Echinocandin Susceptibility Testing of Candida Species: Comparison of EUCAST EDef 7.1, CLSI M27-A3, Etest, Disk Diffusion, and Agar Dilution Methods with RPMI and IsoSensitest Media▿

PubMed Central

Arendrup, Maiken Cavling; Garcia-Effron, Guillermo; Lass-Flörl, Cornelia; Lopez, Alicia Gomez; Rodriguez-Tudela, Juan-Luis; Cuenca-Estrella, Manuel; Perlin, David S.

2010-01-01

This study compared nine susceptibility testing methods and 12 endpoints for anidulafungin, caspofungin, and micafungin with the same collection of blinded FKS hot spot mutant (n = 29) and wild-type isolates (n = 94). The susceptibility tests included EUCAST Edef 7.1, agar dilution, Etest, and disk diffusion with RPMI-1640 plus 2% glucose (2G) and IsoSensitest-2G media and CLSI M27A-3. Microdilution plates were read after 24 and 48 h. The following test parameters were evaluated: fks hot spot mutants overlapping the wild-type distribution, distance between the two populations, number of very major errors (VMEs; fks mutants misclassified as susceptible), and major errors (MEs; wild-type isolates classified as resistant) using a wild-type-upper-limit value (WT-UL) (two twofold-dilutions higher than the MIC50) as the susceptibility breakpoint. The methods with the lowest number of errors (given as VMEs/MEs) across the three echinocandins were CLSI (12%/1%), agar dilution with RPMI-2G medium (14%/0%), and Etest with RPMI-2G medium (8%/3%). The fewest errors overall were observed for anidulafungin (4%/1% for EUCAST, 4%/3% for CLSI, and 3%/9% for Etest with RPMI-2G). For micafungin, VME rates of 10 to 71% were observed. For caspofungin, agar dilution with either medium was superior (VMEs/MEs of 0%/1%), while CLSI, EUCAST with IsoSensitest-2G medium, and Etest were less optimal (VMEs of 7%, 10%, and 10%, respectively). Applying the CLSI breakpoint (S ≤ 2 μg/ml) for CLSI results, 89.2% fks hot spot mutants were classified as anidulafungin susceptible, 60.7% as caspofungin susceptible, and 92.9% as micafungin susceptible. In conclusion, no test was perfect, but anidulafungin susceptibility testing using the WT-UL to define susceptibility reliably identified fks hot spot mutants. PMID:19884370

Sarcolipin overexpression improves muscle energetics and reduces fatigue

PubMed Central

Sopariwala, Danesh H.; Pant, Meghna; Shaikh, Sana A.; Goonasekera, Sanjeewa A.; Molkentin, Jeffery D.; Weisleder, Noah; Ma, Jianjie; Pan, Zui

2015-01-01

Sarcolipin (SLN) is a regulator of sarcoendoplasmic reticulum calcium ATPase in skeletal muscle. Recent studies using SLN-null mice have identified SLN as a key player in muscle thermogenesis and metabolism. In this study, we exploited a SLN overexpression (SlnOE) mouse model to determine whether increased SLN level affected muscle contractile properties, exercise capacity/fatigue, and metabolic rate in whole animals and isolated muscle. We found that SlnOE mice are more resistant to fatigue and can run significantly longer distances than wild-type (WT). Studies with isolated extensor digitorum longus (EDL) muscles showed that SlnOE EDL produced higher twitch force than WT. The force-frequency curves were not different between WT and SlnOE EDLs, but at lower frequencies the pyruvate-induced potentiation of force was significantly higher in SlnOE EDL. SLN overexpression did not alter the twitch and force-frequency curve in isolated soleus muscle. However, during a 10-min fatigue protocol, both EDL and soleus from SlnOE mice fatigued significantly less than WT muscles. Interestingly, SlnOE muscles showed higher carnitine palmitoyl transferase-1 protein expression, which could enhance fatty acid metabolism. In addition, lactate dehydrogenase expression was higher in SlnOE EDL, suggesting increased glycolytic capacity. We also found an increase in store-operated calcium entry (SOCE) in isolated flexor digitorum brevis fibers of SlnOE compared with WT mice. These data allow us to conclude that increased SLN expression improves skeletal muscle performance during prolonged muscle activity by increasing SOCE and muscle energetics. PMID:25701006

