG-rich articular cartilage (Eisenberg SR, and Grodzinsky AJ; J Orthop Res 3: 148, 1985) are compared to the predictions of two models. Each model is a representation of electrostatic forces arising from charge present on spatially fixed macromolecules and spatially mobile micro-ions. The first is a macroscopic continuum model based on Donnan equilibrium that includes no molecular-level structure and assumes that the electrical potential is spatially invariant within the polyelectrolyte medium (i.e. zero electric field). The second model is based on a microstructural, molecular-level solution of the Poisson-Boltzmann (PB) equation within a unit cell containing a charged glycosaminoglycan (GAG) molecule and its surrounding atmosphere of mobile ions. This latter approach accounts for the space-varying electrical potential and electrical field between the GAG constituents of the PG. In computations involving no adjustable parameters, the PB-cell model agrees with the measured pressure of PG solutions to within experimental error (10%), whereas the ideal Donnan model overestimates the pressure by up to 3-fold. In computations involving one adjustable parameter for each model, the PB-cell model predicts the ionic strength dependence of the equilibrium modulus of articular cartilage. Near physiological ionic strength, the Donnan model overpredicts the modulus data by 2-fold, but the two models coincide for low ionic strengths (C0 < 0.025M) where the spatially invariant Donnan potential is a closer approximation to the PB potential distribution. The PB-cell model result indicates that electrostatic forces between adjacent GAGs predominate in determining the swelling pressure of PG in the concentration range found in articular cartilage (20-80 mg/ml). The PB-cell model is also consistent with data (Eisenberg and Grodzinsky, 1985, Lai WM, Hou JS, and Mow VC; J Biomech Eng 113: 245, 1991) showing that these electrostatic forces account for approximately 1/2 (290kPa) the equilibrium modulus of cartilage at physiological ionic strength while absolute swelling pressures may be as low as approximately 25-100kPa. This important property of electrostatic repulsion between GAGs that are highly charged but spaced a few Debye lengths apart allows cartilage to resist compression (high modulus) without generating excessive intratissue swelling pressures.

Neuropeptides CRH, SP, HK-1, and Inflammatory Cytokines IL-6 and TNF Are Increased in Serum of Patients with Fibromyalgia Syndrome, Implicating Mast Cells

PubMed Central

Tsilioni, Irene; Russell, Irwin J.; Stewart, Julia M.; Gleason, Rae M.

2016-01-01

Fibromyalgia syndrome (FMS) is a chronic, idiopathic condition of widespread musculoskeletal pain affecting more women than men. Even though clinical studies have provided evidence of altered central pain pathways, the lack of definitive pathogenesis or reliable objective markers has hampered development of effective treatments. Here we report that the neuropeptides corticotropin-releasing hormone (CRH), substance P (SP), and SP-structurally-related hemokinin-1 (HK-1) were significantly (P = 0.026, P < 0.0001, and P = 0.002, respectively) elevated (0.82 ± 0.57 ng/ml, 0.39 ± 0.18 ng/ml, and 7.98 ± 3.12 ng/ml, respectively) in the serum of patients with FMS compared with healthy controls (0.49 ± 0.26 ng/ml, 0.12 ± 0.1 ng/ml, and 5.71 ± 1.08 ng/ml, respectively). Moreover, SP and HK-1 levels were positively correlated (Pearson r = 0.45, P = 0.002) in FMS. The serum concentrations of the inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF) were also significantly (P = 0.029 and P = 0.006, respectively) higher (2.97 ± 2.35 pg/ml and 0.92 ± 0.31 pg/ml, respectively) in the FMS group compared with healthy subjects (1.79 ± 0.62 pg/ml and 0.69 ± 0.16 pg/ml, respectively). In contrast, serum IL-31 and IL-33 levels were significantly lower (P = 0.0001 and P = 0.044, respectively) in the FMS patients (849.5 ± 1005 pg/ml and 923.2 ± 1284 pg/ml, respectively) in comparison with healthy controls (1281 ± 806.4 pg/ml and 3149 ± 4073 pg/ml, respectively). FMS serum levels of neurotensin were not different from controls. We had previously shown that CRH and SP stimulate IL-6 and TNF release from mast cells (MCs). Our current results indicate that neuropeptides could stimulate MCs to secrete inflammatory cytokines that contribute importantly to the symptoms of FMS. Treatment directed at preventing the secretion or antagonizing these elevated neuroimmune markers, both centrally and peripherally, may prove to be useful in the management of FMS. PMID:26763911

Bovine colostrum supplementation does not affect plasma I-FABP concentrations following exercise in a hot and humid environment.

PubMed

McKenna, Zachary; Berkemeier, Quint; Naylor, Ashley; Kleint, Austin; Gorini, Felipe; Ng, Jason; Kim, Jong-Kyung; Sullivan, Sean; Gillum, Trevor

2017-12-01

To quantify the impact of a 14-day bovine colostrum (BC) supplementation on intestinal cell damage following exercise in a hot and humid environment. Ten male participants (20 ± 2 years, VO 2max 55.80 ± 3.79 mL kg -1  min -1 , 11.81 ± 2.71% body fat) ran for 46 ± 7.75 min at 95% of ventiliatory threshold in 40 °C and 50% RH following a 14-day double-blinded supplementation with either BC or placebo (Plac). Core temperature, skin temperature, heart rate, and rating of perceived exertion were recorded every 5 min during exercise. Blood was taken pre, post, 1 h, and 4 h post exercise. Intestinal cell damage was assessed via intestinal fatty acid binding protein (I-FABP). I-FABP concentrations were similar between conditions at all time points [pre 989.39 ± 490.88 pg ml -1 (BC) 851.35 ± 450.71 pg ml -1 (Plac) post 1505.10 ± 788.63 pg ml -1 (BC) 1267.12 ± 521.51 pg ml -1 (Plac) 1-h, 1087.77 ± 397.06 pg ml -1 (BC) 997.25 ± 524.74 pg ml -1 (Plac) 4-h, 511.35 ± 243.10 pg ml -1 (BC) 501.46 ± 222.54 pg ml -1 (Plac)]. I-FABP was elevated pre to post exercise for both BC (162 ± 50%) and Plac (162 ± 56%) (p < 0.05). BC had no effect on mean body temperature [beginning 36.11 ± 0.30 °C, ending: 39.52 ± 0.28 °C (BC); beginning:35.96 ± 0.43 °C, ending:39.42 ± 0.38 °C (Plac)]. While BC supplementation may protect against enterocyte damage during exercise in thermonuetral environments, our data suggest that BC supplementation may not be an effective technique for preventing enterocyte damage during exercise when core temperature exceeds 39 °C.

Wall effects in continuous microfluidic magneto-affinity cell separation.

PubMed

Wu, Liqun; Zhang, Yong; Palaniapan, Moorthi; Roy, Partha

2010-05-01

Continuous microfluidic magneto-affinity cell separator combines unique microscale flow phenomenon with advantageous nanobead properties, to isolate cells with high specificity. Owing to the comparable size of the cell-bead complexes and the microchannels, the walls of the microchannel exert a strong influence on the separation of cells by this method. We present a theoretical and experimental study that provides a quantitative description of hydrodynamic wall interactions and wall rolling velocity of cells. A transient convection model describes the transport of cells in two-phase microfluidic flow under the influence of an external magnetic field. Transport of cells along the microchannel walls is also considered via an additional equation. Results show the variation of cell flux in the fluid phases and the wall as a function of a dimensionless parameter arising in the equations. Our results suggest that conditions may be optimized to maximize cell separation while minimizing contact with the wall surfaces. Experimentally measured cell rolling velocities on the wall indicate the presence of other near-wall forces in addition to fluid shear forces. Separation of a human colon carcinoma cell line from a mixture of red blood cells, with folic acid conjugated 1 microm and 200 nm beads, is reported.

Comparison of 0.3% Hypotonic and Isotonic Sodium Hyaluronate Eye Drops in the Treatment of Experimental Dry Eye.

PubMed

Li, Ying; Cui, Lian; Lee, Hyo Seok; Kang, Yeon Soo; Choi, Won; Yoon, Kyung Chul

2017-08-01

To compare the efficacy of 0.3% hypotonic and isotonic sodium hyaluronate (SH) eye drops in the treatment of experimental dry eye. Experimental dry eye was established in female C57BL/6 mice by subcutaneous scopolamine injection and an air draft. The mice were divided into three groups (n = 15): control, preservative-free 0.3% isotonic SH, and preservative-free 0.3% hypotonic SH. The tear volume, tear film break-up time, and corneal fluorescein staining scores were measured 5 and 10 days after treatment. After conjunctival tissues were excised at 10 days, the levels of interleukin (IL)-6, IL-17, interferon (IFN)-γ, and IFN-γ inducible protein-10 were determined using the multiplex immunobead assay. In addition, PAS staining and flow cytometry were performed to evaluate the counts of conjunctival goblet cells and CD4+ IFN-γ+ T cells. Mice treated with 0.3% hypotonic SH showed a significant decrease in corneal staining scores (P = 0.04) and the levels of IL-6 (16.7 ± 1.4 pg/mL, P = 0.02) and IFN-γ (46.5 ± 11.5 pg/mL, P = 0.02) compared to mice treated with 0.3% isotonic SH (IL-6; 32.5 ± 8.8 pg/mL, IFN-γ; 92.0 ± 16.0 pg/mL) at day 10. Although no significant difference in CD4+ IFN-γ+ T cell numbers was observed, goblet cell counts were higher in the hyopotonic SH group than in the isotonic SH group (P = 0.02). When compared to 0.3% isotonic SH eye drops, 0.3% hypotonic SH eye drops can be more effective by improving corneal staining scores, decreasing inflammatory molecules, and increasing goblet cell counts for experimental dry eye. These data suggest that hypotonic artificial tears may be useful as an adjunctive treatment for inflammatory dry eye.

B-type natriuretic peptide and plasma hemoglobin levels following transfusion of shorter-storage versus longer-storage Red Blood Cells: results from the TOTAL randomized trial

PubMed Central

Dhabangi, Aggrey; Ainomugisha, Brenda; Cserti-Gazdewich, Christine; Ddungu, Henry; Kyeyune, Dorothy; Musisi, Ezra; Opoka, Robert; Stowell, Christopher P.; Dzik, Walter H

2016-01-01

Background Prior studies have suggested that transfusion of stored RBCs with increased levels of cell free hemoglobin might reduce the bioavailability of recipient nitric oxide (NO) and cause myocardial strain. Methods Ugandan children (ages 6 to 60 months) with severe anemia and lactic acidosis were randomly assigned to receive RBCs stored 1-10 days versus 25-35 days. B-type natriuretic peptide (BNP), vital signs, renal function tests, and plasma hemoglobin were measured. Most children had either malaria or sickle cell disease and were thus at risk for reduced NO bioavailability. Results 70 patients received RBCs stored 1-10 days and 77 received RBCs stored 25-35 days. The median (IQR) cell free hemoglobin was nearly three times higher in longer-storage RBCs (26.4 [15.5-43.4] μmol/L) than in shorter-storage RBCs (10.8 [7.8-18.6] μmol/L), p<0.0001. Median (IQR) BNP 2 hours post-transfusion was 156 (59-650) pg/mL (shorter-storage) versus 158 (59-425) pg/mL (longer-storage), p=0.76. BNP values 22 hours post-transfusion were 110 (46-337) pg/mL (shorter-storage) versus 96 (49-310) pg/mL (longer-storage), p=0.76. Changes in BNP within individuals from pre-transfusion to 2-hour (or 22-hour) post-transfusion were not significantly different between the study groups. BNP change following transfusion did not correlate with the concentration of cell free hemoglobin in the RBC supernatant. Blood pressure, BUN, creatinine, and change in plasma hemoglobin were not significantly different in the two groups. Conclusion In a randomized trial among children at risk for reduced NO bioavailability, we found that BNP, blood pressure, creatinine, and plasma hemoglobin were not higher in patients receiving RBCs stored for 25-35 days versus 1-10 days. PMID:27302626

Role of Rifampin in Reducing Inflammation and Neuronal Damage in Childhood Bacterial Meningitis: A Pilot Randomized Controlled Trial.

PubMed

Uppal, Lipi; Singhi, Sunit; Singhi, Pratibha; Aggarwal, Ritu

2017-06-01

Treatment of acute bacterial meningitis in children with bactericidal antibiotics causes cell wall lysis and a surge in inflammatory cascade, which in turn contributes to neuronal damage and morbidity. Pretreatment with a nonbacteriolytic antibiotic, such as rifampin, has been shown to attenuate the inflammatory response in experimental models of bacterial meningitis. In a pilot study, in children with bacterial meningitis, we have studied markers of inflammatory response and neuronal damage in 2 groups of children with bacterial meningitis; one group received rifampin pretreatment with ceftriaxone and the other group received ceftriaxone alone. Forty children with bacterial meningitis, who were 3 months to 12 years of age, were randomly assigned to receive either a single dose rifampin (20 mg/kg) 30 minutes before ceftriaxone or ceftriaxone alone was given. The primary outcome variables were cerebrospinal fluid (CSF) concentrations of tumor necrosis factor alpha (TNFα), S100B and neuron-specific enolase on day 1 and day 5, and secondary outcome variables were the values of TNFα and interleukin 6 in serum on day 1 and day 5; hearing and neurologic sequelae at 3 months after recovery from the illness. Children in rifampin pretreatment group had significantly lower CSF TNFα concentrations [median (interquartile range [IQR]): 15.5 (7.2-22.0) vs. 53.0 (9.0-87.5) pg/mL, P = 0.019] and S100B [median (IQR): 145.0 (54.7-450.0) vs. 447.5 (221.0-804.6) pg/mL, P = 0.033] on day 1 and S100B [median (IQR): 109.7 (64.0-287.0) vs. 322 (106.7-578.0) pg/mL, P = 0.048] and neuron-specific enolase [median (IQR): 8.6 (5-14.75) vs. 18.2 (7.0-28.75) ng/mL, P = 0.035] on day 5 when compared with ceftriaxone alone group. The rifampin-treated group also had reduced morbidity and neurologic sequelae; however, these were not statistically significant. Pretreatment with single dose rifampin 30 minutes before ceftriaxone administration reduced the CSF concentrations of markers of inflammation and neuronal damage in children with bacterial meningitis.

Ubiquitin-proteasomal degradation of COX-2 in TGF-β stimulated human endometrial cells is mediated through endoplasmic reticulum mannosidase I.

PubMed

Singh, Mohan; Chaudhry, Parvesh; Parent, Sophie; Asselin, Eric

2012-01-01

Cyclooxygenase (COX)-2 is a key regulatory enzyme in the production of prostaglandins (PG) during various physiological processes. Mechanisms of COX-2 regulation in human endometrial stromal cells (human endometrial stromal cells) are not fully understood. In this study, we investigate the role of TGF-β in the regulation of COX-2 in human uterine stromal cells. Each TGF-β isoform decreases COX-2 protein level in human uterine stromal cells in Smad2/3-dependent manner. The decrease in COX-2 is accompanied by a decrease in PG synthesis. Knockdown of Smad4 using specific small interfering RNA prevents the decrease in COX-2 protein, confirming that Smad pathway is implicated in the regulation of COX-2 expression in human endometrial stromal cells. Pretreatment with 26S proteasome inhibitor, MG132, significantly restores COX-2 protein and PG synthesis, indicating that COX-2 undergoes proteasomal degradation in the presence of TGF-β. In addition, each TGF-β isoform up-regulates endoplasmic reticulum (ER)-mannosidase I (ERManI) implying that COX-2 degradation is mediated through ER-associated degradation pathway in these cells. Furthermore, inhibition of ERManI activity using the mannosidase inhibitor (kifunensine), or small interfering RNA-mediated knockdown of ERManI, prevents TGF-β-induced COX-2 degradation. Taken together, these studies suggest that TGF-β promotes COX-2 degradation in a Smad-dependent manner by up-regulating the expression of ERManI and thereby enhancing ER-associated degradation and proteasomal degradation pathways.

Antioxidant, anti-inflammatory, antiproliferative and antimycobacterial activities of the essential oil of Psidium guineense Sw. and spathulenol.

PubMed

do Nascimento, Kamilla Felipe; Moreira, Flora Martinez Figueira; Alencar Santos, Joyce; Kassuya, Candida Aparecida Leite; Croda, Julio Henrique Rosa; Cardoso, Claudia Andrea Lima; Vieira, Maria do Carmo; Góis Ruiz, Ana Lúcia Tasca; Ann Foglio, Mary; de Carvalho, João Ernesto; Formagio, Anelise Samara Nazari

2018-01-10

Leaves from Psidium guineense Sw. are used in popular medicine for the treatment of inflammatory disease. However, there is no scientific evidence demonstrating this activity. To evaluate the antioxidant, anti-inflammatory, antiproliferative and antimycobacterial activities of the essential oil of P. guineense and spathulenol (a major constituent). The study was conducted in part to provide evidence supporting the ethnobotanical use of the leaves of this species. The essential oil (EOPG) was extracted from the leaves of P. guineense by hydrodistillation and analysed by gas chromatography-mass spectrometry (GC-MS). The major compound, spathulenol (PG-1), was isolated in a chromatographic column and characterized by nuclear magnetic resonance (NMR). EOPG and PG-1 were evaluated in vitro for antioxidant activity by DPPH, ABTS and MDA methods; anti-inflammatory potential was assessed using two models, including pleurisy and oedema, in mice. The impact of EOPG and PG-1 on cell proliferation was determined via spectrophotometric quantification of the cellular protein content using a sulforhodamine B assay, and anti-Mycobacterium tuberculosis activity was determined using the REMA method. A total of 38 components were identified from the EOPG, with the sesquiterpenic alcohol spathulenol (PG-1) (80.7%) being the major constituent. EOPG and PG-1 exhibited the highest antioxidant activities in the DPPH and MDA system compared with reference standard, with IC 50 values ranging from 26.13 to 85.60μg/mL. Oral administration of EOPG and PG-1 showed significant inhibition in the Cg-induced mice paw oedema and pleurisy model. The EOPG (GI 50 = 0.89μg/mL) and PG-1 (GI 50 = 49.30μg/mL) were particularly effective against the ovarian cancer cell line. Both showed moderate antimycobacterial activity. For the first time, this study demonstrated the antioxidant, anti-inflammatory, antiproliferative and antimycobacterial properties of the essential oil of P. guineense (leaves were collected in Dourados-MS) and spathulenol, collaborating the etnhopharmacologycal use of this plant due to its an anti-inflammatory effect. Copyright © 2017. Published by Elsevier B.V.

Structure, function, and biosynthesis of plant cell walls: proceedings of the seventh annual symposium in botany

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dugger, W.M.; Bartnicki-Garcia, S.