Isolation of Novel Synthetic Prion Strains by Amplification in Transgenic Mice Coexpressing Wild-Type and Anchorless Prion Proteins

PubMed Central

Raymond, Gregory J.; Race, Brent; Hollister, Jason R.; Offerdahl, Danielle K.; Moore, Roger A.; Kodali, Ravindra; Raymond, Lynne D.; Hughson, Andrew G.; Rosenke, Rebecca; Long, Dan; Dorward, David W.

2012-01-01

Mammalian prions are thought to consist of misfolded aggregates (protease-resistant isoform of the prion protein [PrPres]) of the cellular prion protein (PrPC). Transmissible spongiform encephalopathy (TSE) can be induced in animals inoculated with recombinant PrP (rPrP) amyloid fibrils lacking mammalian posttranslational modifications, but this induction is inefficient in hamsters or transgenic mice overexpressing glycosylphosphatidylinositol (GPI)-anchored PrPC. Here we show that TSE can be initiated by inoculation of misfolded rPrP into mice that express wild-type (wt) levels of PrPC and that synthetic prion strain propagation and selection can be affected by GPI anchoring of the host's PrPC. To create prions de novo, we fibrillized mouse rPrP in the absence of molecular cofactors, generating fibrils with a PrPres-like protease-resistant banding profile. These fibrils induced the formation of PrPres deposits in transgenic mice coexpressing wt and GPI-anchorless PrPC (wt/GPI−) at a combined level comparable to that of PrPC expression in wt mice. Secondary passage into mice expressing wt, GPI−, or wt plus GPI− PrPC induced TSE disease with novel clinical, histopathological, and biochemical phenotypes. Contrary to laboratory-adapted mouse scrapie strains, the synthetic prion agents exhibited a preference for conversion of GPI− PrPC and, in one case, caused disease only in GPI− mice. Our data show that novel TSE agents can be generated de novo solely from purified mouse rPrP after amplification in mice coexpressing normal levels of wt and anchorless PrPC. These observations provide insight into the minimal elements required to create prions in vitro and suggest that the PrPC GPI anchor can modulate the propagation of synthetic TSE strains. PMID:22915801

Isolation and characterization of dental epithelial cells derived from amelogenesis imperfecta rat.

PubMed

Adiningrat, A; Tanimura, A; Miyoshi, K; Hagita, H; Yanuaryska, R D; Arinawati, D Y; Horiguchi, T; Noma, T

2016-03-01

Disruption of the third zinc finger domain of specificity protein 6 (SP6) presents an enamel-specific defect in a rat model of amelogenesis imperfecta (AMI rats). To understand the molecular basis of amelogenesis imperfecta caused by the Sp6 mutation, we established and characterized AMI-derived rat dental epithelial (ARE) cells. ARE cell clones were isolated from the mandibular incisors of AMI rats, and amelogenesis-related gene expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR). Localization of wild-type SP6 (SP6WT) and mutant-type SP6 (SP6AMI) was analyzed by immunocytochemistry. SP6 transcriptional activity was monitored by rho-associated protein kinase 1 (Rock1) promoter activity with its specific binding to the promoter region in dental (G5 and ARE) and non-dental (COS-7) epithelial cells. Isolated ARE cells were varied in morphology and gene expression. Both SP6WT and SP6AMI were mainly detected in nuclei. The promoter analysis revealed that SP6WT and SP6AMI enhanced Rock1 promoter activity in G5 cells but that enhancement by SP6AMI was weaker, whereas no enhancement was observed in the ARE and COS-7 cells, even though SP6WT and SP6AMI bound to the promoter in all instances. ARE cell clones can provide a useful in vitro model to study the mechanism of SP6-mediated amelogenesis imperfecta. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Expression of Biologically Active Human Butyrylcholinesterase in the Cabbage Looper (Trichoplusia ni)