Papers in the following areas were included in these symposium proceedings: (1) cell wall chemistry and biosynthesis; (2) cell wall hydrolysis and associated physiology; (3) cellular events associated with cell wall biosynthesis; and (4) interactions of plant cell walls with pathogens and related responses. Papers have been individually abstracted for the data base. (ACR)

Comparison of CCL28, interleukin-8, interleukin-1β and tumor necrosis factor-alpha in subjects with gingivitis, chronic periodontitis and generalized aggressive periodontitis.

PubMed

Ertugrul, A S; Sahin, H; Dikilitas, A; Alpaslan, N; Bozoglan, A

2013-02-01

Cytokines produced by various cells are strong local mediators of inflammation. Mucosa-associated epithelial chemokine (CCL28), interleukin-8 (IL-8), interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) are major cytokines that play important roles in the periodontal inflammatory process. In this study we aimed to compare the levels of CCL28, IL-8, IL-1β and TNF-α in the gingival crevicular fluid of both periodontally healthy subjects and in subjects diagnosed with gingivitis, chronic periodontitis and generalized aggressive periodontitis. A total of 84 subjects participated in the study: 21 subjects had gingivitis, 21 subjects had chronic periodontitis, 21 subjects had generalized aggressive periodontitis and 21 were periodontally healthy. The levels of CCL28, IL-8, IL-1β and TNF-α were analyzed using enzyme-linked immune sorbent assay (ELISA). The total levels of CCL28 and IL-8 in the gingival crevicular fluid of the generalized aggressive periodontitis group (324.74 ± 42.62 pg/30 s, 487.62 ± 49.21 pg/30 s) were significantly higher than those of the chronic periodontitis group (268.81 ± 28.64 pg/30 s, 423.65 ± 35.24 pg/30 s), the gingivitis group (146.35 ± 17.46 pg/30 s, 310.24 ± 48.20 pg/30 s) and the periodontally healthy group (92.46 ± 22.04 pg/30 s, 148.41 ± 24.64 pg/30 s). Similarly, the total levels of IL-1β and TNF-α in the generalized aggressive periodontitis group (110.23 ± 9.20 pg/30 s, 1284.46 ± 86.32 pg/30 s) were significantly higher than those in the chronic periodontitis group (423.65 ± 35.24 pg/30 s, 82.64 ± 9.12 pg/30 s), the gingivitis group (52.10 ± 7.15 pg/30 s, 824.24 ± 44.68 pg/30 s) and the periodontally healthy group (36.44 ± 8.86 pg/30 s, 628.26 ± 34.61 pg/30 s). CCL28, IL-8, IL-1β and TNF-α may play key roles in the host response to inflammation in periodontal diseases. As the severity of periodontal diseases increases, destruction of periodontal tissues also increases. Inflammation is one among many factors that trigger periodontal tissue destruction. Identification of the mediators that influence the development and progression of inflammation in periodontal diseases may be very important in understanding the prognoses of periodontal diseases. © 2012 John Wiley & Sons A/S.

Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

PubMed Central

Orlean, Peter

2012-01-01

The wall gives a Saccharomyces cerevisiae cell its osmotic integrity; defines cell shape during budding growth, mating, sporulation, and pseudohypha formation; and presents adhesive glycoproteins to other yeast cells. The wall consists of β1,3- and β1,6-glucans, a small amount of chitin, and many different proteins that may bear N- and O-linked glycans and a glycolipid anchor. These components become cross-linked in various ways to form higher-order complexes. Wall composition and degree of cross-linking vary during growth and development and change in response to cell wall stress. This article reviews wall biogenesis in vegetative cells, covering the structure of wall components and how they are cross-linked; the biosynthesis of N- and O-linked glycans, glycosylphosphatidylinositol membrane anchors, β1,3- and β1,6-linked glucans, and chitin; the reactions that cross-link wall components; and the possible functions of enzymatic and nonenzymatic cell wall proteins. PMID:23135325

Retention of Proanthocyanidin in Wine-like Solution Is Conferred by a Dynamic Interaction between Soluble and Insoluble Grape Cell Wall Components.

PubMed

Bindon, Keren A; Li, Sijing; Kassara, Stella; Smith, Paul A

2016-11-09

For better understanding of the factors that impact proanthocyanidin (PA) adsorption by insoluble cell walls or interaction with soluble cell wall-derived components, application of a commercial polygalacturonase enzyme preparation was investigated to modify grape cell wall structure. Soluble and insoluble cell wall material was isolated from the skin and mesocarp components of Vitis vinifera Shiraz grapes. It was observed that significant depolymerization of the insoluble grape cell wall occurred following enzyme application to both grape cell wall fractions, with increased solubilization of rhamnogalacturonan-enriched, low molecular weight polysaccharides. However, in the case of grape mesocarp, the solubilization of protein from cell walls (in buffer) was significant and increased only slightly by the enzyme treatment. Enzyme treatment significantly reduced the adsorption of PA by insoluble cell walls, but this effect was observed only when material solubilized from grape cell walls had been removed. The loss of PA through interaction with the soluble cell wall fraction was observed to be greater for mesocarp than skin cell walls. Subsequent experiments on the soluble mesocarp cell wall fraction confirmed a role for protein in the precipitation of PA. This identified a potential mechanism by which extracted grape PA may be lost from wine during vinification, as a precipitate with solubilized grape mesocarp proteins. Although protein was a minor component in terms of total concentration, losses of PA via precipitation with proteins were in the order of 50% of available PA. PA-induced precipitation could proceed until all protein was removed from solution and may account for the very low levels of residual protein observed in red wines. The results point to a dynamic interaction of grape insoluble and soluble components in modulating PA retention in wine.

The transcription factor Rap1p is required for tolerance to cell-wall perturbing agents and for cell-wall maintenance in Saccharomyces cerevisiae.

PubMed

Azad, Gajendra Kumar; Singh, Vikash; Baranwal, Shivani; Thakare, Mayur Jankiram; Tomar, Raghuvir S

2015-01-02

Yeast repressor activator protein (Rap1p) is involved in genomic stability and transcriptional regulation. We explored the function of Rap1p in yeast physiology using Rap1p truncation mutants. Our results revealed that the N-terminal truncation of Rap1p (Rap1ΔN) leads to hypersensitivity towards elevated temperature and cell-wall perturbing agents. Cell wall analysis showed an increase in the chitin and glucan content in Rap1ΔN cells as compared with wild type cells. Accordingly, mutant cells had a twofold thicker cell wall, as observed by electron microscopy. Furthermore, Rap1ΔN cells had increased levels of phosphorylated Slt2p, a MAP kinase of the cell wall integrity pathway. Mutant cells also had elevated levels of cell wall integrity response transcripts. Taken together, our findings suggest a connection between Rap1p and cell wall homeostasis. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Bacterial cell-wall recycling

PubMed Central

Johnson, Jarrod W.; Fisher, Jed F.; Mobashery, Shahriar

2012-01-01

Many Gram-negative and Gram-positive bacteria recycle a significant proportion of the peptidoglycan components of their cell walls during their growth and septation. In many—and quite possibly all—bacteria, the peptidoglycan fragments are recovered and recycled. While cell-wall recycling is beneficial for the recovery of resources, it also serves as a mechanism to detect cell-wall–targeting antibiotics and to regulate resistance mechanisms. In several Gram-negative pathogens, anhydro-MurNAc-peptide cell-wall fragments regulate AmpC β-lactamase induction. In some Gram-positive organisms, short peptides derived from the cell wall regulate the induction of both β-lactamase and β-lactam-resistant penicillin-binding proteins. The involvement of peptidoglycan recycling with resistance regulation suggests that inhibitors of the enzymes involved in the recycling might synergize with cell-wall-targeted antibiotics. Indeed, such inhibitors improve the potency of β-lactams in vitro against inducible AmpC β-lactamase-producing bacteria. We describe the key steps of cell-wall remodeling and recycling, the regulation of resistance mechanisms by cell-wall recycling, and recent advances toward the discovery of cell-wall recycling inhibitors. PMID:23163477

Characteristic Thickened Cell Walls of the Bracts of the ‘Eternal Flower’ Helichrysum bracteatum

PubMed Central

Nishikawa, Kuniko; Ito, Hiroaki; Awano, Tatsuya; Hosokawa, Munetaka; Yazawa, Susumu

2008-01-01

Background and Aims Helichrysum bracteatum is called an ‘eternal flower’ and has large, coloured, scarious bracts. These maintain their aesthetic value without wilting or discoloration for many years. There have been no research studies of cell death or cell morphology of the scarious bract, and hence the aim of this work was to elucidate these characteristics for the bract of H. bracteatum. Methods DAPI (4'6-diamidino-2-phenylindol dihydrochloride) staining and fluorescence microscopy were used for observation of cell nuclei. Light microscopy (LM), transmission electron microscopy (TEM) and polarized light microscopy were used for observation of cells, including cell wall morphology. Key Results Cell death occurred at the bract tip during the early stage of flower development. The cell wall was the most prominent characteristic of H. bracteatum bract cells. Characteristic thickened secondary cell walls on the inside of the primary cell walls were observed in both epidermal and inner cells. In addition, the walls of all cells exhibited birefringence. Characteristic thickened secondary cell walls have orientated cellulose microfibrils as well as general secondary cell walls of the tracheary elements. For comparison, these characters were not observed in the petal and bract tissues of Chrysanthemum morifolium. Conclusions Bracts at anthesis are composed of dead cells. Helichrysum bracteatum bracts have characteristic thickened secondary cell walls that have not been observed in the parenchyma of any other flowers or leaves. The cells of the H. bracteatum bract differ from other tissues with secondary cell walls, suggesting that they may be a new cell type. PMID:18436550

Mechanical feedback coordinates cell wall expansion and assembly in yeast mating morphogenesis

PubMed Central

2018-01-01

The shaping of individual cells requires a tight coordination of cell mechanics and growth. However, it is unclear how information about the mechanical state of the wall is relayed to the molecular processes building it, thereby enabling the coordination of cell wall expansion and assembly during morphogenesis. Combining theoretical and experimental approaches, we show that a mechanical feedback coordinating cell wall assembly and expansion is essential to sustain mating projection growth in budding yeast (Saccharomyces cerevisiae). Our theoretical results indicate that the mechanical feedback provided by the Cell Wall Integrity pathway, with cell wall stress sensors Wsc1 and Mid2 increasingly activating membrane-localized cell wall synthases Fks1/2 upon faster cell wall expansion, stabilizes mating projection growth without affecting cell shape. Experimental perturbation of the osmotic pressure and cell wall mechanics, as well as compromising the mechanical feedback through genetic deletion of the stress sensors, leads to cellular phenotypes that support the theoretical predictions. Our results indicate that while the existence of mechanical feedback is essential to stabilize mating projection growth, the shape and size of the cell are insensitive to the feedback. PMID:29346368

Wall relaxation and the driving forces for cell expansive growth

NASA Technical Reports Server (NTRS)

Cosgrove, D. J.

1987-01-01

When water uptake by growing cells is prevented, the turgor pressure and the tensile stress in the cell wall are reduced by continued wall loosening. This process, termed in vivo stress relaxation, provides a new way to study the dynamics of wall loosening and to measure the wall yield threshold and the physiological wall extensibility. Stress relaxation experiments indicate that wall stress supplies the mechanical driving force for wall yielding. Cell expansion also requires water absorption. The driving force for water uptake during growth is created by wall relaxation, which lowers the water potential of the expanding cells. New techniques for measuring this driving force show that it is smaller than believed previously; in elongating stems it is only 0.3 to 0.5 bar. This means that the hydraulic resistance of the water transport pathway is small and that rate of cell expansion is controlled primarily by wall loosening and yielding.

Ozone and haze pollution weakens net primary productivity in China

NASA Astrophysics Data System (ADS)

Yue, Xu; Unger, Nadine; Harper, Kandice; Xia, Xiangao; Liao, Hong; Zhu, Tong; Xiao, Jingfeng; Feng, Zhaozhong; Li, Jing

2017-05-01

Atmospheric pollutants have both beneficial and detrimental effects on carbon uptake by land ecosystems. Surface ozone (O3) damages leaf photosynthesis by oxidizing plant cells, while aerosols promote carbon uptake by increasing diffuse radiation and exert additional influences through concomitant perturbations to meteorology and hydrology. China is currently the world's largest emitter of both carbon dioxide and short-lived air pollutants. The land ecosystems of China are estimated to provide a carbon sink, but it remains unclear whether air pollution acts to inhibit or promote carbon uptake. Here, we employ Earth system modeling and multiple measurement datasets to assess the separate and combined effects of anthropogenic O3 and aerosol pollution on net primary productivity (NPP) in China. In the present day, O3 reduces annual NPP by 0.6 Pg C (14 %) with a range from 0.4 Pg C (low O3 sensitivity) to 0.8 Pg C (high O3 sensitivity). In contrast, aerosol direct effects increase NPP by 0.2 Pg C (5 %) through the combination of diffuse radiation fertilization, reduced canopy temperatures, and reduced evaporation leading to higher soil moisture. Consequently, the net effects of O3 and aerosols decrease NPP by 0.4 Pg C (9 %) with a range from 0.2 Pg C (low O3 sensitivity) to 0.6 Pg C (high O3 sensitivity). However, precipitation inhibition from combined aerosol direct and indirect effects reduces annual NPP by 0.2 Pg C (4 %), leading to a net air pollution suppression of 0.8 Pg C (16 %) with a range from 0.6 Pg C (low O3 sensitivity) to 1.0 Pg C (high O3 sensitivity). Our results reveal strong dampening effects of air pollution on the land carbon uptake in China today. Following the current legislation emission scenario, this suppression will be further increased by the year 2030, mainly due to a continuing increase in surface O3. However, the maximum technically feasible reduction scenario could drastically relieve the current level of NPP damage by 70 % in 2030, offering protection of this critical ecosystem service and the mitigation of long-term global warming.

Two endogenous proteins that induce cell wall extension in plants

NASA Technical Reports Server (NTRS)

McQueen-Mason, S.; Durachko, D. M.; Cosgrove, D. J.

1992-01-01

Plant cell enlargement is regulated by wall relaxation and yielding, which is thought to be catalyzed by elusive "wall-loosening" enzymes. By employing a reconstitution approach, we found that a crude protein extract from the cell walls of growing cucumber seedlings possessed the ability to induce the extension of isolated cell walls. This activity was restricted to the growing region of the stem and could induce the extension of isolated cell walls from various dicot stems and the leaves of amaryllidaceous monocots, but was less effective on grass coleoptile walls. Endogenous and reconstituted wall extension activities showed similar sensitivities to pH, metal ions, thiol reducing agents, proteases, and boiling in methanol or water. Sequential HPLC fractionation of the active wall extract revealed two proteins with molecular masses of 29 and 30 kD associated with the activity. Each protein, by itself, could induce wall extension without detectable hydrolytic breakdown of the wall. These proteins appear to mediate "acid growth" responses of isolated walls and may catalyze plant cell wall extension by a novel biochemical mechanism.

Pectinous cell wall thickenings formation - A common defense strategy of plants to cope with Pb.

PubMed

Krzesłowska, Magdalena; Rabęda, Irena; Basińska, Aneta; Lewandowski, Michał; Mellerowicz, Ewa J; Napieralska, Anna; Samardakiewicz, Sławomir; Woźny, Adam

2016-07-01

Lead, one of the most abundant and hazardous trace metals affecting living organisms, has been commonly detected in plant cell walls including some tolerant plants, mining ecotypes and hyperaccumulators. We have previously shown that in tip growing Funaria sp. protonemata cell wall is remodeled in response to lead by formation of thickenings rich in low-methylesterified pectins (pectin epitope JIM5 - JIM5-P) able to bind metal ions, which accumulate large amounts of Pb. Hence, it leads to the increase of cell wall capacity for Pb compartmentalization. Here we show that diverse plant species belonging to different phyla (Arabidopsis, hybrid aspen, star duckweed), form similar cell wall thickenings in response to Pb. These thickenings are formed in tip growing cells such as the root hairs, and in diffuse growing cells such as meristematic and root cap columella cells of root apices in hybrid aspen and Arabidopsis and in mesophyll cells in star duckweed fronds. Notably, all analyzed cell wall thickenings were abundant in JIM5-P and accumulated high amounts of Pb. In addition, the co-localization of JIM5-P and Pb commonly occurred in these cells. Hence, cell wall thickenings formed the extra compartment for Pb accumulation. In this way plant cells increased cell wall capacity for compartmentalization of this toxic metal, protecting protoplast from its toxicity. As cell wall thickenings occurred in diverse plant species and cell types differing in the type of growth we may conclude that pectinous cell wall thickenings formation is a widespread defense strategy of plants to cope with Pb. Moreover, detection of natural defense strategy, increasing plant cell walls capacity for metal accumulation, reveals a promising direction for enhancing plant efficiency in phytoremediation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Use of negative pressure wound therapy in the management of infected abdominal wounds containing mesh: an analysis of outcomes.

PubMed

Baharestani, Mona Mylene; Gabriel, Allen

2011-04-01

The purpose of this study was to examine the clinical outcomes of negative pressure wound therapy (NPWT) using reticulated open-cell foam (ROCF) in the adjunctive management of abdominal wounds with exposed and known infected synthetic mesh. A non randomised, retrospective review of medical records for 21 consecutive patients with infected abdominal wounds treated with NPWT was conducted. All abdominal wounds contained exposed synthetic mesh [composite, polypropylene (PP), or knitted polyglactin 910 (PG) mesh]. Demographic and bacteriological data, wound history, pre-NPWT and comparative post-NPWT, operative procedures and complications, hospital length of stay (LOS) and wound healing outcomes were all analysed. Primary endpoints measured were (1) hospital LOS prior to initiation of NPWT, (2) total time on NPWT, (3) hospital LOS from NPWT initiation to discharge and (4) wound closure status at discharge. A total of 21 patients with abdominal wounds with exposed, infected mesh were treated with NPWT. Aetiology of the wounds was ventral hernia repair (n = 11) and acute abdominal wall defect (n = 10). Prior to NPWT initiation, the mean hospital LOS for the composite, PP and PG meshes were 76 days (range: 21-171 days), 51 days (range: 32-62 days) and 19 days (range: 12-39 days), respectively. The mean hospital LOS following initiation of NPWT for wounds with exposed composite, PP and PG mesh were 28, 31 and 32 days, respectively. Eighteen of the 21 wounds (86%) reached full closure after a mean time of 26 days of NPWT and a mean hospital LOS of 30 days postinitiation of NPWT. Three wounds, all with composite mesh left in situ, did not reach full closure, although all exhibited decreased wound dimensions, granulating beds and decreased surface area exposure of mesh. During NPWT/ROCF, one hypoalbuminemic patient with exposed PP mesh developed an enterocutaneous fistula over a prior enterotomy site. This patient subsequently underwent total mesh extraction, takedown of the fistula and PP mesh replacement followed by reinstitution of NPWT and flap closure. In addition to appropriate systemic antibiotics and nutritional optimisation, the adjunctive use of NPWT resulted in successful closure of 86% of infected abdominal wounds with exposed prosthetic mesh. Patient hospital LOS (except those with PG mesh), operative procedures and readmissions were decreased during NPWT compared with treatment prior to NPWT. Future multi-site prospective, controlled studies would provide a strong evidence base from which treatment decisions could be made in the management of these challenging and costly cases. © 2010 The Authors. © 2010 Blackwell Publishing Ltd and Medicalhelplines.com Inc.

Genetic and biochemical characterization of the GH72 family of cell wall transglycosylases in Neurospora crassa.

PubMed

Ao, Jie; Free, Stephen J

2017-04-01

The Neurospora crassa genome encodes five GH72 family transglycosylases, and four of these enzymes (GEL-1, GEL-2, GEL-3 and GEL-5) have been found to be present in the cell wall proteome. We carried out an extensive genetic analysis on the role of these four transglycosylases in cell wall biogenesis and demonstrated that the transglycosylases are required for the formation of a normal cell wall. As suggested by the proteomic analysis, we found that multiple transglycosylases were being expressed in N. crassa cells and that different combinations of the enzymes are required in different cell types. The combination of GEL-1, GEL-2 and GEL-5 is required for the growth of vegetative hyphae, while the GEL-1, GEL-2, GEL-3 combination is needed for the production of aerial hyphae and conidia. Our data demonstrates that the enzymes are redundant with partially overlapping enzymatic activities, which provides the fungus with a robust cell wall biosynthetic system. Characterization of the transglycosylase-deficient mutants demonstrated that the incorporation of cell wall proteins was severely compromised. Interestingly, we found that the transglycosylase-deficient mutant cell walls contained more β-1,3-glucan than the wild type cell wall. Our results demonstrate that the GH72 transglycosylases are not needed for the incorporation of β-1,3-glucan into the cell wall, but they are required for the incorporation of cell wall glycoprotein into the cell wall. Copyright © 2017 Elsevier Inc. All rights reserved.

A Cell Wall Proteome and Targeted Cell Wall Analyses Provide Novel Information on Hemicellulose Metabolism in Flax.

PubMed

Chabi, Malika; Goulas, Estelle; Leclercq, Celine C; de Waele, Isabelle; Rihouey, Christophe; Cenci, Ugo; Day, Arnaud; Blervacq, Anne-Sophie; Neutelings, Godfrey; Duponchel, Ludovic; Lerouge, Patrice; Hausman, Jean-François; Renaut, Jenny; Hawkins, Simon

2017-09-01

Experimentally-generated (nanoLC-MS/MS) proteomic analyses of four different flax organs/tissues (inner-stem, outer-stem, leaves and roots) enriched in proteins from 3 different sub-compartments (soluble-, membrane-, and cell wall-proteins) was combined with publically available data on flax seed and whole-stem proteins to generate a flax protein database containing 2996 nonredundant total proteins. Subsequent multiple analyses (MapMan, CAZy, WallProtDB and expert curation) of this database were then used to identify a flax cell wall proteome consisting of 456 nonredundant proteins localized in the cell wall and/or associated with cell wall biosynthesis, remodeling and other cell wall related processes. Examination of the proteins present in different flax organs/tissues provided a detailed overview of cell wall metabolism and highlighted the importance of hemicellulose and pectin remodeling in stem tissues. Phylogenetic analyses of proteins in the cell wall proteome revealed an important paralogy in the class IIIA xyloglucan endo-transglycosylase/hydrolase (XTH) family associated with xyloglucan endo-hydrolase activity.Immunolocalisation, FT-IR microspectroscopy, and enzymatic fingerprinting indicated that flax fiber primary/S1 cell walls contained xyloglucans with typical substituted side chains as well as glucuronoxylans in much lower quantities. These results suggest a likely central role of xyloglucans and endotransglucosylase/hydrolase activity in flax fiber formation and cell wall remodeling processes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Immunosuppression in patients who die of sepsis and multiple organ failure.

PubMed

Boomer, Jonathan S; To, Kathleen; Chang, Kathy C; Takasu, Osamu; Osborne, Dale F; Walton, Andrew H; Bricker, Traci L; Jarman, Stephen D; Kreisel, Daniel; Krupnick, Alexander S; Srivastava, Anil; Swanson, Paul E; Green, Jonathan M; Hotchkiss, Richard S

2011-12-21

Severe sepsis is typically characterized by initial cytokine-mediated hyperinflammation. Whether this hyperinflammatory phase is followed by immunosuppression is controversial. Animal studies suggest that multiple immune defects occur in sepsis, but data from humans remain conflicting. To determine the association of sepsis with changes in host innate and adaptive immunity and to examine potential mechanisms for putative immunosuppression. Rapid postmortem spleen and lung tissue harvest was performed at the bedsides of 40 patients who died in intensive care units (ICUs) of academic medical centers with active severe sepsis to characterize their immune status at the time of death (2009-2011). Control spleens (n = 29) were obtained from patients who were declared brain-dead or had emergent splenectomy due to trauma; control lungs (n = 20) were obtained from transplant donors or from lung cancer resections. Cytokine secretion assays and immunophenotyping of cell surface receptor-ligand expression profiles were performed to identify potential mechanisms of immune dysfunction. Immunohistochemical staining was performed to evaluate the loss of immune effector cells. The mean ages of patients with sepsis and controls were 71.7 (SD, 15.9) and 52.7 (SD, 15.0) years, respectively. The median number of ICU days for patients with sepsis was 8 (range, 1-195 days), while control patients were in ICUs for 4 or fewer days. The median duration of sepsis was 4 days (range, 1-40 days). Compared with controls, anti-CD3/anti-CD28-stimulated splenocytes from sepsis patients had significant reductions in cytokine secretion at 5 hours: tumor necrosis factor, 5361 (95% CI, 3327-7485) pg/mL vs 418 (95% CI, 98-738) pg/mL; interferon γ, 1374 (95% CI, 550-2197) pg/mL vs 37.5 (95% CI, -5 to 80) pg/mL; interleukin 6, 3691 (95% CI, 2313-5070) vs 365 (95% CI, 87-642) pg/mL; and interleukin 10, 633 (95% CI, -269 to 1534) vs 58 (95% CI, -39 to 156) pg/mL; (P < .001 for all). There were similar reductions in 5-hour lipopolysaccharide-stimulated cytokine secretion. Cytokine secretion in sepsis patients was generally less than 10% that in controls, independent of age, duration of sepsis, corticosteroid use, and nutritional status. Although differences existed between spleen and lung, flow cytometric analysis showed increased expression of selected inhibitory receptors and ligands and expansion of suppressor cell populations in both organs. Unique differences in cellular inhibitory molecule expression existed in immune cells isolated from lungs of sepsis patients vs cancer patients and vs transplant donors. Immunohistochemical staining showed extensive depletion of splenic CD4, CD8, and HLA-DR cells and expression of ligands for inhibitory receptors on lung epithelial cells. Patients who die in the ICU following sepsis compared with patients who die of nonsepsis etiologies have biochemical, flow cytometric, and immunohistochemical findings consistent with immunosuppression. Targeted immune-enhancing therapy may be a valid approach in selected patients with sepsis.

Developmental and Tissue-Specific Structural Alterations of the Cell-Wall Polysaccharides of Arabidopsis thaliana Roots.

PubMed Central

Freshour, G.; Clay, R. P.; Fuller, M. S.; Albersheim, P.; Darvill, A. G.; Hahn, M. G.

1996-01-01

The plant cell wall is a dynamic structure that plays important roles in growth and development and in the interactions of plants with their environment and other organisms. We have used monoclonal antibodies that recognize different carbohydrate epitopes present in plant cell-wall polysaccharides to locate these epitopes in roots of developing Arabidopsis thaliana seedlings. An epitope in the pectic polysaccharide rhamnogalacturonan I is observed in the walls of epidermal and cortical cells in mature parts of the root. This epitope is inserted into the walls in a developmentally regulated manner. Initially, the epitope is observed in atrichoblasts and later appears in trichoblasts and simultaneously in cortical cells. A terminal [alpha]-fucosyl-containing epitope is present in almost all of the cell walls in the root. An arabinosylated (1->6)-[beta]-galactan epitope is also found in all of the cell walls of the root with the exception of lateral root-cap cell walls. It is striking that these three polysaccharide epitopes are not uniformly distributed (or accessible) within the walls of a given cell, nor are these epitopes distributed equally across the two walls laid down by adjacent cells. Our results further suggest that the biosynthesis and differentiation of primary cell walls in plants are precisely regulated in a temporal, spatial, and developmental manner. PMID:12226270

Atomic force microscopy stiffness tomography on living Arabidopsis thaliana cells reveals the mechanical properties of surface and deep cell-wall layers during growth.

PubMed

Radotić, Ksenija; Roduit, Charles; Simonović, Jasna; Hornitschek, Patricia; Fankhauser, Christian; Mutavdžić, Dragosav; Steinbach, Gabor; Dietler, Giovanni; Kasas, Sandor

2012-08-08

Cell-wall mechanical properties play a key role in the growth and the protection of plants. However, little is known about genuine wall mechanical properties and their growth-related dynamics at subcellular resolution and in living cells. Here, we used atomic force microscopy (AFM) stiffness tomography to explore stiffness distribution in the cell wall of suspension-cultured Arabidopsis thaliana as a model of primary, growing cell wall. For the first time that we know of, this new imaging technique was performed on living single cells of a higher plant, permitting monitoring of the stiffness distribution in cell-wall layers as a function of the depth and its evolution during the different growth phases. The mechanical measurements were correlated with changes in the composition of the cell wall, which were revealed by Fourier-transform infrared (FTIR) spectroscopy. In the beginning and end of cell growth, the average stiffness of the cell wall was low and the wall was mechanically homogenous, whereas in the exponential growth phase, the average wall stiffness increased, with increasing heterogeneity. In this phase, the difference between the superficial and deep wall stiffness was highest. FTIR spectra revealed a relative increase in the polysaccharide/lignin content. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

A computational approach for inferring the cell wall properties that govern guard cell dynamics.

PubMed

Woolfenden, Hugh C; Bourdais, Gildas; Kopischke, Michaela; Miedes, Eva; Molina, Antonio; Robatzek, Silke; Morris, Richard J

2017-10-01

Guard cells dynamically adjust their shape in order to regulate photosynthetic gas exchange, respiration rates and defend against pathogen entry. Cell shape changes are determined by the interplay of cell wall material properties and turgor pressure. To investigate this relationship between turgor pressure, cell wall properties and cell shape, we focused on kidney-shaped stomata and developed a biomechanical model of a guard cell pair. Treating the cell wall as a composite of the pectin-rich cell wall matrix embedded with cellulose microfibrils, we show that strong, circumferentially oriented fibres are critical for opening. We find that the opening dynamics are dictated by the mechanical stress response of the cell wall matrix, and as the turgor rises, the pectinaceous matrix stiffens. We validate these predictions with stomatal opening experiments in selected Arabidopsis cell wall mutants. Thus, using a computational framework that combines a 3D biomechanical model with parameter optimization, we demonstrate how to exploit subtle shape changes to infer cell wall material properties. Our findings reveal that proper stomatal dynamics are built on two key properties of the cell wall, namely anisotropy in the form of hoop reinforcement and strain stiffening. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd and Society for Experimental Biology.

Virus-induced gene silencing offers a functional genomics platform for studying plant cell wall formation.

PubMed

Zhu, Xiaohong; Pattathil, Sivakumar; Mazumder, Koushik; Brehm, Amanda; Hahn, Michael G; Dinesh-Kumar, S P; Joshi, Chandrashekhar P

2010-09-01

Virus-induced gene silencing (VIGS) is a powerful genetic tool for rapid assessment of plant gene functions in the post-genomic era. Here, we successfully implemented a Tobacco Rattle Virus (TRV)-based VIGS system to study functions of genes involved in either primary or secondary cell wall formation in Nicotiana benthamiana plants. A 3-week post-VIGS time frame is sufficient to observe phenotypic alterations in the anatomical structure of stems and chemical composition of the primary and secondary cell walls. We used cell wall glycan-directed monoclonal antibodies to demonstrate that alteration of cell wall polymer synthesis during the secondary growth phase of VIGS plants has profound effects on the extractability of components from woody stem cell walls. Therefore, TRV-based VIGS together with cell wall component profiling methods provide a high-throughput gene discovery platform for studying plant cell wall formation from a bioenergy perspective.

Plant cell wall signalling and receptor-like kinases.

PubMed

Wolf, Sebastian

2017-02-15

Communication between the extracellular matrix and the cell interior is essential for all organisms as intrinsic and extrinsic cues have to be integrated to co-ordinate development, growth, and behaviour. This applies in particular to plants, the growth and shape of which is governed by deposition and remodelling of the cell wall, a rigid, yet dynamic, extracellular network. It is thus generally assumed that cell wall surveillance pathways exist to monitor the state of the wall and, if needed, elicit compensatory responses such as altered expression of cell wall remodelling and biosynthesis genes. Here, I highlight recent advances in the field of cell wall signalling in plants, with emphasis on the role of plasma membrane receptor-like kinase complexes. In addition, possible roles for cell wall-mediated signalling beyond the maintenance of cell wall integrity are discussed. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Mutagen and Oncogen Study on JP-4

DTIC Science & Technology

1978-09-01

random bred mice were killed by cranial blow , decapitated and bled. The liver was immediately dissected from the animal using aseptic technique and placed...chemical. Positive Controls--N-methylnitrosoguanidine (MNNG) at a concentration of 10 pg/ml was used as the positive control agent in nonactivation tests...The positive control agent in activation tests was 3,4-benzo(u)pyrene (BiP) at a concentration of 10 pg/ml. EXPERIMENTAL DESIGN Cell Preparation

The Specific Nature of Plant Cell Wall Polysaccharides 1

PubMed Central

Nevins, Donald J.; English, Patricia D.; Albersheim, Peter

1967-01-01

Polysaccharide compositions of cell walls were assessed by quantitative analyses of the component sugars. Cell walls were hydrolyzed in 2 n trifluoroacetic acid and the liberated sugars reduced to their respective alditols. The alditols were acetylated and the resulting alditol acetates separated by gas chromatography. Quantitative assay of the alditol acetates was accomplished by electronically integrating the detector output of the gas chromatograph. Myo-inositol, introduced into the sample prior to hydrolysis, served as an internal standard. The cell wall polysaccharide compositions of plant varieties within a given species are essentially identical. However, differences in the sugar composition were observed in cell walls prepared from different species of the same as well as of different genera. The fact that the wall compositions of different varieties of the same species are the same indicates that the biosynthesis of cell wall polysaccharides is genetically regulated. The cell walls of various morphological parts (roots, hypocotyls, first internodes and primary leaves) of bean plants were each found to have a characteristic sugar composition. It was found that the cell wall sugar composition of suspension-cultured sycamore cells could be altered by growing the cells on different carbon sources. This demonstrates that the biosynthesis of cell wall polysaccharides can be manipulated without fatal consequences. PMID:16656594

Dynamic changes in transcriptome and cell wall composition underlying brassinosteroid-mediated lignification of switchgrass suspension cells.

PubMed

Rao, Xiaolan; Shen, Hui; Pattathil, Sivakumar; Hahn, Michael G; Gelineo-Albersheim, Ivana; Mohnen, Debra; Pu, Yunqiao; Ragauskas, Arthur J; Chen, Xin; Chen, Fang; Dixon, Richard A

2017-01-01

Plant cell walls contribute the majority of plant biomass that can be used to produce transportation fuels. However, the complexity and variability in composition and structure of cell walls, particularly the presence of lignin, negatively impacts their deconstruction for bioenergy. Metabolic and genetic changes associated with secondary wall development in the biofuel crop switchgrass ( Panicum virgatum ) have yet to be reported. Our previous studies have established a cell suspension system for switchgrass, in which cell wall lignification can be induced by application of brassinolide (BL). We have now collected cell wall composition and microarray-based transcriptome profiles for BL-induced and non-induced suspension cultures to provide an overview of the dynamic changes in transcriptional reprogramming during BL-induced cell wall modification. From this analysis, we have identified changes in candidate genes involved in cell wall precursor synthesis, cellulose, hemicellulose, and pectin formation and ester-linkage generation. We have also identified a large number of transcription factors with expression correlated with lignin biosynthesis genes, among which are candidates for control of syringyl (S) lignin accumulation. Together, this work provides an overview of the dynamic compositional changes during brassinosteroid-induced cell wall remodeling, and identifies candidate genes for future plant genetic engineering to overcome cell wall recalcitrance.

Remarkable proanthocyanidin adsorption properties of monastrell pomace cell wall material highlight its potential use as an alternative fining agent in red wine production.

PubMed

Bautista-Ortín, Ana Belén; Ruiz-García, Yolanda; Marín, Fátima; Molero, Noelia; Apolinar-Valiente, Rafael; Gómez-Plaza, Encarna

2015-01-21

The existence of interactions between the polysaccharides of vegetal cell walls and proanthocyanins makes this cell wall material an interesting option for its use as a fining agent to reduce the level of proanthocyanins in wines. Pomace wastes from the winery are widely available and a source of cell wall material, and the identification of varieties whose pomace cell walls present high proanthocyanin binding capacity and of processing methods that could enhance their adsorption properties could be of great interest. This study compared the proanthocyanin adsorption properties of pomace cell wall material from three different grape varieties (Monastrell, Cabernet Sauvignon, and Syrah), and the results were compared with those obtained using fresh grape cell walls. Also, the effect of the vinification method has been studied. Analysis of the proanthocyanidins in the solution after reaction with the cell wall material, using phloroglucinolysis and size exclusion chromatography, provided quantitative and qualitative information on the adsorbed and nonadsorbed compounds. A highlight of this study was the observation that Monastrell pomace cell wall material showed a strong affinity for proanthocyanidins, with values similar to that obtained for fresh grapes cell walls, and a preferential binding of high molecular mass proanthocyanidins, so these pomace cell walls could be used in wines to reduce astringency. The use of maceration enzymes during vinification had little effect on the retention capacity of the pomace cell walls obtained from this vinification, although an increase in the retention of low molecular mass proanthocyanidins was observed, and this might have implications for wine sensory properties.

The relationship of Chlamydophila pneumoniae with schizophrenia: The role of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in this relationship.