DTIC Science & Technology

2000-01-01

recombinant human BUChE; Sf, Spodoptera frugiperda ; VX, 0-ethyl S-[2-[bis(I -methylethyl)amino]ethyl]methyl phosphonothiolate; wt, wild-type. 1 To whom...ATCC (Rockville, MD, U.S.A.). Insect cells ( Spodoptera frugiperda Sf9 cells and T. ni High 5 cells) and wild-type (wt)-AcNPV were purchased from

Differential effect of T-type voltage-gated Ca2+ channel disruption on renal plasma flow and glomerular filtration rate in vivo.

PubMed

Thuesen, Anne D; Andersen, Henrik; Cardel, Majken; Toft, Anja; Walter, Steen; Marcussen, Niels; Jensen, Boye L; Bie, Peter; Hansen, Pernille B L

2014-08-15

Voltage-gated Ca(2+) (Cav) channels play an essential role in the regulation of renal blood flow and glomerular filtration rate (GFR). Because T-type Cav channels are differentially expressed in pre- and postglomerular vessels, it was hypothesized that they impact renal blood flow and GFR differentially. The question was addressed with the use of two T-type Cav knockout (Cav3.1(-/-) and Cav3.2(-/-)) mouse strains. Continuous recordings of blood pressure and heart rate, para-aminohippurate clearance (renal plasma flow), and inulin clearance (GFR) were performed in conscious, chronically catheterized, wild-type (WT) and Cav3.1(-/-) and Cav3.2(-/-) mice. The contractility of afferent and efferent arterioles was determined in isolated perfused blood vessels. Efferent arterioles from Cav3.2(-/-) mice constricted significantly more in response to a depolarization compared with WT mice. GFR was increased in Cav3.2(-/-) mice with no significant changes in renal plasma flow, heart rate, and blood pressure. Cav3.1(-/-) mice had a higher renal plasma flow compared with WT mice, whereas GFR was indistinguishable from WT mice. No difference in the concentration response to K(+) was observed in isolated afferent and efferent arterioles from Cav3.1(-/-) mice compared with WT mice. Heart rate was significantly lower in Cav3.1(-/-) mice compared with WT mice with no difference in blood pressure. T-type antagonists significantly inhibited the constriction of human intrarenal arteries in response to a small depolarization. In conclusion, Cav3.2 channels support dilatation of efferent arterioles and affect GFR, whereas Cav3.1 channels in vivo contribute to renal vascular resistance. It is suggested that endothelial and nerve localization of Cav3.2 and Cav3.1, respectively, may account for the observed effects. Copyright © 2014 the American Physiological Society.

[Genetic characteristics of hemagglutinin in measles viruses isolated in Henan Province, China].

PubMed

Feng, Da-Xing; Seng, Ming-Hua; Liu, Qian; Zhang, Zhen-Ying

2014-03-01

This study aims to investigate the genetic characteristics of hemagglutinin in wild-type measles viruses in Henan Province, China and to provide a basis for measles control and elimination. Specimens were collected from suspected measles cases in Henan during 2008-2012. Cell culture was performed for virus isolation, and RT-PCR was used to amplify hemagglutinin gene. The PCR products were sequenced and analyzed, including construction of phylogenetic tree and analysis of the distance between the isolated virus and the reference virus; then, the variations in predicted amino acids were analyzed. The results showed that 12 measles viruses were isolated in Henan Province and identified as H1a genotype; the nucleotide and amino acid homologies were 98.0%-100% and 97.2%-99.8%, respectively. One glycosylation site changed in all the 12 sequences because of the amino acid mutation from serine to asparagine at the 240th site, as compared with Edmonston-wt. USA/54/A. Overall, the wild-type measles virus genotype circulating in Henan Province from 2008 to 2012 was H1a, with high homology between strains; there were some variations in amino acid sequences, resulting in glycosylation site deletion.