PubMed

Kalayci, Fatma; Ozdemir, Armagan; Saribas, Suat; Yuksel, Pelin; Ergin, Sevgi; Kuskucu, Ali Mert; Poyraz, Cana Aksoy; Balcioglu, Ibrahim; Alpay, Nihat; Kurt, Aykut; Sezgin, Zeynep; Kocak, Banu Tufan; Icel, Rana Sucu; Can, Gunay; Tokman, Hrisi Bahar; Kocazeybek, Bekir

Several pathogens have been suspected of playing a role in the pathogenesis of schizophrenia. Chronic inflammation has been proposed to occur as a result of persistent infection caused by Chlamydophila pneumoniae cells that reside in brain endothelial cells for many years. It was recently hypothesized that brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) may play prominent roles in the development of schizophrenia. NT-3 and BDNF levels have been suggested to change in response to various manifestations of infection. Therefore, we aimed to elucidate the roles of BDNF and NT3 in the schizophrenia-C. pneumoniae infection relationship. RT-PCR, immunofluorescence and ELISA methods were used. Fifty patients suffering from schizophrenia and 35 healthy individuals were included as the patient group (PG) and the healthy control group (HCG), respectively. We detected persistent infection in 14 of the 50 individuals in the PG and in 1 of the 35 individuals in the HCG. A significant difference was found between the two groups (p<0.05). Twenty-two individuals in the PG and 13 in the HCG showed seropositivity for past C. pneumoniae infection, and no difference was observed between the groups (p>0.05). C. pneumoniae DNA was not detected in any group. A significant difference in NT-3 levels was observed between the groups, with very low levels in the PG (p<0.001). A significant difference in BDNF levels was also found, with lower levels in the PG (p<0.05). The mean serum NT-3 level was higher in the PG cases with C. pneumoniae seropositivity than in seronegative cases; however, this difference was not statistically significant (p>0.05). In conclusion, we suggest that NT-3 levels during persistent C. pneumoniae infection may play a role in this relationship. Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Pomegranate polyphenolics reduce inflammation and ulceration in intestinal colitis-involvement of the miR-145/p70S6K1/HIF1α axis in vivo and in vitro.

PubMed

Kim, Hyemee; Banerjee, Nivedita; Sirven, Maritza A; Minamoto, Yasushi; Markel, Melissa E; Suchodolski, Jan S; Talcott, Stephen T; Mertens-Talcott, Susanne U

2017-05-01

This study investigated the potential role of the p70S6K1/HIF1α axis in the anti-inflammatory activities of pomegranate (Punica granatum L.) polyphenolics in dextran sodium sulfate (DSS)-induced colitis in Sprague-Dawley rats and in lipopolysaccharide (LPS)-treated CCD-18Co colon-myofibroblastic cells. Rats were administered either control (CT) or pomegranate beverage (PG), containing ellagic acid and ellagitannins, then exposed to three cycles of 3% DSS followed by a 2-week recovery period. PG protected against DSS-induced colon inflammation and ulceration (50% and 66.7%, P=.05 and .045, respectively), and decreased the Ki-67 proliferative index in the central and basal regions compared to the control. PG also significantly reduced the expression of proinflammatory cytokines (TNF-α and IL-1β), COX-2, and iNOS at mRNA and protein levels. In addition, the expression of p70S6K1 and HIF1α was reduced, while the tumor suppressor miR-145 was induced by PG. The intestinal microbiota of rats treated with PG showed a significant increase in Ruminococcaceae that include several butyrate producing bacteria (P=.03). In vitro, PG reduced the expression of p70S6K1 and HIF1α and induced miR-145 in a dose-dependent manner. The involvement of miR-145/p70S6K1 was confirmed by treating LPS-treated CCD-18Co cells with miR-145 antagomiR, where the pomegranate polyphenolics reversed the effects of the antagomiR for p70S6K1 mRNA and protein levels. These results suggest that pomegranate polyphenols attenuated DSS-induced colitis by modulating the miR-145/p70S6K/HIF1α axis, indicating potential use in therapeutic treatment of ulcerative colitis. Copyright © 2017 Elsevier Inc. All rights reserved.

Visualizing chemical functionality in plant cell walls

DOE PAGES

Zeng, Yining; Himmel, Michael E.; Ding, Shi-You

2017-11-30

Understanding plant cell wall cross-linking chemistry and polymeric architecture is key to the efficient utilization of biomass in all prospects from rational genetic modification to downstream chemical and biological conversion to produce fuels and value chemicals. In fact, the bulk properties of cell wall recalcitrance are collectively determined by its chemical features over a wide range of length scales from tissue, cellular to polymeric architectures. Microscopic visualization of cell walls from the nanometer to the micrometer scale offers an in situ approach to study their chemical functionality considering its spatial and chemical complexity, particularly the capabilities of characterizing biomass non-destructivelymore » and in real-time during conversion processes. Microscopic characterization has revealed heterogeneity in the distribution of chemical features, which would otherwise be hidden in bulk analysis. Key microscopic features include cell wall type, wall layering, and wall composition - especially cellulose and lignin distributions. Microscopic tools, such as atomic force microscopy, stimulated Raman scattering microscopy, and fluorescence microscopy, have been applied to investigations of cell wall structure and chemistry from the native wall to wall treated by thermal chemical pretreatment and enzymatic hydrolysis. While advancing our current understanding of plant cell wall recalcitrance and deconstruction, microscopic tools with improved spatial resolution will steadily enhance our fundamental understanding of cell wall function.« less

Visualizing chemical functionality in plant cell walls

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zeng, Yining; Himmel, Michael E.; Ding, Shi-You

Understanding plant cell wall cross-linking chemistry and polymeric architecture is key to the efficient utilization of biomass in all prospects from rational genetic modification to downstream chemical and biological conversion to produce fuels and value chemicals. In fact, the bulk properties of cell wall recalcitrance are collectively determined by its chemical features over a wide range of length scales from tissue, cellular to polymeric architectures. Microscopic visualization of cell walls from the nanometer to the micrometer scale offers an in situ approach to study their chemical functionality considering its spatial and chemical complexity, particularly the capabilities of characterizing biomass non-destructivelymore » and in real-time during conversion processes. Microscopic characterization has revealed heterogeneity in the distribution of chemical features, which would otherwise be hidden in bulk analysis. Key microscopic features include cell wall type, wall layering, and wall composition - especially cellulose and lignin distributions. Microscopic tools, such as atomic force microscopy, stimulated Raman scattering microscopy, and fluorescence microscopy, have been applied to investigations of cell wall structure and chemistry from the native wall to wall treated by thermal chemical pretreatment and enzymatic hydrolysis. While advancing our current understanding of plant cell wall recalcitrance and deconstruction, microscopic tools with improved spatial resolution will steadily enhance our fundamental understanding of cell wall function.« less

Visualizing chemical functionality in plant cell walls.

PubMed

Zeng, Yining; Himmel, Michael E; Ding, Shi-You

2017-01-01

Understanding plant cell wall cross-linking chemistry and polymeric architecture is key to the efficient utilization of biomass in all prospects from rational genetic modification to downstream chemical and biological conversion to produce fuels and value chemicals. In fact, the bulk properties of cell wall recalcitrance are collectively determined by its chemical features over a wide range of length scales from tissue, cellular to polymeric architectures. Microscopic visualization of cell walls from the nanometer to the micrometer scale offers an in situ approach to study their chemical functionality considering its spatial and chemical complexity, particularly the capabilities of characterizing biomass non-destructively and in real-time during conversion processes. Microscopic characterization has revealed heterogeneity in the distribution of chemical features, which would otherwise be hidden in bulk analysis. Key microscopic features include cell wall type, wall layering, and wall composition-especially cellulose and lignin distributions. Microscopic tools, such as atomic force microscopy, stimulated Raman scattering microscopy, and fluorescence microscopy, have been applied to investigations of cell wall structure and chemistry from the native wall to wall treated by thermal chemical pretreatment and enzymatic hydrolysis. While advancing our current understanding of plant cell wall recalcitrance and deconstruction, microscopic tools with improved spatial resolution will steadily enhance our fundamental understanding of cell wall function.

Comparison of Glaucoma Progression Detection by Optical Coherence Tomography and Visual Field.

PubMed

Zhang, Xinbo; Dastiridou, Anna; Francis, Brian A; Tan, Ou; Varma, Rohit; Greenfield, David S; Schuman, Joel S; Huang, David

2017-12-01

To compare longitudinal glaucoma progression detection using optical coherence tomography (OCT) and visual field (VF). Validity assessment. We analyzed subjects with more than 4 semi-annual follow-up visits (every 6 months) in the multicenter Advanced Imaging for Glaucoma Study. Fourier-domain optical coherence tomography (OCT) was used to map the thickness of the peripapillary retinal nerve fiber layer (NFL) and ganglion cell complex (GCC). OCT-based progression detection was defined as a significant negative trend for either NFL or GCC. VF progression was reached if either the event or trend analysis reached significance. The analysis included 356 glaucoma suspect/preperimetric glaucoma (GS/PPG) eyes and 153 perimetric glaucoma (PG) eyes. Follow-up length was 54.1 ± 16.2 months for GS/PPG eyes and 56.7 ± 16.0 for PG eyes. Progression was detected in 62.1% of PG eyes and 59.8% of GS/PPG eyes by OCT, significantly (P < .001) more than the detection rate of 41.8% and 27.3% by VF. In severity-stratified analysis of PG eyes, OCT had significantly higher detection rate than VF in mild PG (63.1% vs. 38.7%, P < .001), but not in moderate and advanced PG. The rate of NFL thinning slowed dramatically in advanced PG, but GCC thinning rate remained relatively steady and allowed good progression detection even in advanced disease. The Kaplan-Meier time-to-event analyses showed that OCT detected progression earlier than VF in both PG and GS/PPG groups. OCT is more sensitive than VF for the detection of progression in early glaucoma. While the utility of NFL declines in advanced glaucoma, GCC remains a sensitive progression detector from early to advanced stages. Copyright © 2017 Elsevier Inc. All rights reserved.

Formation of wood secondary cell wall may involve two type cellulose synthase complexes in Populus.

PubMed

Xi, Wang; Song, Dongliang; Sun, Jiayan; Shen, Junhui; Li, Laigeng

2017-03-01

Cellulose biosynthesis is mediated by cellulose synthases (CesAs), which constitute into rosette-like cellulose synthase complexe (CSC) on the plasma membrane. Two types of CSCs in Arabidopsis are believed to be involved in cellulose synthesis in the primary cell wall and secondary cell walls, respectively. In this work, we found that the two type CSCs participated cellulose biosynthesis in differentiating xylem cells undergoing secondary cell wall thickening in Populus. During the cell wall thickening process, expression of one type CSC genes increased while expression of the other type CSC genes decreased. Suppression of different type CSC genes both affected the wall-thickening and disrupted the multilaminar structure of the secondary cell walls. When CesA7A was suppressed, crystalline cellulose content was reduced, which, however, showed an increase when CesA3D was suppressed. The CesA suppression also affected cellulose digestibility of the wood cell walls. The results suggest that two type CSCs are involved in coordinating the cellulose biosynthesis in formation of the multilaminar structure in Populus wood secondary cell walls.

Mechanosensation Dynamically Coordinates Polar Growth and Cell Wall Assembly to Promote Cell Survival.

PubMed

Davì, Valeria; Tanimoto, Hirokazu; Ershov, Dmitry; Haupt, Armin; De Belly, Henry; Le Borgne, Rémi; Couturier, Etienne; Boudaoud, Arezki; Minc, Nicolas

2018-04-23

How growing cells cope with size expansion while ensuring mechanical integrity is not known. In walled cells, such as those of microbes and plants, growth and viability are both supported by a thin and rigid encasing cell wall (CW). We deciphered the dynamic mechanisms controlling wall surface assembly during cell growth, using a sub-resolution microscopy approach to monitor CW thickness in live rod-shaped fission yeast cells. We found that polar cell growth yielded wall thinning and that thickness negatively influenced growth. Thickness at growing tips exhibited a fluctuating behavior with thickening phases followed by thinning phases, indicative of a delayed feedback promoting thickness homeostasis. This feedback was mediated by mechanosensing through the CW integrity pathway, which probes strain in the wall to adjust synthase localization and activity to surface growth. Mutants defective in thickness homeostasis lysed by rupturing the wall, demonstrating its pivotal role for walled cell survival. Copyright © 2018 Elsevier Inc. All rights reserved.

The Cell Wall-Associated Proteins in the Dimorphic Pathogenic Species of Paracoccidioides.

PubMed

Puccia, Rosana; Vallejo, Milene C; Longo, Larissa V G

2017-01-01

Paracoccidioides brasiliensis and P. lutzii cause human paracoccidioidomycosis (PCM). They are dimorphic ascomycetes that grow as filaments at mild temperatures up to 28oC and as multibudding pathogenic yeast cells at 37oC. Components of the fungal cell wall have an important role in the interaction with the host because they compose the cell outermost layer. The Paracoccidioides cell wall is composed mainly of polysaccharides, but it also contains proportionally smaller rates of proteins, lipids, and melanin. The polysaccharide cell wall composition and structure of Paracoccidioides yeast cells, filamentous and transition phases were studied in detail in the past. Other cell wall components have been better analyzed in the last decades. The present work gives to the readers a detailed updated view of cell wall-associated proteins. Proteins that have been localized at the cell wall compartment using antibodies are individually addressed. We also make an overview about PCM, the Paracoccidioides cell wall structure, secretion mechanisms, and fungal extracellular vesicles. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Unexpected features of exponentially growing Tobacco Bright Yellow-2 cell suspension culture in relation to excreted extracellular polysaccharides and cell wall composition.

PubMed

Issawi, Mohammad; Muhieddine, Mohammad; Girard, Celine; Sol, Vincent; Riou, Catherine

2017-10-01

This article presents a new insight about TBY-2 cells; from extracellular polysaccharides secretion to cell wall composition during cell suspension culture. In the medium of cells taken 2 days after dilution (end of lag phase), a two unit pH decrease from 5.38 to 3.45 was observed and linked to a high uronic acid (UA) amount secretion (47.8%) while, in 4 and 7 day-old spent media, pH increased and UA amounts decreased 35.6 and 42.3% UA, respectively. To attain deeper knowledge of the putative link between extracellular polysaccharide excretion and cell wall composition, we determined cell wall UA and neutral sugar composition of cells from D2 to D12 cultures. While cell walls from D2 and D3 cells contained a large amount of uronic acid (twice as much as the other analysed cell walls), similar amounts of neutral sugar were detected in cells from lag to end of exponential phase cells suggesting an enriched pectin network in young cultures. Indeed, monosaccharide composition analysis leads to an estimated percentage of pectins of 56% for D3 cell wall against 45% D7 cell walls indicating that the cells at the mid-exponential growth phase re-organized their cell wall linked to a decrease in secreted UA that finally led to a stabilization of the spent medium pH to 5.4. In conclusion, TBY-2 cell suspension from lag to stationary phase showed cell wall remodeling that could be of interest in drug interaction and internalization study.

Investigation of Plant Cell Wall Properties: A Study of Contributions from the Nanoscale to the Macroscale Impacting Cell Wall Recalcitrance

NASA Astrophysics Data System (ADS)

Crowe, Jacob Dillon

Biochemical conversion of lignocellulosic biomass to fuel ethanol is one of a few challenging, yet opportune technologies that can reduce the consumption of petroleum-derived transportation fuels, while providing parallel reductions in greenhouse gas emissions. Biomass recalcitrance, or resistance to deconstruction, is a major technical challenge that limits effective conversion of biomass to fermentable sugars, often requiring a costly thermochemical pretreatment step to improve biomass deconstruction. Biomass recalcitrance is imparted largely by the secondary cell wall, a complex polymeric matrix of cell wall polysaccharides and aromatic heteropolymers, that provides structural stability to cells and enables plant upright growth. Polymers within the cell wall can vary both compositionally and structurally depending upon plant species and anatomical fraction, and have varied responses to thermochemical pretreatments. Cell wall properties impacting recalcitrance are still not well understood, and as a result, the goal of this dissertation is to investigate structural features of the cell wall contributing to recalcitrance (1) in diverse anatomical fractions of a single species, (2) in response to diverse pretreatments, and (3) resulting from genetic modification. In the first study, feedstock cell wall heterogeneity was investigated in anatomical (stem, leaf sheaths, and leaf blades) and internode fractions of switchgrass at varying tissue maturities. Lignin content was observed as the key contributor to recalcitrance in maturing stem tissues only, with non-cellulosic substituted glucuronoarabinoxylans and pectic polysaccharides contributing to cell wall recalcitrance in leaf sheath and leaf blades. Hydroxycinnamate (i.e., saponifiable p-coumarate and ferulate) content along with xylan and pectin extractability decreased with tissue maturity, suggesting lignification is only one component imparting maturity specific cell wall recalcitrance. In the second study, alkaline hydrogen peroxide and liquid hot water pretreatments were shown to alter structural properties impacting nanoscale porosity in corn stover. Delignification by alkaline hydrogen peroxide pretreatment decreased cell wall rigidity, with subsequent cell wall swelling resulting in increased nanoscale porosity and improved enzymatic hydrolysis compared to limited swelling and increased accessible surface areas observed in liquid hot water pretreated biomass. The volume accessible to a 90 A dextran probe within the cell wall was found to be positively correlated to both enzyme binding and glucose hydrolysis yields, indicating cell wall porosity is a key contributor to effective hydrolysis yields. In the third study, the effect of altered xylan content and structure was investigated in irregular xylem (irx) Arabidopsis thaliana mutants to understand the role xylan plays in secondary cell wall development and organization. Higher xylan extractability and lower cellulose crystallinity observed in irx9 and irx15 irx15-L mutants compared to wild type indicated altered xylan integration into the secondary cell wall. Nanoscale cell wall organization observed using multiple microscopy techniques was impacted to some extent in all irx mutants, with disorganized cellulose microfibril layers in sclerenchyma secondary cell walls likely resulting from irregular xylan structure and content. Irregular secondary cell wall microfibril layers showed heterogeneous nanomechanical properties compared to wild type, which translated to mechanical deficiencies observed in stem tensile tests. These results suggest nanoscale defects in cell wall strength can correspond to macroscale phenotypes.