Host-induced aneuploidy and phenotypic diversification in the Sudden Oak Death pathogen Phytophthora ramorum.

PubMed

Kasuga, Takao; Bui, Mai; Bernhardt, Elizabeth; Swiecki, Tedmund; Aram, Kamyar; Cano, Liliana M; Webber, Joan; Brasier, Clive; Press, Caroline; Grünwald, Niklaus J; Rizzo, David M; Garbelotto, Matteo

2016-05-20

Aneuploidy can result in significant phenotypic changes, which can sometimes be selectively advantageous. For example, aneuploidy confers resistance to antifungal drugs in human pathogenic fungi. Aneuploidy has also been observed in invasive fungal and oomycete plant pathogens in the field. Environments conducive to the generation of aneuploids, the underlying genetic mechanisms, and the contribution of aneuploidy to invasiveness are underexplored. We studied phenotypic diversification and associated genome changes in Phytophthora ramorum, a highly destructive oomycete pathogen with a wide host-range that causes Sudden Oak Death in western North America and Sudden Larch Death in the UK. Introduced populations of the pathogen are exclusively clonal. In California, oak (Quercus spp.) isolates obtained from trunk cankers frequently exhibit host-dependent, atypical phenotypes called non-wild type (nwt), apparently without any host-associated population differentiation. Based on a large survey of genotypes from different hosts, we previously hypothesized that the environment in oak cankers may be responsible for the observed phenotypic diversification in P. ramorum. We show that both normal wild type (wt) and nwt phenotypes were obtained when wt P. ramorum isolates from the foliar host California bay (Umbellularia californica) were re-isolated from cankers of artificially-inoculated canyon live oak (Q. chrysolepis). We also found comparable nwt phenotypes in P. ramorum isolates from a bark canker of Lawson cypress (Chamaecyparis lawsoniana) in the UK; previously nwt was not known to occur in this pathogen population. High-throughput sequencing-based analyses identified major genomic alterations including partial aneuploidy and copy-neutral loss of heterozygosity predominantly in nwt isolates. Chromosomal breakpoints were located at or near transposons. This work demonstrates that major genome alterations of a pathogen can be induced by its host species. This is an undocumented type of plant-microbe interaction, and its contribution to pathogen evolution is yet to be investigated, but one of the potential collateral effects of nwt phenotypes may be host survival.

Deficiency of Cholesteryl Ester Transfer Protein Protects Against Atherosclerosis in Rabbits.

PubMed

Zhang, Jifeng; Niimi, Manabu; Yang, Dongshan; Liang, Jingyan; Xu, Jie; Kimura, Tokuhide; Mathew, Anna V; Guo, Yanhong; Fan, Yanbo; Zhu, Tianqing; Song, Jun; Ackermann, Rose; Koike, Yui; Schwendeman, Anna; Lai, Liangxue; Pennathur, Subramaniam; Garcia-Barrio, Minerva; Fan, Jianglin; Chen, Y Eugene

2017-06-01

CETP (cholesteryl ester transfer protein) plays an important role in lipoprotein metabolism; however, whether inhibition of CETP activity can prevent cardiovascular disease remains controversial. We generated CETP knockout (KO) rabbits by zinc finger nuclease gene editing and compared their susceptibility to cholesterol diet-induced atherosclerosis to that of wild-type (WT) rabbits. On a chow diet, KO rabbits showed higher plasma levels of high-density lipoprotein (HDL) cholesterol than WT controls, and HDL particles of KO rabbits were essentially rich in apolipoprotein AI and apolipoprotein E contents. When challenged with a cholesterol-rich diet for 18 weeks, KO rabbits not only had higher HDL cholesterol levels but also lower total cholesterol levels than WT rabbits. Analysis of plasma lipoproteins revealed that reduced plasma total cholesterol in KO rabbits was attributable to decreased apolipoprotein B-containing particles, while HDLs remained higher than that in WT rabbits. Both aortic and coronary atherosclerosis was significantly reduced in KO rabbits compared with WT rabbits. Apolipoprotein B-depleted plasma isolated from CETP KO rabbits showed significantly higher capacity for cholesterol efflux from macrophages than that from WT rabbits. Furthermore, HDLs isolated from CETP KO rabbits suppressed tumor necrosis factor-α-induced vascular cell adhesion molecule 1 and E-selectin expression in cultured endothelial cells. These results provide evidence that genetic ablation of CETP activity protects against cholesterol diet-induced atherosclerosis in rabbits. © 2017 American Heart Association, Inc.