Proteomic Analysis of Kveim Reagent Identifies Targets of Cellular Immunity in Sarcoidosis

PubMed Central

Parker, Robert; Siddiqui, Nazneen; Potiphar, Lee; Goldin, Rob; Timms, John F.; Wells, Athol U.; Kon, Onn M.; Wickremasinghe, Melissa; Mitchell, Donald; Weeks, Mark E.; Lalvani, Ajit

2017-01-01

Background Kveim-reagent (Kv) skin testing was a historical method of diagnosing sarcoidosis. Intradermal injection of treated sarcoidosis spleen tissue resulted in a granuloma response at injection site by 4–6 weeks. Previous work indicates proteins as the possible trigger of this reaction. We aimed to identify Kv-specific proteins and characterise the ex vivo response of Peripheral Blood Mononuclear Cells (PBMCs) from sarcoidosis, tuberculosis and healthy control patients when stimulated with both Kv and selected Kv-specific proteins. Methods Kv extracts were separated by 1D-SDS-PAGE and 2D-DIGE and then underwent mass spectrometric analysis for protein identification. Sarcoidosis and control PBMCs were first stimulated with Kv and then with three selected recombinant protein candidates which were identified from the proteomic analysis. PBMC secreted cytokines were subsequently measured by Multiplex Cytokine Assay. Results We observed significantly increased IFN-γ and TNF-α secretion from Kv-stimulated PBMCs of sarcoidosis patients vs. PBMCs from healthy volunteers (IFN-γ: 207.2 pg/mL vs. 3.86 pg/mL, p = 0.0018; TNF-α: 2375 pg/mL vs. 42.82 pg/mL, p = 0.0003). Through proteomic approaches we then identified 74 sarcoidosis tissue-specific proteins. Of these, 3 proteins (vimentin, tubulin and alpha-actinin-4) were identified using both 1D-SDS-PAGE and 2D-DIGE. Data are available via ProteomeXchange with identifier PXD005150. Increased cytokine secretion was subsequently observed with vimentin stimulation of sarcoidosis PBMCs vs. tuberculosis PBMCs (IFN-γ: 396.6 pg/mL vs 0.1 pg/mL, p = 0.0009; TNF-α: 1139 pg/mL vs 0.1 pg/mL, p<0.0001). This finding was also observed in vimentin stimulation of sarcoidosis PBMCs compared to PBMCs from healthy controls (IFN-γ: 396.6 pg/mL vs. 0.1 pg/mL, p = 0.014; TNF-α: 1139 pg/mL vs 42.29 pg/mL, p = 0.027). No difference was found in cytokine secretion between sarcoidosis and control PBMCs when stimulated with either tubulin or alpha-actinin-4. Conclusions Stimulation with both Kveim reagent and vimentin induces a specific pro-inflammatory cytokine secretion from sarcoidosis PBMCs. Further investigation of cellular immune responses to Kveim-specific proteins may identify novel biomarkers to assist the diagnosis of sarcoidosis. PMID:28114394

The Acid Growth Theory of auxin-induced cell elongation is alive and well

NASA Technical Reports Server (NTRS)

Rayle, D. L.; Cleland, R. E.

1992-01-01

Plant cells elongate irreversibly only when load-bearing bonds in the walls are cleaved. Auxin causes the elongation of stem and coleoptile cells by promoting wall loosening via cleavage of these bonds. This process may be coupled with the intercalation of new cell wall polymers. Because the primary site of auxin action appears to be the plasma membrane or some intracellular site, and wall loosening is extracellular, there must be communication between the protoplast and the wall. Some "wall-loosening factor" must be exported from auxin-impacted cells, which sets into motion the wall loosening events. About 20 years ago, it was suggested that the wall-loosening factor is hydrogen ions. This idea and subsequent supporting data gave rise to the Acid Growth Theory, which states that when exposed to auxin, susceptible cells excrete protons into the wall (apoplast) at an enhanced rate, resulting in a decrease in apoplastic pH. The lowered wall pH then activates wall-loosening processes, the precise nature of which is unknown. Because exogenous acid causes a transient (1-4 h) increase in growth rate, auxin must also mediate events in addition to wall acidification for growth to continue for an extended period of time. These events may include osmoregulation, cell wall synthesis, and maintenance of the capacity of walls to undergo acid-induced wall loosening. At present, we do not know if these phenomena are tightly coupled to wall acidification or if they are the products of multiple independent signal transduction pathways.

PopZ identifies the new pole, and PodJ identifies the old pole during polar growth in Agrobacterium tumefaciens

PubMed Central

Grangeon, Romain; Zupan, John R.; Anderson-Furgeson, James; Zambryski, Patricia C.

2015-01-01

Agrobacterium tumefaciens elongates by addition of peptidoglycan (PG) only at the pole created by cell division, the growth pole, whereas the opposite pole, the old pole, is inactive for PG synthesis. How Agrobacterium assigns and maintains pole asymmetry is not understood. Here, we investigated whether polar growth is correlated with novel pole-specific localization of proteins implicated in a variety of growth and cell division pathways. The cell cycle of A. tumefaciens was monitored by time-lapse and superresolution microscopy to image the localization of A. tumefaciens homologs of proteins involved in cell division, PG synthesis and pole identity. FtsZ and FtsA accumulate at the growth pole during elongation, and improved imaging reveals FtsZ disappears from the growth pole and accumulates at the midcell before FtsA. The L,D-transpeptidase Atu0845 was detected mainly at the growth pole. A. tumefaciens specific pole-organizing protein (Pop) PopZAt and polar organelle development (Pod) protein PodJAt exhibited dynamic yet distinct behavior. PopZAt was found exclusively at the growing pole and quickly switches to the new growth poles of both siblings immediately after septation. PodJAt is initially at the old pole but then also accumulates at the growth pole as the cell cycle progresses suggesting that PodJAt may mediate the transition of the growth pole to an old pole. Thus, PopZAt is a marker for growth pole identity, whereas PodJAt identifies the old pole. PMID:26324921

Cell Wall Ultrastructure of Stem Wood, Roots, and Needles of a Conifer Varies in Response to Moisture Availability

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pattathil, Sivakumar; Ingwers, Miles W.; Victoriano, Olivia L.

The composition, integrity, and architecture of the macromolecular matrix of cell walls, collectively referred to as cell wall ultrastructure, exhibits variation across species and organs and among cell types within organs. Indirect approaches have suggested that modifications to cell wall ultrastructure occur in response to abiotic stress; however, modifications have not been directly observed. Glycome profiling was used to study cell wall ultrastructure by examining variation in composition and extractability of non-cellulosic glycans in cell walls of stem wood, roots, and needles of loblolly pine saplings exposed to high and low soil moisture. Soil moisture influenced physiological processes and themore » overall composition and extractability of cell wall components differed as a function of soil moisture treatments. The strongest response of cell wall ultrastructure to soil moisture was increased extractability of pectic backbone epitopes in the low soil moisture treatment. The higher abundance of these pectic backbone epitopes in the oxalate extract indicate that the loosening of cell wall pectic components could be associated with the release of pectic signals as a stress response. The increased extractability of pectic backbone epitopes in response to low soil moisture availability was more pronounced in stem wood than in roots or needles. Additional responses to low soil moisture availability were observed in lignin associated carbohydrates released in chlorite extracts of stem wood, including an increased abundance of pectic arabinogalactan epitopes. Overall, these results indicate that cell walls of loblolly pine organs undergo changes in their ultrastructural composition and extractability as a response to soil moisture availability and that cell walls of the stem wood are more responsive to low soil moisture availability compared to cell walls of roots and needles. In conclusion, to our knowledge, this is the first direct evidence, delineated by glycomic analyses, that abiotic stress affects cell wall ultrastructure. This study is also unique in that glycome profiling of pine needles has never before been reported.« less

Cell Wall Ultrastructure of Stem Wood, Roots, and Needles of a Conifer Varies in Response to Moisture Availability.

PubMed

Pattathil, Sivakumar; Ingwers, Miles W; Victoriano, Olivia L; Kandemkavil, Sindhu; McGuire, Mary Anne; Teskey, Robert O; Aubrey, Doug P

2016-01-01

The composition, integrity, and architecture of the macromolecular matrix of cell walls, collectively referred to as cell wall ultrastructure, exhibits variation across species and organs and among cell types within organs. Indirect approaches have suggested that modifications to cell wall ultrastructure occur in response to abiotic stress; however, modifications have not been directly observed. Glycome profiling was used to study cell wall ultrastructure by examining variation in composition and extractability of non-cellulosic glycans in cell walls of stem wood, roots, and needles of loblolly pine saplings exposed to high and low soil moisture. Soil moisture influenced physiological processes and the overall composition and extractability of cell wall components differed as a function of soil moisture treatments. The strongest response of cell wall ultrastructure to soil moisture was increased extractability of pectic backbone epitopes in the low soil moisture treatment. The higher abundance of these pectic backbone epitopes in the oxalate extract indicate that the loosening of cell wall pectic components could be associated with the release of pectic signals as a stress response. The increased extractability of pectic backbone epitopes in response to low soil moisture availability was more pronounced in stem wood than in roots or needles. Additional responses to low soil moisture availability were observed in lignin-associated carbohydrates released in chlorite extracts of stem wood, including an increased abundance of pectic arabinogalactan epitopes. Overall, these results indicate that cell walls of loblolly pine organs undergo changes in their ultrastructural composition and extractability as a response to soil moisture availability and that cell walls of the stem wood are more responsive to low soil moisture availability compared to cell walls of roots and needles. To our knowledge, this is the first direct evidence, delineated by glycomic analyses, that abiotic stress affects cell wall ultrastructure. This study is also unique in that glycome profiling of pine needles has never before been reported.

Cell Wall Ultrastructure of Stem Wood, Roots, and Needles of a Conifer Varies in Response to Moisture Availability

DOE PAGES

Pattathil, Sivakumar; Ingwers, Miles W.; Victoriano, Olivia L.; ...

2016-06-24

The composition, integrity, and architecture of the macromolecular matrix of cell walls, collectively referred to as cell wall ultrastructure, exhibits variation across species and organs and among cell types within organs. Indirect approaches have suggested that modifications to cell wall ultrastructure occur in response to abiotic stress; however, modifications have not been directly observed. Glycome profiling was used to study cell wall ultrastructure by examining variation in composition and extractability of non-cellulosic glycans in cell walls of stem wood, roots, and needles of loblolly pine saplings exposed to high and low soil moisture. Soil moisture influenced physiological processes and themore » overall composition and extractability of cell wall components differed as a function of soil moisture treatments. The strongest response of cell wall ultrastructure to soil moisture was increased extractability of pectic backbone epitopes in the low soil moisture treatment. The higher abundance of these pectic backbone epitopes in the oxalate extract indicate that the loosening of cell wall pectic components could be associated with the release of pectic signals as a stress response. The increased extractability of pectic backbone epitopes in response to low soil moisture availability was more pronounced in stem wood than in roots or needles. Additional responses to low soil moisture availability were observed in lignin associated carbohydrates released in chlorite extracts of stem wood, including an increased abundance of pectic arabinogalactan epitopes. Overall, these results indicate that cell walls of loblolly pine organs undergo changes in their ultrastructural composition and extractability as a response to soil moisture availability and that cell walls of the stem wood are more responsive to low soil moisture availability compared to cell walls of roots and needles. In conclusion, to our knowledge, this is the first direct evidence, delineated by glycomic analyses, that abiotic stress affects cell wall ultrastructure. This study is also unique in that glycome profiling of pine needles has never before been reported.« less

Dual delivery systems based on polyamine analog BENSpm as prodrug and gene delivery vectors

NASA Astrophysics Data System (ADS)

Zhu, Yu

Combination drug and gene therapy shows promise in cancer treatment. However, the success of such strategy requires careful selection of the therapeutic agents, as well as development of efficient delivery vectors. BENSpm (N 1, N11-bisethylnorspermine), a polyamine analogue targeting the intracellular polyamine pathway, draws our special attention because of the following reasons: (1) polyamine pathway is frequently dysregulated in cancer; (2) BENSpm exhibits multiple functions to interfere with the polyamine pathway, such as to up-regulate polyamine metabolism enzymes and down-regulate polyamine biosynthesis enzymes. Therefore BENSpm depletes all natural polyamines and leads to apoptosis and cell growth inhibition in a wide range of cancers; (3) preclinical studies proved that BENSpm can act synergistically with various chemotherapy agents, making it a promising candidate in combination therapy; (4) multiple positive charges in BENSpm enable it as a suitable building block for cationic polymers, which can be further applied to gene delivery. In this dissertation, our goal was to design dual-function delivery vector based on BENSpm that can function as a gene delivery vector and, after intracellular degradation, as an active anticancer agent targeting dysregulated polyamine metabolism. We first demonstrated strong synergism between BENSpm and a potential therapeutic gene product TRAIL. Strong synergism was obtained in both estrogen-dependent MCF-7 breast cancer cells and triple-negative MDA-MB-231 breast cancer cells. Significant dose reduction of TRAIL in combination with BENSpm in MDA-MB-231 cells, together with the fact that BENSpm rendered MCF-7 cells more sensitive to TRAIL treatment verified our rationale of designing BENSpm-based delivery platform. This was expected to be beneficial for overcoming drug resistance in chemotherapy, as well as boosting the therapeutic effect of therapeutic genes. We first designed a lipid-based BENSpm dual vector (Lipo-SS-BEN) capable of intracellular release of BENSpm using thiolytically sensitive dithiobenzyl carbamate linker. Similar activity on SSAT enzyme induction by Lipo-SS-BEN compared with BENSpm free drug verified the success of this prodrug design. Biodegradability of Lipo-SS-BEN contributed to decreased toxicity compared with nondegradable control LipoBEN. However, decreased enhancement of TRAIL activity was observed for Lipo-SS-BEN when compared with BENSpm, indicating that the lipid-related toxicity diminished the synergism. In addition, compared with LipoBEN and DOTAP, decreased transfection efficiency of Lipo-SS-BEN demonstrated instability of Lipo-SS-BEN in extracellular environment. In order to design a dual delivery vector with reduced vector toxicity and improved linker stability, we employed dendritic polyglycerol (PG) as a safe carrier backbone, onto which BENSpm was conjugated through carbamate linkage (PG-BEN). Polymers with norspermine (PG-Nor) shell and amine-terminated PG (PG-NH2) were synthesized as controls. The BENSpm dual vector PG-BEN demonstrated superior gene delivery function, and showed decreased toxicity compared with the control polymers. However, compared with BENSpm, which depleted all natural polyamines, PG-BEN only down-regulated intracellular putrescine levels. In addition, no free BENSpm was detected in PG-BEN treated cells. These results suggested that in order to take full advantage of BENSpm anticancer activity, alternative linker chemistry needs to be further explored. We then incorporated bis(2-hydroxyethyl) disulfide as a self-immolative linker to synthesize polymer prodrugs of BENSpm (DSS-BEN). The proposed mechanism of BENSpm release from DSS-BEN contains two steps: disulfide bond is first cleaved in the reducing intracellular space, then the intermediate further undergoes slow intramolecular cyclization to release free BENSpm. Cell line-dependent BENSpm release after DSS-BEN treatment was observed using HPLC analysis, demonstrating the success of our linker strategy. DSS-BEN showed comparable transfection efficiency as polyethylenimine and showed decreased toxicity in several cell lines compared with the nondegradable control DCC-BEN. We further demonstrated that DSS-BEN could act synergistically with several therapeutic agents, making it a promising delivery platform for combination therapy in cancer. In all, we have successfully developed a dual delivery vector based on BENSpm, which fulfills its function as a gene delivery vector as well as a prodrug of BENSpm, and possesses synergistic potential to augment the effect of the co-delivered agents.

Vesicles between plasma membrane and cell wall prior to visible senescence of Iris and Dendrobium flowers.

PubMed

Kamdee, Channatika; Kirasak, Kanjana; Ketsa, Saichol; van Doorn, Wouter G

2015-09-01

Cut Iris flowers (Iris x hollandica, cv. Blue Magic) show visible senescence about two days after full opening. Epidermal cells of the outer tepals collapse due to programmed cell death (PCD). Transmission electron microscopy (TEM) showed irregular swelling of the cell walls, starting prior to cell collapse. Compared to cells in flowers that had just opened, wall thickness increased up to tenfold prior to cell death. Fibrils were visible in the swollen walls. After cell death very little of the cell wall remained. Prior to and during visible wall swelling, vesicles (paramural bodies) were observed between the plasma membrane and the cell walls. The vesicles were also found in groups and were accompanied by amorphous substance. They usually showed a single membrane, and had a variety of diameters and electron densities. Cut Dendrobium hybrid cv. Lucky Duan flowers exhibited visible senescence about 14 days after full flower opening. Paramural bodies were also found in Dendrobium tepal epidermis and mesophyll cells, related to wall swelling and degradation. Although alternative explanations are well possible, it is hypothesized that paramural bodies carry enzymes involved in cell wall breakdown. The literature has not yet reported such bodies in association with senescence/PCD. Copyright © 2015 Elsevier GmbH. All rights reserved.

Changes in cell wall polysaccharide composition, gene transcription and alternative splicing in germinating barley embryos.

PubMed

Zhang, Qisen; Zhang, Xiaoqi; Pettolino, Filomena; Zhou, Gaofeng; Li, Chengdao

2016-02-01

Barley (Hordeum vulgare L.) seed germination initiates many important biological processes such as DNA, membrane and mitochondrial repairs. However, little is known on cell wall modifications in germinating embryos. We have investigated cell wall polysaccharide composition change, gene transcription and alternative splicing events in four barley varieties at 24h and 48 h germination. Cell wall components in germinating barley embryos changed rapidly, with increases in cellulose and (1,3)(1,4)-β-D-glucan (20-100%) within 24h, but decreases in heteroxylan and arabinan (3-50%). There were also significant changes in the levels of type I arabinogalactans and heteromannans. Alternative splicing played very important roles in cell wall modifications. At least 22 cell wall transcripts were detected to undergo either alternative 3' splicing, alternative 5' splicing or intron retention type of alternative splicing. These genes coded enzymes catalyzing synthesis and degradation of cellulose, heteroxylan, (1,3)(1,4)-β-D-glucan and other cell wall polymers. Furthermore, transcriptional regulation also played very important roles in cell wall modifications. Transcript levels of primary wall cellulase synthase, heteroxylan synthesizing and nucleotide sugar inter-conversion genes were very high in germinating embryos. At least 50 cell wall genes changed transcript levels significantly. Expression patterns of many cell wall genes coincided with changes in polysaccharide composition. Our data showed that cell wall polysaccharide metabolism was very active in germinating barley embryos, which was regulated at both transcriptional and post-transcriptional levels. Copyright © 2015 Elsevier GmbH. All rights reserved.