Wild-Type MIC Distributions and Epidemiological Cutoff Values for Caspofungin and Aspergillus spp. for the CLSI Broth Microdilution Method (M38-A2 Document)▿

PubMed Central

Espinel-Ingroff, A.; Fothergill, A.; Fuller, J.; Johnson, E.; Pelaez, T.; Turnidge, J.

2011-01-01

Clinical breakpoints have not been established for mold testing. Epidemiologic cutoff values (ECVs) are available for six Aspergillus spp. and the triazoles, but not for caspofungin. Wild-type (WT) minimal effective concentration (MEC) distributions (organisms in a species-drug combination with no acquired resistance mechanisms) were defined in order to establish ECVs for six Aspergillus spp. and caspofungin. The number of available isolates was as follows: 1,691 A. fumigatus, 432 A. flavus, 192 A. nidulans, 440 A. niger, 385 A. terreus, and 75 A. versicolor isolates. CLSI broth microdilution MEC data gathered in five independent laboratories in Canada, Europe, and the United States were aggregated for the analyses. ECVs expressed in μg/ml that captured 95% and 99% of the modeled wild-type population were for A. fumigatus 0.5 and 1, A. flavus 0.25 and 0.5, A. nidulans 0.5 and 0.5, A. niger 0.25 and 0.25, A. terreus 0.25 and 0.5, and A. versicolor 0.25 and 0.5. Although caspofungin ECVs are not designed to predict the outcome of therapy, they may aid in the detection of strains with reduced antifungal susceptibility to this agent and acquired resistance mechanisms. PMID:21422219

Lack of angiopoietin-like-2 expression limits the metabolic stress induced by a high-fat diet and maintains endothelial function in mice.

PubMed

Yu, Carol; Luo, Xiaoyan; Farhat, Nada; Daneault, Caroline; Duquette, Natacha; Martel, Cécile; Lambert, Jean; Thorin-Trescases, Nathalie; Rosiers, Christine Des; Thorin, Eric

2014-08-15

Angiopoietin-like-2 (angptl2) is produced by several cell types including endothelial cells, adipocytes and macrophages, and contributes to the inflammatory process in cardiovascular diseases. We hypothesized that angptl2 impairs endothelial function, and that lowering angptl2 levels protects the endothelium against high-fat diet (HFD)-induced fat accumulation and hypercholesterolemia. Acute recombinant angptl2 reduced (P<0.05) acetylcholine-mediated vasodilation of isolated wild-type (WT) mouse femoral artery, an effect reversed (P<0.05) by the antioxidant N-acetylcysteine. Accordingly, in angptl2 knockdown (KD) mice, ACh-mediated endothelium-dependent vasodilation was greater (P<0.05) than in WT mice. In arteries from KD mice, prostacyclin contributed to the overall dilation unlike in WT mice. After a 3-month HFD, overall vasodilation was not altered, but dissecting out the endothelial intrinsic pathways revealed that NO production was reduced in arteries isolated from HFD-fed WT mice (P<0.05), while NO release was maintained in KD mice. Similarly, endothelium-derived hyperpolarizing factor (EDHF) was preserved in mesenteric arteries from HFD-fed KD mice but not in those from WT mice. Finally, the HFD increased (P<0.05) total cholesterol-to-high-density lipoprotein ratios, low-density lipoprotein-to-high-density lipoprotein ratios, and leptin levels in WT mice only, while glycemia remained similar in the 2 strains. KD mice displayed less triglyceride accumulation in the liver (P<0.05 versus WT), and adipocyte diameters in mesenteric and epididymal white adipose tissues were smaller (P<0.05) in KD than in WT fed an HFD, while inflammatory gene expression increased (P<0.05) in the fat of WT mice only. Lack of angptl2 expression limits the metabolic stress induced by an HFD and maintains endothelial function in mice. © 2014 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Characterization of a new dog isolate of canine distemper virus from China.

PubMed

Qiao, J; Meng, Q; Chen, C; Xia, X; Cai, X; Ren, Y; Zhang, H

2011-01-01

Canine distemper virus (CDV) is a highly contagious pathogen of dogs. Vaccination is an effective way to protect dogs from CDV infection, but occasionally fails. In the present study, a wild type (wt) CDV, named XJ2, was isolated from a dead vaccinated dog. The hemagglutinin (H) gene of the XJ2 was amplified and analyzed for the molecular characteristics including N-glycosylation sites, phylogenesis, hydrophobicity and epitopes. The data indicated that XJ2 was a genetic variant strain of CDV. CDV-sero-negative dogs were inoculated intranasally with XJ2, developed severe clinical symptoms and died, suggesting high virulence.

Reduced Innate Immune Response to a Staphylococcus aureus Small Colony Variant Compared to Its Wild-Type Parent Strain

PubMed Central

Ou, Judy J. J.; Drilling, Amanda J.; Cooksley, Clare; Bassiouni, Ahmed; Kidd, Stephen P.; Psaltis, Alkis J.; Wormald, Peter J.; Vreugde, Sarah

2016-01-01

Background: Staphylococcus aureus (S. aureus) small colony variants (SCVs) can survive within the host intracellular milieu and are associated with chronic relapsing infections. However, it is unknown whether host invasion rates and immune responses differ between SCVs and their wild-type counterparts. This study used a stable S. aureus SCV (WCH-SK2SCV) developed from a clinical isolate (WCH-SK2WT) in inflammation-relevant conditions. Intracellular infection rates as well as host immune responses to WCH-SK2WT and WCH-SK2SCV infections were investigated. Method: NuLi-1 cells were infected with either WCH-SK2WT or WCH-SK2SCV, and the intracellular infection rate was determined over time. mRNA expression of cells infected with each strain intra- and extra-cellularly was analyzed using a microfluidic qPCR array to generate an expression profile of thirty-nine genes involved in the host immune response. Results: No difference was found in the intracellular infection rate between WCH-SK2WT and WCH-SK2SCV. Whereas, extracellular infection induced a robust pro-inflammatory response, intracellular infection elicited a modest response. Intracellular WCH-SK2WT infection induced mRNA expression of TLR2, pro-inflammatory cytokines (IL1B, IL6, and IL12) and tissue remodeling factors (MMP9). In contrast, intracellular WCH-SK2SCV infection induced up regulation of only TLR2. Conclusions: Whereas, host intracellular infection rates of WCH-SK2SCV and WCH-SK2WT were similar, WCH-SK2SCV intracellular infection induced a less widespread up regulation of pro-inflammatory and tissue remodeling factors in comparison to intracellular WCH-SK2WT infection. These findings support the current view that SCVs are able to evade host immune detection to allow their own survival. PMID:28083514

The therapeutic effect of tigecycline, unlike that of Ceftazidime, is not influenced by whether the Klebsiella pneumoniae strain produces extended-spectrum β-lactamases in experimental pneumonia in rats.