Ovarian-Cell-Like Cells from Skin Stem Cells Restored Estradiol Production and Estrus Cycling in Ovariectomized Mice

PubMed Central

Park, Bong-Wook; Pan, Bo; Toms, Derek; Huynh, Evanna; Byun, June-Ho; Lee, Yeon-Mi; Shen, Wei

2014-01-01

Reduction of estradiol production and high serum concentrations of follicular stimulating hormone (FSH) are endocrine disorders associated with premature ovarian failure. Here, we report that transplantation of ovarian-like cells differentiated from stem cells restored endogenous serum estradiol levels. Stem cells were isolated from postnatal mouse skin and differentiated into ovarian-cell-like cells that are consistent with female germ, and ovarian follicle somatic cells. The ovarian-cell-like cells were transplanted into ovariectomized mice (Cell Trans), whereas control mice were subjected to bilateral ovariectomies without cell transplantation (OVX). Using vaginal cytology analysis, it was revealed that in 13 out of 19 Cell Trans mice, estrus cycles were restored around 8 weeks after cell transplantation and were maintained until 16 weeks post-transplantation, whereas in the OVX group, all mice were arrested at metestrus/diestrus of the estrus cycle. The uterine weight in the Cell Trans group was similar to sham operation mice (Sham OP), while severe uterine atrophy and a decreased uterine weight were observed in the OVX group. Histologically, ectopic follicle-like structures and blood vessels were found within and around the transplants. At 12–14 weeks after cell transplantation, mean serum estradiol level in Cell Trans mice (178.0±35 pg/mL) was comparable to that of the Sham OP group (188.9±29 pg/mL), whereas it was lower in the OVX group (59.0±4 pg/mL). Serum FSH concentration increased in the OVX group (1.62±0.32 ng/mL) compared with the Sham OP group (0.39±0.34 ng/mL). Cell Trans mice had a similar FSH level (0.94±0.23 ng/mL; P<0.05) to Sham OP mice. Our results suggest that ovarian somatic cells differentiated from stem cells are functional in vivo. In addition to providing insights into the function of ovarian somatic cells derived from stem cells, our study may offer potential therapeutic means for patients with hypo-estradiol levels like those encountered in premature ovarian failure. PMID:24593690

An integrated vector system for cellular studies of phage display-derived peptides.

PubMed

Voss, Stephan D; DeGrand, Alec M; Romeo, Giulio R; Cantley, Lewis C; Frangioni, John V

2002-09-15

Peptide phage display is a method by which large numbers of diverse peptides can be screened for binding to a target of interest. Even when successful, the rate-limiting step is usually validation of peptide bioactivity using living cells. In this paper, we describe an integrated system of vectors that expedites both the screening and the characterization processes. Library construction and screening is performed using an optimized type 3 phage display vector, mJ(1), which is shown to accept peptide libraries of at least 23 amino acids in length. Peptide coding sequences are shuttled from mJ(1) into one of three families of mammalian expression vectors for cell physiological studies. The vector pAL(1) expresses phage display-derived peptides as Gal4 DNA binding domain fusion proteins for transcriptional activation studies. The vectors pG(1), pG(1)N, and pG(1)C express phage display-derived peptides as green fluorescent protein fusions targeted to the entire cell, nucleus, or cytoplasm, respectively. The vector pAP(1) expresses phage display-derived peptides as fusions to secreted placental alkaline phosphatase. Such enzyme fusions can be used as highly sensitive affinity reagents for high-throughput assays and for cloning of peptide-binding cell surface receptors. Taken together, this system of vectors should facilitate the development of phage display-derived peptides into useful biomolecules.

Modelling cell wall growth using a fibre-reinforced hyperelastic-viscoplastic constitutive law

NASA Astrophysics Data System (ADS)

Huang, R.; Becker, A. A.; Jones, I. A.

2012-04-01

A fibre-reinforced hyperelastic-viscoplastic model using a finite strain Finite Element (FE) analysis is presented to study the expansive growth of cell walls. Based on the connections between biological concepts and plasticity theory, e.g. wall-loosening and plastic yield, wall-stiffening and plastic hardening, the modelling of cell wall growth is established within a framework of anisotropic viscoplasticity aiming to represent the corresponding biology-controlled behaviour of a cell wall. In order to model in vivo growth, special attention is paid to the differences between a living cell and an isolated wall. The proposed hyperelastic-viscoplastic theory provides a unique framework to clarify the interplay between cellulose microfibrils and cell wall matrix and how this interplay regulates sustainable growth in a particular direction while maintaining the mechanical strength of the cell walls by new material deposition. Moreover, the effect of temperature is taken into account. A numerical scheme is suggested and FE case studies are presented and compared with experimental data.

Microbacterium horti sp. nov., a bacterium isolated from Cucurbita maxima cultivating soil.

PubMed

Akter, Shahina; Park, Jae Hee; Yin, Chang Shik

2016-04-01

A novel bacterial strain THG-SL1(T) was isolated from a soil sample of Cucurbita maxima garden and was characterized by using a polyphasic approach. Cells were Gram-reaction-positive, non-motile and rod-shaped. The strain was aerobic, catalase positive and weakly positive for oxidase. Phylogenetic analysis based on 16S rRNA gene sequence analysis but it shared highest similarity with Microbacterium ginsengisoli KCTC 19189(T) (96.6 %), indicating that strain THG-SL1(T) belongs to the genus Microbacterium. The DNA G + C content of the isolate was 68.9 mol %. The major fatty acids were anteiso-C15: 0 (39.7 %), anteiso-C17: 0 (24.4 %) and iso-C16: 0 (18.5 %). The major polar lipids of strain THG-SL1(T) were phosphatidylglycerol (PG) and an unidentified glycolipid (GL). The predominant respiratory isoprenoid quinones were menaquinone-11 and menaquinone-12. The diamino acid in the cell-wall peptidoglycan was ornithine. Based on the results of polyphasic characterization, strain THG-SL1(T) represented a novel species within the genus Microbacterium, for which the name Microbacterium horti sp. nov. is proposed. The type strain is THG-SL1(T) (=KACC 18286(T)=CCTCC AB 2015117(T)).

Effect of alternative peritoneal dialysis solutions on cell viability, apoptosis/necrosis and cytokine expression in human monocytes.

PubMed

Plum, J; Lordnejad, M R; Grabensee, B

1998-07-01

Cellular function, cell viability and the cytokine network of human monocytes are influenced by the specific composition of peritoneal dialysis (PD) fluids. In an in vitro study using isolated human blood monocytes, we investigated the effect of peritoneal dialysates containing amino acids (Amino) or glucose polymer (Glu-poly) instead of glucose (Glu) as the osmotic agent, and bicarbonate (Bic) or PBS instead of lactate (Lac) as a buffer. The following parameters were studied: mitochondrial dehydrogenase activity (using the MTT assay), interleukin (IL)-6 and IL-8 release (ELISA) and cellular IL-6 mRNA expression after lipopolysaccharide (LPS) stimulation (using RT-PCR). FACS flow cytometry with annexin V and propidium iodide as markers and fluorescence microscopic methods were used to study the effects of the test fluids on cell necrosis and apoptosis. Glu/Lac pH 5.5 and Glu-poly/PBS pH 7.4 both significantly reduced mitochondrial dehydrogenase activity by more than 50% after 60 minutes of incubation (30.5 +/- 7.6%, 42.5 +/- 6.5%, referred to RPMI 1640 as 100%). Amino/Bic and Glu/Bic were both superior (Mtt assay > 63%). The rate of necrotic cells after 15 minutes of incubation measured by FACS was mostly increased with Glu/Lac pH 5.5 (29.9 +/- 4.0%). The rate of apoptotic cells, however, was not significantly different between the test solutions. The concentration of IL-6 in the supernatant of stimulated monocytes was highest with Glu/Bic (1023 +/- 278 pg/ml) and Amino/Bic (776 +/- 296 pg/ml) an lowest with Glu/lac pH 5.5 (46 +/- 22 pg/ml) and Glu-poly/PBS (32 +/- 13 pg/ml). IL-8 release from stimulated monocytes showed a similar pattern. Glu-poly/PBS showed a suppressive effect on IL-6 mRNA expression (ratio IL-6/beta-Actin, 0.4 +/- 0.25 vs. RPMI 1.5 +/- 3.6). Bicarbonate buffered solutions both with glucose or amino acids as osmotic agents were superior when regarding cell metabolism, viability and cytokine release, while lactate buffered solutions and Glu-poly/PBS showed some reduced biocompatibility pattern for monocytes in vitro.

(Hydroxyproline-rich glycoproteins of the plant cell wall)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Varner, J.E.

1990-01-01

We are studying the chemistry and architecture of plant cells walls, the extracellular matrices that taken together shape the plant and provide mechanical support for the plant. Cell walls are dynamic structures that regulate, or are the site of, many physiological processes, in addition to being the cells' first line of defense against invading pathogens. In the past year we have examined the role of the cell wall enzyme ascorbic acid oxidase as related to the structure of the wall and its possible interactions with hydroxyproline-rich glycoproteins of the wall.

A Structurally Specialized Uniform Wall Layer is Essential for Constructing Wall Ingrowth Papillae in Transfer Cells

PubMed Central

Xia, Xue; Zhang, Hui-Ming; Offler, Christina E.; Patrick, John W.

2017-01-01

Transfer cells are characterized by wall labyrinths with either a flange or reticulate architecture. A literature survey established that reticulate wall ingrowth papillae ubiquitously arise from a modified component of their wall labyrinth, termed the uniform wall layer; a structure absent from flange transfer cells. This finding sparked an investigation of the deposition characteristics and role of the uniform wall layer using a Vicia faba cotyledon culture system. On transfer of cotyledons to culture, their adaxial epidermal cells spontaneously trans-differentiate to a reticulate architecture comparable to their abaxial epidermal transfer cell counterparts formed in planta. Uniform wall layer construction commenced once adaxial epidermal cell expansion had ceased to overlay the original outer periclinal wall on its inner surface. In contrast to the dense ring-like lattice of cellulose microfibrils in the original primary wall, the uniform wall layer was characterized by a sparsely dispersed array of linear cellulose microfibrils. A re-modeled cortical microtubule array exerted no influence on uniform wall layer formation or on its cellulose microfibril organization. Surprisingly, formation of the uniform wall layer was not dependent upon depositing a cellulose scaffold. In contrast, uniform wall cellulose microfibrils were essential precursors for constructing wall ingrowth papillae. On converging to form wall ingrowth papillae, the cellulose microfibril diameters increased 3-fold. This event correlated with up-regulated differential, and transfer-cell specific, expression of VfCesA3B while transcript levels of other cellulose biosynthetic-related genes linked with primary wall construction were substantially down-regulated. PMID:29259611

A Structurally Specialized Uniform Wall Layer is Essential for Constructing Wall Ingrowth Papillae in Transfer Cells.

PubMed

Xia, Xue; Zhang, Hui-Ming; Offler, Christina E; Patrick, John W

2017-01-01

Transfer cells are characterized by wall labyrinths with either a flange or reticulate architecture. A literature survey established that reticulate wall ingrowth papillae ubiquitously arise from a modified component of their wall labyrinth, termed the uniform wall layer; a structure absent from flange transfer cells. This finding sparked an investigation of the deposition characteristics and role of the uniform wall layer using a Vicia faba cotyledon culture system. On transfer of cotyledons to culture, their adaxial epidermal cells spontaneously trans -differentiate to a reticulate architecture comparable to their abaxial epidermal transfer cell counterparts formed in planta . Uniform wall layer construction commenced once adaxial epidermal cell expansion had ceased to overlay the original outer periclinal wall on its inner surface. In contrast to the dense ring-like lattice of cellulose microfibrils in the original primary wall, the uniform wall layer was characterized by a sparsely dispersed array of linear cellulose microfibrils. A re-modeled cortical microtubule array exerted no influence on uniform wall layer formation or on its cellulose microfibril organization. Surprisingly, formation of the uniform wall layer was not dependent upon depositing a cellulose scaffold. In contrast, uniform wall cellulose microfibrils were essential precursors for constructing wall ingrowth papillae. On converging to form wall ingrowth papillae, the cellulose microfibril diameters increased 3-fold. This event correlated with up-regulated differential, and transfer-cell specific, expression of VfCesA3B while transcript levels of other cellulose biosynthetic-related genes linked with primary wall construction were substantially down-regulated.

Isolation of a novel cell wall architecture mutant of rice with defective Arabidopsis COBL4 ortholog BC1 required for regulated deposition of secondary cell wall components.

PubMed

Sato, Kanna; Suzuki, Ryu; Nishikubo, Nobuyuki; Takenouchi, Sachi; Ito, Sachiko; Nakano, Yoshimi; Nakaba, Satoshi; Sano, Yuzou; Funada, Ryo; Kajita, Shinya; Kitano, Hidemi; Katayama, Yoshihiro

2010-06-01

The plant secondary cell wall is a highly ordered structure composed of various polysaccharides, phenolic components and proteins. Its coordinated regulation of a number of complex metabolic pathways and assembly has not been resolved. To understand the molecular mechanisms that regulate secondary cell wall synthesis, we isolated a novel rice mutant, cell wall architecture1 (cwa1), that exhibits an irregular thickening pattern in the secondary cell wall of sclerenchyma, as well as culm brittleness and reduced cellulose content in mature internodes. Light and transmission electron microscopy revealed that the cwa1 mutant plant has regions of local aggregation in the secondary cell walls of the cortical fibers in its internodes, showing uneven thickness. Ultraviolet microscopic observation indicated that localization of cell wall phenolic components was perturbed and that these components abundantly deposited at the aggregated cell wall regions in sclerenchyma. Therefore, regulation of deposition and assembly of secondary cell wall materials, i.e. phenolic components, appear to be disturbed by mutation of the cwa1 gene. Genetic analysis showed that cwa1 is allelic to brittle culm1 (bc1), which encodes the glycosylphosphatidylinositol-anchored COBRA-like protein specifically in plants. BC1 is known as a regulator that controls the culm mechanical strength and cellulose content in the secondary cell walls of sclerenchyma, but the precise function of BC1 has not been resolved. Our results suggest that CWA1/BC1 has an essential role in assembling cell wall constituents at their appropriate sites, thereby enabling synthesis of solid and flexible internodes in rice.

Biomarkers in Acute Heart Failure – Cardiac And Kidney

PubMed Central

2015-01-01

Natriuretic peptides (NP) are well-validated aids in the diagnosis of acute decompensated heart failure (ADHF). In acute presentations, both brain natriuretic peptide (BNP) and N-terminal of the prohormone brain natriuretic peptide (NT-proBNP) offer high sensitivity (>90 %) and negative predictive values (>95 %) for ruling out ADHF at thresholds of 100 and 300 pg/ml, respectively. Plasma NP rise with age. For added rule-in performance age-adjusted thresholds (450 pg/ml for under 50 years, 900 pg/ml for 50–75 years and 1,800 pg/ml for those >75 years) can be applied to NT-proBNP results. Test performance (specificity and accuracy but not sensitivity) is clearly reduced by renal dysfunction and atrial fibrillation. Obesity offsets the threshold downwards (to ~50 pg/ml for BNP), but overall discrimination is preserved. Reliable markers for impending acute kidney injury in ADHF constitute an unmet need, with candidates, such as kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin, failing to perform sufficiently well, and new possibilities, including the cell cycle markers insulin growth factor binding protein 7 and tissue inhibitor of metalloproteinases type 2, remain the subject of research. PMID:28785442

Mechanical stimulation of skeletal muscle generates lipid-related second messengers by phospholipase activation

NASA Technical Reports Server (NTRS)

Vandenburgh, H. H.; Shansky, J.; Karlisch, P.; Solerssi, R. L.

1993-01-01

Repetitive mechanical stimulation of cultured avian skeletal muscle increases the synthesis of prostaglandins (PG) E2 and F2 alpha which regulate protein turnover rates and muscle cell growth. These stretch-induced PG increases are reduced in low extracellular calcium medium and by specific phospholipase inhibitors. Mechanical stimulation increases the breakdown rate of 3H-arachidonic acid labelled phospholipids, releasing free 3H-arachidonic acid, the rate-limiting precursor of PG synthesis. Mechanical stimulation also increases 3H-arachidonic acid labelled diacylglycerol formation and intracellular levels of inositol phosphates from myo-[2-3H]inositol labelled phospholipids. Phospholipase A2 (PLA2), phosphatidylinositol-specific phospholipase C (PLC), and phospholipase D (PLD) are all activated by stretch. The stretch-induced increases in PG production, 3H-arachidonic acid labelled phospholipid breakdown, and 3H-arachidonic acid labelled diacylglycerol formation occur independently of cellular electrical activity (tetrodotoxin insensitive) whereas the formation of inositol phosphates from myo-[2-3H]inositol labelled phospholipids is dependent on cellular electrical activity. These results indicate that mechanical stimulation increases the lipid-related second messengers arachidonic acid, diacylglycerol, and PG through activation of specific phospholipases such as PLA2 and PLD, but not by activation of phosphatidylinositol-specific PLC.

Cell Wall Remodeling by a Synthetic Analog Reveals Metabolic Adaptation in Vancomycin Resistant Enterococci.

PubMed

Pidgeon, Sean E; Pires, Marcos M

2017-07-21

Drug-resistant bacterial infections threaten to overburden our healthcare system and disrupt modern medicine. A large class of potent antibiotics, including vancomycin, operate by interfering with bacterial cell wall biosynthesis. Vancomycin-resistant enterococci (VRE) evade the blockage of cell wall biosynthesis by altering cell wall precursors, rendering them drug insensitive. Herein, we reveal the phenotypic plasticity and cell wall remodeling of VRE in response to vancomycin in live bacterial cells via a metabolic probe. A synthetic cell wall analog was designed and constructed to monitor cell wall structural alterations. Our results demonstrate that the biosynthetic pathway for vancomycin-resistant precursors can be hijacked by synthetic analogs to track the kinetics of phenotype induction. In addition, we leveraged this probe to interrogate the response of VRE cells to vancomycin analogs and a series of cell wall-targeted antibiotics. Finally, we describe a proof-of-principle strategy to visually inspect drug resistance induction. Based on our findings, we anticipate that our metabolic probe will play an important role in further elucidating the interplay among the enzymes involved in the VRE biosynthetic rewiring.

Do plant cell walls have a code?

PubMed

Tavares, Eveline Q P; Buckeridge, Marcos S

2015-12-01

A code is a set of rules that establish correspondence between two worlds, signs (consisting of encrypted information) and meaning (of the decrypted message). A third element, the adaptor, connects both worlds, assigning meaning to a code. We propose that a Glycomic Code exists in plant cell walls where signs are represented by monosaccharides and phenylpropanoids and meaning is cell wall architecture with its highly complex association of polymers. Cell wall biosynthetic mechanisms, structure, architecture and properties are addressed according to Code Biology perspective, focusing on how they oppose to cell wall deconstruction. Cell wall hydrolysis is mainly focused as a mechanism of decryption of the Glycomic Code. Evidence for encoded information in cell wall polymers fine structure is highlighted and the implications of the existence of the Glycomic Code are discussed. Aspects related to fine structure are responsible for polysaccharide packing and polymer-polymer interactions, affecting the final cell wall architecture. The question whether polymers assembly within a wall display similar properties as other biological macromolecules (i.e. proteins, DNA, histones) is addressed, i.e. do they display a code? Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Bacterial glycobiology: rhamnose-containing cell wall polysaccharides in Gram-positive bacteria

PubMed Central

Mistou, Michel-Yves; Sutcliffe, Iain C.; van Sorge, Nina M.

2016-01-01

The composition of the Gram-positive cell wall is typically described as containing peptidoglycan, proteins and essential secondary cell wall structures called teichoic acids, which comprise approximately half of the cell wall mass. The cell walls of many species within the genera Streptococcus, Enterococcus and Lactococcus contain large amounts of the sugar rhamnose, which is incorporated in cell wall-anchored polysaccharides (CWP) that possibly function as homologues of well-studied wall teichoic acids (WTA). The presence and chemical structure of many rhamnose-containing cell wall polysaccharides (RhaCWP) has sometimes been known for decades. In contrast to WTA, insight into the biosynthesis and functional role of RhaCWP has been lacking. Recent studies in human streptococcal and enterococcal pathogens have highlighted critical roles for these complex polysaccharides in bacterial cell wall architecture and pathogenesis. In this review, we provide an overview of the RhaCWP with regards to their biosynthesis, genetics and biological function in species most relevant to human health. We also briefly discuss how increased knowledge in this field can provide interesting leads for new therapeutic compounds and improve biotechnological applications. PMID:26975195

Bacterial glycobiology: rhamnose-containing cell wall polysaccharides in Gram-positive bacteria.

PubMed

Mistou, Michel-Yves; Sutcliffe, Iain C; van Sorge, Nina M

2016-07-01

The composition of the Gram-positive cell wall is typically described as containing peptidoglycan, proteins and essential secondary cell wall structures called teichoic acids, which comprise approximately half of the cell wall mass. The cell walls of many species within the genera Streptococcus, Enterococcus and Lactococcus contain large amounts of the sugar rhamnose, which is incorporated in cell wall-anchored polysaccharides (CWP) that possibly function as homologues of well-studied wall teichoic acids (WTA). The presence and chemical structure of many rhamnose-containing cell wall polysaccharides (RhaCWP) has sometimes been known for decades. In contrast to WTA, insight into the biosynthesis and functional role of RhaCWP has been lacking. Recent studies in human streptococcal and enterococcal pathogens have highlighted critical roles for these complex polysaccharides in bacterial cell wall architecture and pathogenesis. In this review, we provide an overview of the RhaCWP with regards to their biosynthesis, genetics and biological function in species most relevant to human health. We also briefly discuss how increased knowledge in this field can provide interesting leads for new therapeutic compounds and improve biotechnological applications. © FEMS 2016.

Synchrotron Time-Lapse Imaging of Lignocellulosic Biomass Hydrolysis: Tracking Enzyme Localization by Protein Autofluorescence and Biochemical Modification of Cell Walls by Microfluidic Infrared Microspectroscopy

PubMed Central

Devaux, Marie-Françoise; Jamme, Frédéric; André, William; Bouchet, Brigitte; Alvarado, Camille; Durand, Sylvie; Robert, Paul; Saulnier, Luc; Bonnin, Estelle; Guillon, Fabienne

2018-01-01

Tracking enzyme localization and following the local biochemical modification of the substrate should help explain the recalcitrance of lignocellulosic plant cell walls to enzymatic degradation. Time-lapse studies using conventional imaging require enzyme labeling and following the biochemical modifications of biopolymers found in plant cell walls, which cannot be easily achieved. In the present work, synchrotron facilities have been used to image the enzymatic degradation of lignocellulosic biomass without labeling the enzyme or the cell walls. Multichannel autofluorescence imaging of the protein and phenolic compounds after excitation at 275 nm highlighted the presence or absence of enzymes on cell walls and made it possible to track them during the reaction. Image analysis was used to quantify the fluorescence intensity variations. Consistent variations in the enzyme concentration were found locally for cell cavities and their surrounding cell walls. Microfluidic FT-IR microspectroscopy allowed for time-lapse tracking of local changes in the polysaccharides in cell walls during degradation. Hemicellulose degradation was found to occur prior to cellulose degradation using a Celluclast® preparation. Combining the fluorescence and FT-IR information yielded the conclusion that enzymes did not bind to lignified cell walls, which were consequently not degraded. Fluorescence multiscale imaging and FT-IR microspectroscopy showed an unexpected variability both in the initial biochemical composition and the degradation pattern, highlighting micro-domains in the cell wall of a given cell. Fluorescence intensity quantification showed that the enzymes were not evenly distributed, and their amount increased progressively on degradable cell walls. During degradation, adjacent cells were separated and the cell wall fragmented until complete degradation. PMID:29515611

Cell wall staining with Trypan blue enables quantitative analysis of morphological changes in yeast cells.

PubMed

Liesche, Johannes; Marek, Magdalena; Günther-Pomorski, Thomas

2015-01-01

Yeast cells are protected by a cell wall that plays an important role in the exchange of substances with the environment. The cell wall structure is dynamic and can adapt to different physiological states or environmental conditions. For the investigation of morphological changes, selective staining with fluorescent dyes is a valuable tool. Furthermore, cell wall staining is used to facilitate sub-cellular localization experiments with fluorescently-labeled proteins and the detection of yeast cells in non-fungal host tissues. Here, we report staining of Saccharomyces cerevisiae cell wall with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain Calcofluor White. Trypan Blue staining facilitated quantification of cell size and cell wall volume when utilizing the optical sectioning capacity of a confocal microscope. This enabled the quantification of morphological changes during growth under anaerobic conditions and in the presence of chemicals, demonstrating the potential of this approach for morphological investigations or screening assays.

Skin deposition and permeation of finasteride in vitro: effects of propylene glycol, ethanol and sodium lauryl sulfate.

PubMed

Limpongsa, Ekapol; Jaipakdee, Napaphak; Pongjanyakul, Thaned

2014-08-27

Abstract The objective of this study was to investigate the effects of propylene glycol (PG), ethanol (EtOH) and sodium lauryl sulfate (SLS) on the in vitro deposition and permeation of finasteride (FNS). A side-by-side diffusion cell mounted with a pig ear skin and a saturated solution of FNS in PG (10, 20% v/v), EtOH (10, 20% v/v) or SLS (0.5, 1% w/v) vehicles were used. Incorporation of PG, EtOH or SLS caused a significant increase in FNS solubility both in the solution and on the skin with SLS > EtOH > PG. The results obtained from skin deposition studies showed that the FNS deposition rate and time increased in the same order as that of the solubility. The deposition kinetics of FNS solubilized in PG, EtOH and SLS vehicles followed either zero-order, square-root-of-time or pseudo-first-order kinetic models depending on the type and concentration of the enhancer. The permeation studies demonstrated that FNS permeation fluxes were enhanced only by EtOH vehicles. These results suggest that PG and SLS could be used as deposition enhancers, while EtOH could be the effective permeation enhancer of FNS. The obtained results can be used as the considerable insights for formulating the topical and transdermal products of FNS.

A unified wall function for compressible turbulence modelling

NASA Astrophysics Data System (ADS)

Ong, K. C.; Chan, A.

2018-05-01

Turbulence modelling near the wall often requires a high mesh density clustered around the wall and the first cells adjacent to the wall to be placed in the viscous sublayer. As a result, the numerical stability is constrained by the smallest cell size and hence requires high computational overhead. In the present study, a unified wall function is developed which is valid for viscous sublayer, buffer sublayer and inertial sublayer, as well as including effects of compressibility, heat transfer and pressure gradient. The resulting wall function applies to compressible turbulence modelling for both isothermal and adiabatic wall boundary conditions with the non-zero pressure gradient. Two simple wall function algorithms are implemented for practical computation of isothermal and adiabatic wall boundary conditions. The numerical results show that the wall function evaluates the wall shear stress and turbulent quantities of wall adjacent cells at wide range of non-dimensional wall distance and alleviate the number and size of cells required.

Growth and cell wall changes in stem organs under microgravity and hypergravity conditions

NASA Astrophysics Data System (ADS)

Hoson, Takayuki; Soga, Kouichi; Wakabayashi, Kazuyuki; Kamisaka, Seiichiro

Gravity strongly influences plant growth and development, which is fundamentally brought about by modifications to the properties of the cell wall. We have examined the changes in growth and cell wall properties in seedling organs under hypergravity conditions produced by centrifugation and under microgravity conditions in space. Hypergravity stimuli have been shown to decrease the growth rate of various seedling organs. When hypergravity suppressed elongation growth, a decrease in cell wall extensibility (an increase in cell wall rigidity) was induced. Hypergravity has also been shown to increase cell wall thickness in various mate-rials. In addition, a polymerization of certain matrix polysaccharides was brought about by hypergravity: in dicotyledons hypergravity increased the molecular size of xyloglucans, whereas hypergravity increased that of 1,3,1,4-β-glucans in monocotyledonous Gramineae. These mod-ifications to cell wall metabolism may be responsible for a decrease in cell wall extensibility, leading to growth suppression under hypergravity conditions. How then does microgravity in-fluence growth and cell wall properties? Here, there was a possibility that microgravity might induce changes similar to those by hypergravity, because plants have evolved and adapted to 1 g condition for more than 400 million years. However, the changes observed under microgravity conditions in space were just opposite to those induced by hypergravity: stimulation of elonga-tion growth, an increase in cell wall extensibility, and a decrease in cell wall thickness as well as depolymerization of cell wall polysaccharides were brought about in space. Furthermore, growth and cell wall properties varied in proportion to the logarithm of the magnitude of grav-ity in the range from microgravity to hypergravity, as shown in the dose-response relation in light and hormonal responses. Thus, microgravity may be a `stress-less' environment for plant seedlings to grow and develop. Preliminary results obtained by recent Space Seed experiment in the Kibo Module on the International Space Station (PI: S. Kamisaka) suggest that this hypothesis is also applicable to mature Arabidopsis plants.

Indomethacin Inhibits Circulating PGE2 and Reverses Postexercise Suppression of Natural Killer Cell Activity

DTIC Science & Technology

1999-01-01

after the oral administration of a placebo, the PG inhibitor indomethacin (75 mg/day for 5 days), or naltrexone (reported elsewhere). Circulating...which blocks PGE2 biosynthe- sis via inhibition of cyclooxygenase activity (57). Maxi- mal suppression of PG production occurs with doses between 50...and 150 mg (1). In addition to the indepen- dent effects of PGE2 on NKCA, low circulating levels of PGE2 can synergize with endogenous glucocorticoids

Profiling the Hydrolysis of Isolated Grape Berry Skin Cell Walls by Purified Enzymes.

PubMed

Zietsman, Anscha J J; Moore, John P; Fangel, Jonatan U; Willats, William G T; Vivier, Melané A

2015-09-23

The unraveling of crushed grapes by maceration enzymes during winemaking is difficult to study because of the complex and rather undefined nature of both the substrate and the enzyme preparations. In this study we simplified both the substrate, by using isolated grape skin cell walls, and the enzyme preparations, by using purified enzymes in buffered conditions, to carefully follow the impact of the individual and combined enzymes on the grape skin cell walls. By using cell wall profiling techniques we could monitor the compositional changes in the grape cell wall polymers due to enzyme activity. Extensive enzymatic hydrolysis, achieved with a preparation of pectinases or pectinases combined with cellulase or hemicellulase enzymes, completely removed or drastically reduced levels of pectin polymers, whereas less extensive hydrolysis only opened up the cell wall structure and allowed extraction of polymers from within the cell wall layers. Synergistic enzyme activity was detectable as well as indications of specific cell wall polymer associations.

Outside-in control -Does plant cell wall integrity regulate cell cycle progression?

PubMed

Gigli-Bisceglia, Nora; Hamann, Thorsten

2018-04-13

During recent years it has become accepted that plant cell walls are not inert objects surrounding all plant cells but are instead highly dynamic, plastic structures. They are involved in a large number of cell biological processes and contribute actively to plant growth, development and interaction with environment. Therefore, it is not surprising that cellular processes can control plant cell wall integrity while, simultaneously, cell wall integrity can influence cellular processes. In yeast and animal cells such a bi-directional relationship also exists between the yeast/animal extra-cellular matrices and the cell cycle. In yeast, the cell wall integrity maintenance mechanism and a dedicated plasmamembrane integrity checkpoint are mediating this relationship. Recent research has yielded insights into the mechanism controlling plant cell wall metabolism during cytokinesis. However, knowledge regarding putative regulatory pathways controlling adaptive modifications in plant cell cycle activity in response to changes in the state of the plant cell wall are not yet identified. In this review, we summarize similarities and differences in regulatory mechanisms coordinating extra cellular matrices and cell cycle activity in animal and yeast cells, discuss the available evidence supporting the existence of such a mechanism in plants and suggest that the plant cell wall integrity maintenance mechanism might also control cell cycle activity in plant cells. This article is protected by copyright. All rights reserved.

Phenotype-Based Screening of Small Molecules to Modify Plant Cell Walls Using BY-2 Cells.

PubMed

Okubo-Kurihara, Emiko; Matsui, Minami

2018-01-01

The plant cell wall is an important and abundant biomass with great potential for use as a modern recyclable resource. For effective utilization of this cellulosic biomass, its ability to degrade efficiently is key point. With the aim of modifying the cell wall to allow easy decomposition, we used chemical biological technology to alter its structure. As a first step toward evaluating the chemicals in the cell wall we employed a phenotype-based approach using high-throughput screening. As the plant cell wall is essential in determining cell morphology, phenotype-based screening is particularly effective in identifying compounds that bring about alterations in the cell wall. For rapid and reproducible screening, tobacco BY-2 cell is an excellent system in which to observe cell morphology. In this chapter, we provide a detailed chemical biological methodology for studying cell morphology using tobacco BY-2 cells.

The plant cell wall in the feeding sites of cyst nematodes.

PubMed

Bohlmann, Holger; Sobczak, Miroslaw

2014-01-01

Plant parasitic cyst nematodes (genera Heterodera and Globodera) are serious pests for many crops. They enter the host roots as migratory second stage juveniles (J2) and migrate intracellularly toward the vascular cylinder using their stylet and a set of cell wall degrading enzymes produced in the pharyngeal glands. They select an initial syncytial cell (ISC) within the vascular cylinder or inner cortex layers to induce the formation of a multicellular feeding site called a syncytium, which is the only source of nutrients for the parasite during its entire life. A syncytium can consist of more than hundred cells whose protoplasts are fused together through local cell wall dissolutions. While the nematode produces a cocktail of cell wall degrading and modifying enzymes during migration through the root, the cell wall degradations occurring during syncytium development are due to the plants own cell wall modifying and degrading proteins. The outer syncytial cell wall thickens to withstand the increasing osmotic pressure inside the syncytium. Furthermore, pronounced cell wall ingrowths can be formed on the outer syncytial wall at the interface with xylem vessels. They increase the surface of the symplast-apoplast interface, thus enhancing nutrient uptake into the syncytium. Processes of cell wall degradation, synthesis and modification in the syncytium are facilitated by a variety of plant proteins and enzymes including expansins, glucanases, pectate lyases and cellulose synthases, which are produced inside the syncytium or in cells surrounding the syncytium.

Cell wall of pathogenic yeasts and implications for antimycotic therapy.

PubMed

Cassone, A

1986-01-01

Yeast cell wall is a complex, multilayered structure where amorphous, granular and fibrillar components interact with each other to confer both the specific cell shape and osmotic protection against lysis. Thus it is widely recognized that as is the case with bacteria, yeast cell wall is a major potential target for selective chemotherapeutic drugs. Despite intensive research, very few such drugs have been discovered and none has found substantial application in human diseases to date. Among the different cell wall components, beta-glucan and chitin are the fibrillar materials playing a fundamental role in the overall rigidity and resistance of the wall. Inhibition of the metabolism of these polymers, therefore, should promptly lead to lysis. This indeed occurs and aculeacin, echinocandin and polyoxins are examples of agents producing such an action. Particular attention should be focused on chitin synthesis. Although quantitatively a minor cell wall component, chitin is important in the mechanism of dimorphic transition, especially in Candida albicans, a major human opportunistic pathogen. This transition is associated with increased invasiveness and general virulence of the fungus. Yeast cell wall may also limit the effect of antifungals which owe their action to disturbance of the cytoplasmic membrane or of cell metabolism. Indeed, the cell wall may hinder access to the cell interior both under growing conditions and, particularly, during cell ageing in the stationary phase, when important structural changes occur in the cell wall due to unbalanced wall growth (phenotypic drug resistance).

Studies on the asparagine-linked oligosaccharides from cartilage-specific proteoglycan

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cioffi, L.C.

1987-01-01

Chondrocytes synthesize and secrete a cartilage-specific proteoglycan (PG-H) as one of their major products. This proteoglycan has attached to it several types of carbohydrate chains, including chondroitin sulfate, keratan sulfate, O-linked oligosaccharides, and asparagine-linked oligosaccharides. The asparagine-linked oligosaccharides found on PG-H were investigated in these studies. Methodology was developed for the isolation and separation of standard of standard complex and high mannose type oligosaccharides. This included digesting glycoproteins with N-glycanase and separation of the oligosaccharides according to type by concanavalin-A lectin chromatography. The different oligosaccharide types were then analyzed by high pressure liquid chromatography. This methodology was used in themore » subsequent studies on the PG-H asparagine-linked oligosaccharides. Initially, the asparagine-linked oligosaccharides recovered from the culture medium (CM) and cell-associated (Ma) fractions of PG-H from of tibial chondrocytes were labeled with (/sup 3/H)-mannose and the oligosaccharides were isolated and analyzed.« less

Ovarian response to recombinant human follicle-stimulating hormone in luteinizing hormone-depleted women: examination of the two cell, two gonadotropin theory.