PubMed

Goessens, Wil H F; Mouton, Johan W; Ten Kate, Marian T; Sörgel, Fritz; Kinzig, Martina; Bakker-Woudenberg, Irma A J M

2013-01-01

The efficacies of tigecycline and ceftazidime against fatal pneumonia in rats caused by an extended-spectrum β-lactamase (ESBL)-positive Klebsiella pneumoniae strain or its wild-type (WT) progenitor were compared. Ceftazidime at 12.5 or 50 mg/kg of body weight twice daily (b.i.d.) was effective (50% or 100% rat survival) in pneumonia caused by the WT isolate but unsuccessful (100% rat mortality) in pneumonia caused by the ESBL-positive variant. In contrast, tigecycline at 6.25, 12.5, or 25 mg/kg b.i.d. showed dosage-dependent efficacy up to 100% rat survival irrespective of the ESBL character of the infecting organism.

Patterns of antimicrobial resistance in Streptococcus suis isolates from pigs with or without streptococcal disease in England between 2009 and 2014.

PubMed

Hernandez-Garcia, Juan; Wang, Jinhong; Restif, Olivier; Holmes, Mark A; Mather, Alison E; Weinert, Lucy A; Wileman, Thomas M; Thomson, Jill R; Langford, Paul R; Wren, Brendan W; Rycroft, Andrew; Maskell, Duncan J; Tucker, Alexander W

2017-08-01

Antimicrobial resistance in Streptococcus suis, a global zoonotic pathogen of pigs, has been mostly studied only in diseased animals using surveys that have not evaluated changes over time. We compared patterns of resistance between S. suis isolates from clinical cases of disease (CC) and non-clinical case (NCC) pigs in England, collected over two discrete periods, 2009-2011 and 2013-2014. Minimum inhibitory concentrations (MIC) of 17 antimicrobials (nine classes) were determined on 405 S. suis isolates categorised by sampling period and disease association to assess changes in resistance over time and association with disease. First, isolates were characterized as resistant or susceptible using published clinical breakpoints. Second, epidemiological cut-offs (ECOFF) were derived from MIC values, and isolates classified as wild type (WT) below the ECOFF and non-wild type (NWT) above the ECOFF. Finally, isolate subsets were analysed for shifts in MIC distribution. NCC isolates were more resistant than CC isolates to cephalosporins, penams, pleuromutilins, potentiated sulphonamides and tetracyclines in both study periods. Resistance levels among CC isolates increased in 2013-2014 relative to 2009-2011 for antimicrobials including aminoglycosides, cephalosporins, fluoroquinolones, pleuromutilins, potentiated sulphonamides and tetracyclines. The prevalence of isolates categorised as NWT for five or more classes of antimicrobials was greater among NCC than CC isolates for both time periods, and increased with time. This study used standardised methods to identify significant shifts in antimicrobial resistance phenotypes of S. suis isolated from pigs in England, not only over time but also between isolates from known clinical cases or disease-free pigs. Copyright © 2017. Published by Elsevier B.V.

TRPA1 channels: expression in non-neuronal murine lung tissues and dispensability for hyperoxia-induced alveolar epithelial hyperplasia.

PubMed

Kannler, Martina; Lüling, Robin; Yildirim, Ali Önder; Gudermann, Thomas; Steinritz, Dirk; Dietrich, Alexander