PubMed

Ben-Chetrit, A; Gotlieb, L; Wong, P Y; Casper, R F

1996-04-01

To evaluate the relative contribution of FSH to ovarian estrogen production. Nonrandomized, prospective study. University of Toronto teaching hospital reproductive biology unit. Five women who had been treated with depot GnRH agonist with hormonal add-back for 4 to 48 months and who were confirmed to be gonadotropin depleted by both bioassay and RIA. Subjects received 75 IU SC recombinant human FSH daily for 7 days followed by 150 IU daily for 7 days and 225 IU daily for the third week. Serum steroid determination and vaginal sonography for follicle size and endometrial thickness were performed serially and follicular fluid hormone levels were measured in two subjects. Bioactive LH and FSH activity were less than the detection limit of the assay (0.1 mIU/mL; conversion factor to SI units, 1.00 for LH and FSH) before recombinant FSH treatment in all five women. In all subjects, at least one preovulatory follicle developed by the end of two to three weeks. Endometrial thickness increased to between 7 and 9 mm in four women. Mean serum E2 in the five subjects increased from 17 pg/mL (range: 5 to 33 pg/mL; conversion factor to SI unit, 3.671) at baseline to 230 pg/mL (range: 37 to 489 pg/mL) at the end of the study. Follicular fluid E2 concentrations ranged from 44,296 to 69,367 pg/mL in the four follicles aspirated. Our results indicate that LH is not necessary for ovarian E2 production. We speculate that the granulosa cells, in the absence of detectable LH bioactivity, can use circulating adrenal androgens or constitutive or FSH-stimulated thecal androgens, to produce intrafollicular E2.

Hepatitis B Virus Capsid Assembly Modulators, but Not Nucleoside Analogs, Inhibit the Production of Extracellular Pregenomic RNA and Spliced RNA Variants

PubMed Central

Ren, Suping; Espiritu, Christine; Kelly, Mollie; Lau, Vincent; Zheng, Lingjie; Hartman, George D.; Flores, Osvaldo A.; Klumpp, Klaus

2017-01-01

ABSTRACT The hepatitis B virus (HBV) core protein serves multiple essential functions in the viral life cycle, and antiviral agents that target the core protein are being developed. Capsid assembly modulators (CAMs) are compounds that target core and misdirect capsid assembly, resulting in the suppression of HBV replication and virion production. Besides HBV DNA, circulating HBV RNA has been detected in patient serum and can be associated with the treatment response. Here we studied the effect of HBV CAMs on the production of extracellular HBV RNA using infected HepaRG cells and primary human hepatocytes. Representative compounds from the sulfonamide carboxamide and heteroaryldihydropyrimidine series of CAMs were evaluated and compared to nucleos(t)ide analogs as inhibitors of the viral polymerase. The results showed that CAMs blocked extracellular HBV RNA with efficiencies similar to those with which they blocked pregenomic RNA (pgRNA) encapsidation, HBV DNA replication, and Dane particle production. Nucleos(t)ide analogs inhibited viral replication and virion production but not encapsidation or production of extracellular HBV RNA. Profiling of HBV RNA from both culture supernatants and patient serum showed that extracellular viral RNA consisted of pgRNA and spliced pgRNA variants with an internal deletion(s) but still retained the sequences at both the 5′ and 3′ ends. Similar variants were detected in the supernatants of infected cells with and without nucleos(t)ide analog treatment. Overall, our data demonstrate that HBV CAMs represent direct antiviral agents with a profile differentiated from that of nucleos(t)ide analogs, including the inhibition of extracellular pgRNA and spliced pgRNA. PMID:28559265

[Association of serum decoy receptor 3 protein level with the clinicopathologic features of bladder transitional cell carcinoma].

PubMed

Wang, Dong; Wang, Jian; Chen, Guojun

2013-12-01

To investigate the association of serum levels of decoy receptor 3(DcR3) protein and the clinicopathologic features of bladder transitional cell carcinoma. Enzyme-linked immunosorbent assay was used to examine the serum levels of DcR3 in patients with bladder transitional cell carcinoma for analysis of its association with the patients' age, gender, clinical stages and pathological classification. The patients with bladder transitional cell carcinoma showed a significantly elevated serum level of DcR3 (183.43 ∓78.45 pg/m1) compared with the normal level (116.65∓97.43 pg/m1, P<0.05). The serum level of DcR3 in the patients showed close correlations with the TNM stage and pathological classification of the tumor (P<0.05) but not with the patients' age or gender (P>0.05). In patients with bladder transitional cell carcinoma, a high serum level of DcR3 suggests a higher malignancy of the tumor.

[Determination of polioksin B in the air environment and in washouts from skin of operators by HPLC].

PubMed

Volkova, V N; Mukhina, L P; Chistova, Zh A; Fedorova, S G

Polyoxin B being an effective inhibitor of synthesis of chitin of the cell wall of many phytopathogenic fungi, is recommended as a fungicide for control of phytopathogenic organisms that cause damage to crop. For the determination of the exposure of employees working with pesticides there was developed the method of the measurement of concentrations of polyoxin B in air of working area, atmospheric air of populated areas and washouts from the operators ’ integuments, based on high performance liquid chromatography with ultraviolet detector (detection wavelength of270 nm), including sampling air environment in the sorption tube ORBO-44, filled with sorbent XAD-2, extraction of the sorbent with polyoxin by a mixture of carbinol-water (in a ratio of 95:5,on volume), washout from the surface of the skin with ethyl alcohol by way of washing, concentrating, quantitative chromatographic analysis. Lower limits of the quantification ofpolyoxin B in the air ofworking area - 0.2 mg/m at the aspiration of 2 dm of air, atmospheric air - 0.016 mg/m at the aspiration of 25 dm of air, in washouts from the operators’ integuments - 0.4 pg/wash, the linear range of the defined concentrations accounted for of 0.2 - 2.4 pg/cm, the total error of measurement of the concentrations of polyoxin B in air is 17%; in washouts from the operators’ integuments - 16%. The developed method was approbated for the determination of polyoxin in samples of air of working zone, atmospheric air within the sanitary gap, washouts from the operators ’ integuments and air drift samples taken under processing of roses in the hothouse and in the monitoring of the phytosanitary condition of the plants every other day after treatment.

Plant cell wall extensibility: connecting plant cell growth with cell wall structure, mechanics, and the action of wall-modifying enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cosgrove, Daniel J.

The advent of user-friendly instruments for measuring force/deflection curves of plant surfaces at high spatial resolution has resulted in a recent outpouring of reports of the ‘Young's modulus’ of plant cell walls. The stimulus for these mechanical measurements comes from biomechanical models of morphogenesis of meristems and other tissues, as well as single cells, in which cell wall stress feeds back to regulate microtubule organization, auxin transport, cellulose deposition, and future growth directionality. In this article I review the differences between elastic modulus and wall extensibility in the context of cell growth. Some of the inherent complexities, assumptions, and potentialmore » pitfalls in the interpretation of indentation force/deflection curves are discussed. Reported values of elastic moduli from surface indentation measurements appear to be 10- to >1000-fold smaller than realistic tensile elastic moduli in the plane of plant cell walls. Potential reasons for this disparity are discussed, but further work is needed to make sense of the huge range in reported values. The significance of wall stress relaxation for growth is reviewed and connected to recent advances and remaining enigmas in our concepts of how cellulose, hemicellulose, and pectins are assembled to make an extensible cell wall. A comparison of the loosening action of α-expansin and Cel12A endoglucanase is used to illustrate two different ways in which cell walls may be made more extensible and the divergent effects on wall mechanics.« less

My body is a cage: mechanisms and modulation of plant cell growth.

PubMed

Braidwood, Luke; Breuer, Christian; Sugimoto, Keiko

2014-01-01

388 I. 388 II. 389 III. 389 IV. 390 V. 391 VI. 393 VII. 394 VIII. 398 399 References 399 SUMMARY: The wall surrounding plant cells provides protection from abiotic and biotic stresses, and support through the action of turgor pressure. However, the presence of this strong elastic wall also prevents cell movement and resists cell growth. This growth can be likened to extending a house from the inside, using extremely high pressures to push out the walls. Plants must increase cell volume in order to explore their environment, acquire nutrients and reproduce. Cell wall material must stretch and flow in a controlled manner and, concomitantly, new cell wall material must be deposited at the correct rate and site to prevent wall and cell rupture. In this review, we examine biomechanics, cell wall structure and growth regulatory networks to provide a 'big picture' of plant cell growth. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

The connection of cytoskeletal network with plasma membrane and the cell wall

PubMed Central

Liu, Zengyu; Persson, Staffan; Zhang, Yi

2015-01-01

The cell wall provides external support of the plant cells, while the cytoskeletons including the microtubules and the actin filaments constitute an internal framework. The cytoskeletons contribute to the cell wall biosynthesis by spatially and temporarily regulating the transportation and deposition of cell wall components. This tight control is achieved by the dynamic behavior of the cytoskeletons, but also through the tethering of these structures to the plasma membrane. This tethering may also extend beyond the plasma membrane and impact on the cell wall, possibly in the form of a feedback loop. In this review, we discuss the linking components between the cytoskeletons and the plasma membrane, and/or the cell wall. We also discuss the prospective roles of these components in cell wall biosynthesis and modifications, and aim to provide a platform for further studies in this field. PMID:25693826

Dietary exposure to polychlorinated dibenzo-p-dioxins and dibenzofurans via fish consumption and dioxin-like activity in fish determined by H4IIE-luc bioassay.

PubMed

Chan, Janet Kit Yan; Man, Yu Bon; Xing, Guan Hua; Wu, Sheng Chun; Murphy, Margaret B; Xu, Ying; Wong, Ming H

2013-10-01

Dietary exposure to polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) via fish consumption in two major electronic (e) waste sites: Guiyu (GY), Guangdong Province and Taizhou (TZ), Zhejiang Province, and dioxin-like activity in fish determined by H4IIE-luc bioassay. In the present study, all fish were below EU's maximum allowable concentration in muscle of fish (4 pg WHO-TEQ/g wet wt), except crucian (4.28 pg WHO-TEQ/g wet wt) and silver carps (7.49 pg WHO-TEQ/g wet wt) collected from GY rivers. Moreover, the residual concentration in bighead carp collected from GY (2.15 pg WHO-TEQ/g wet wt) was close to the EU's action level (3 pg WHO-TEQ/g wet wt) which gives "early warning" to the competent authorities and operators to take measures to eliminate contamination. In addition, results indicated that the maximum human intake of PCDD/Fs via freshwater fish consumption in GY was 4.31 pg WHO-TEQ/kg bw/day, which exceeds the higher end of the tolerable daily intake recommended by the WHO, EC-SCF and JECFA (1-4, 2 and 2.3 pg WHO-TEQ/kg bw/day respectively). Furthermore, H4IIE-luc cell bioassay provides a very sensitive and cost-efficient screening tool for assessing the overall dioxin-like toxicity in the study, and is therefore valuable for high-throughput environmental monitoring studies. Copyright © 2012 Elsevier B.V. All rights reserved.

A global carbon assimilation system based on a dual optimization method

NASA Astrophysics Data System (ADS)

Zheng, H.; Li, Y.; Chen, J. M.; Wang, T.; Huang, Q.; Huang, W. X.; Li, S. M.; Yuan, W. P.; Zheng, X.; Zhang, S. P.; Chen, Z. Q.; Jiang, F.

2014-10-01

Ecological models are effective tools to simulate the distribution of global carbon sources and sinks. However, these models often suffer from substantial biases due to inaccurate simulations of complex ecological processes. We introduce a set of scaling factors (parameters) to an ecological model on the basis of plant functional type (PFT) and latitudes. A global carbon assimilation system (GCAS-DOM) is developed by employing a Dual Optimization Method (DOM) to invert the time-dependent ecological model parameter state and the net carbon flux state simultaneously. We use GCAS-DOM to estimate the global distribution of the CO2 flux on 1° ×1° grid cells for the period from 2000 to 2007. Results show that land and ocean absorb -3.69 ± 0.49 Pg C year-1 and -1.91 ± 0.16 Pg C year-1, respectively. North America, Europe and China contribut -0.96 ± 0.15 Pg C year-1, -0.42 ± 0.08 Pg C year-1 and -0.21 ± 0.28 Pg C year-1, respectively. The uncertainties in the flux after optimization by GCAS-DOM have been remarkably reduced by more than 60%. Through parameter optimization, GCAS-DOM can provide improved estimates of the carbon flux for each PFT. Coniferous forest (-0.97 ± 0.27 Pg C year-1) is the largest contributor to the global carbon sink. Fluxes of once-dominant deciduous forest generated by BEPS is reduced to -0.79 ± 0.22 Pg C year-1, being the third largest carbon sink.

Reduction in serum IL-6 after vacination of breast cancer patients with tumour-associated antigens is related to estrogen receptor status.

PubMed

Jiang, X P; Yang, D C; Elliott, R L; Head, J F

2000-05-01

Elevated serum IL-6 concentrations have been associated with poor prognosis in a variety of cancers, and decreases in serum IL-6 concentrations have been reported after chemotherapy. We have demonstrated that serum IL-6 concentrations are elevated in breast cancer patients [normal women 0.7 +/- 2.5 pg/ml (n=36), breast cancer patients 38.3 +/- 138.7 pg/ml (n = 111)]. After vaccination of breast cancer patients with a combination of tumour-associated antigens and biological adjuvants (IL-2 and GM-CSF), the concentration of IL-6 decreased significantly (P<0.05) to 8.1 +/- 14.6 pg/ml (n=85). Other studies have shown that oestrogen suppresses IL-6 production in oestrogen receptor positive breast cancer cells. We have demonstrated that the decrease in IL-6 associated with vaccination is related to the oestrogen receptor status of the tumours from breast cancer patients, as a decrease in IL-6 from 124.0 +/- 267.5 pg/ml (n=26) to 6.2 +/- 11.0 pg/ml (n=34) only occurs in patients with oestrogen receptor negative tumours. The IL-6 concentration in breast cancer patients with oestrogen receptor positive tumours remained unchanged (9.5 pg/ml before vaccination, and 9.3 pg/ml after vaccination). These results suggest that postmenopausal women with oestrogen receptor negative breast cancers, who do not respond well to either hormonal therapy with tamoxifen or adjuvant chemotherapy, may have a significant response to vaccination with autologous tumour-associated antigens.

Cellulose synthase complexes display distinct dynamic behaviors during xylem transdifferentiation.

PubMed

Watanabe, Yoichiro; Schneider, Rene; Barkwill, Sarah; Gonzales-Vigil, Eliana; Hill, Joseph L; Samuels, A Lacey; Persson, Staffan; Mansfield, Shawn D

2018-06-05

In plants, plasma membrane-embedded CELLULOSE SYNTHASE (CESA) enzyme complexes deposit cellulose polymers into the developing cell wall. Cellulose synthesis requires two different sets of CESA complexes that are active during cell expansion and secondary cell wall thickening, respectively. Hence, developing xylem cells, which first undergo cell expansion and subsequently deposit thick secondary walls, need to completely reorganize their CESA complexes from primary wall- to secondary wall-specific CESAs. Using live-cell imaging, we analyzed the principles underlying this remodeling. At the onset of secondary wall synthesis, the primary wall CESAs ceased to be delivered to the plasma membrane and were gradually removed from both the plasma membrane and the Golgi. For a brief transition period, both primary wall- and secondary wall-specific CESAs coexisted in banded domains of the plasma membrane where secondary wall synthesis is concentrated. During this transition, primary and secondary wall CESAs displayed discrete dynamic behaviors and sensitivities to the inhibitor isoxaben. As secondary wall-specific CESAs were delivered and inserted into the plasma membrane, the primary wall CESAs became concentrated in prevacuolar compartments and lytic vacuoles. This adjustment in localization between the two CESAs was accompanied by concurrent decreased primary wall CESA and increased secondary wall CESA protein abundance. Our data reveal distinct and dynamic subcellular trafficking patterns that underpin the remodeling of the cellulose biosynthetic machinery, resulting in the removal and degradation of the primary wall CESA complex with concurrent production and recycling of the secondary wall CESAs. Copyright © 2018 the Author(s). Published by PNAS.

Identification of potential cell wall component that allows Taka-amylase A adsorption in submerged cultures of Aspergillus oryzae.

PubMed

Sato, Hiroki; Toyoshima, Yoshiyuki; Shintani, Takahiro; Gomi, Katsuya

2011-12-01

We observed that α-amylase (Taka-amylase A; TAA) activity in the culture broth disappeared in the later stage of submerged cultivation of Aspergillus oryzae. This disappearance was caused by adsorption of TAA onto the cell wall of A. oryzae and not due to protein degradation by extracellular proteolytic enzymes. To determine the cell wall component(s) that allows TAA adsorption efficiently, the cell wall was fractionated by stepwise alkali treatment and enzymatic digestion. Consequently, alkali-insoluble cell wall fractions exhibited high levels of TAA adsorption. In addition, this adsorption capacity was significantly enhanced by treatment of the alkali-insoluble fraction with β-glucanase, which resulted in the concomitant increase in the amount of chitin in the resulting fraction. In contrast, the adsorption capacity was diminished by treating the cell wall fraction with chitinase. These results suggest that the major component that allows TAA adsorption is chitin. However, both the mycelium and the cell wall demonstrated the inability to allow TAA adsorption in the early stage of cultivation, despite chitin content in the cell wall being identical in both early and late stages of cultivation. These results suggest the existence of unidentified factor(s) that could prevent the adsorption of TAA onto the cell wall. Such factor(s) is most likely removed or diminished from the cell wall following longer cultivation periods.

A model of cell wall expansion based on thermodynamics of polymer networks

NASA Technical Reports Server (NTRS)

Veytsman, B. A.; Cosgrove, D. J.

1998-01-01

A theory of cell wall extension is proposed. It is shown that macroscopic properties of cell walls can be explained through the microscopic properties of interpenetrating networks of cellulose and hemicellulose. The qualitative conclusions of the theory agree with the existing experimental data. The dependence of the cell wall yield threshold on the secretion of the wall components is discussed.

Bacterial cell wall composition and the influence of antibiotics by cell-wall and whole-cell NMR

PubMed Central

Romaniuk, Joseph A. H.; Cegelski, Lynette

2015-01-01

The ability to characterize bacterial cell-wall composition and structure is crucial to understanding the function of the bacterial cell wall, determining drug modes of action and developing new-generation therapeutics. Solid-state NMR has emerged as a powerful tool to quantify chemical composition and to map cell-wall architecture in bacteria and plants, even in the context of unperturbed intact whole cells. In this review, we discuss solid-state NMR approaches to define peptidoglycan composition and to characterize the modes of action of old and new antibiotics, focusing on examples in Staphylococcus aureus. We provide perspectives regarding the selected NMR strategies as we describe the exciting and still-developing cell-wall and whole-cell NMR toolkit. We also discuss specific discoveries regarding the modes of action of vancomycin analogues, including oritavancin, and briefly address the reconsideration of the killing action of β-lactam antibiotics. In such chemical genetics approaches, there is still much to be learned from perturbations enacted by cell-wall assembly inhibitors, and solid-state NMR approaches are poised to address questions of cell-wall composition and assembly in S. aureus and other organisms. PMID:26370936