2018-05-12

Transient receptor potential A1 (TRPA1) channels were originally characterized in neuronal tissues but also identified in lung epithelium by staining with fluorescently coupled TRPA1 antibodies. Its exact function in non-neuronal tissues, however, is elusive. TRPA1 is activated in vitro by hypoxia and hyperoxia and is therefore a promising TRP candidate for sensing hyperoxia in pulmonary epithelial cells and for inducing alveolar epithelial hyperplasia. Here, we isolated tracheal, bronchial, and alveolar epithelial cells and show low but detectable TRPA1 mRNA levels in all these cells as well as TRPA1 protein by Western blotting in alveolar type II (AT II) cells. We quantified changes in intracellular Ca 2+ ([Ca 2+ ] i ) levels induced by application of hyperoxic solutions in primary tracheal epithelial, bronchial epithelial, and AT II cells isolated from wild-type (WT) and TRPA1-deficient (TRPA1-/-) mouse lungs. In all cell types, we detected hyperoxia-induced rises in [Ca 2+ ] i levels, which were not significantly different in TRPA1-deficient cells compared to WT cells. We also tested TRPA1 function in a mouse model for hyperoxia-induced alveolar epithelial hyperplasia. A characteristic significant increase in thickening of alveolar tissues was detected in mouse lungs after exposure to hyperoxia, but not in normoxic WT and TRPA1-/- controls. Quantification of changes in lung morphology in hyperoxic WT and TRPA1-/- mice, however, again revealed no significant changes. Therefore, TRPA1 expression does neither appear to be a key player for hyperoxia-induced changes in [Ca 2+ ] i levels in primary lung epithelial cells, nor being essential for the development of hyperoxia-induced alveolar epithelial hyperplasia.

AtSRP1, SMALL RUBBER PARTICLE PROTEIN HOMOLOG, functions in pollen growth and development in Arabidopsis.

PubMed

Chi, Yong Hun; Kim, Sun Young; Lee, Eun Seon; Jung, Young Jun; Park, Joung Hun; Paeng, Seol Ki; Oh, Hun Taek; Melencion, Sarah Mae Boyles; Alinapon, Cresilda Vergara; Lee, Sang Yeol

2016-06-24

To identify novel roles of SMALL RUBBER PARTICLE PROTEIN Homolog in the non-rubber-producing plant Arabidopsis (AtSRP1), we isolated a T-DNA-insertion knock-out mutant (FLAG_543A05) and investigated its functional characteristics. AtSRP1 is predominantly expressed in reproductive organs and is localized to lipid droplets and ER. Compared to wild-type (WT) Arabidopsis, atsrp1 plants contain small siliques with a reduced number of heterogeneously shaped seeds. The size of anther and pollen grains in atsrp1 is highly irregular, with a lower grain number than WT. Therefore, AtSRP1 plays a novel role related to pollen growth and development in a non-rubber-producing plant. Copyright © 2016 Elsevier Inc. All rights reserved.

Phylogenetic analysis of Tomato spotted wilt virus (TSWV) NSs protein demonstrates the isolated emergence of resistance-breaking strains in pepper.

PubMed

Almási, Asztéria; Csilléry, Gábor; Csömör, Zsófia; Nemes, Katalin; Palkovics, László; Salánki, Katalin; Tóbiás, István

2015-02-01

Resurgence of Tomato spotted wilt virus (TSWV) worldwide as well as in Hungary causing heavy economic losses directed the attention to the factors contributing to the outbreak of this serious epidemics. The introgression of Tsw resistance gene into various pepper cultivars seemed to solve TSWV control, but widely used resistant pepper cultivars bearing the same, unique resistance locus evoked the rapid emergence of resistance-breaking (RB) TSWV strains. In Hungary, the sporadic appearance of RB strains in pepper-producing region was first observed in 2010-2011, but in 2012 it was detected frequently. Previously, the non-structural protein (NSs) encoded by small RNA (S RNA) of TSWV was verified as the avirulence factor for Tsw resistance, therefore we analyzed the S RNA of the Hungarian RB and wild type (WT) isolates and compared to previously analyzed TSWV strains with RB properties from different geographical origins. Phylogenetic analysis demonstrated that the different RB strains had the closest relationship with the local WT isolates and there is no conserved mutation present in all the NSs genes of RB isolates from different geographical origins. According to these results, we concluded that the RB isolates evolved separately in geographic point of view, and also according to the RB mechanism.

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

Gaczynska, Maria; Karpowicz, Przemyslaw; Stuart, Christine E.

α 1-Proteinase inhibitor (antitrypsin) is a canonical example of the serpin family member that binds and inhibits serine proteases. The natural metastability of serpins is crucial to carry out structural rearrangements necessary for biological activity. However, the enhanced metastability of the mutant Z variant of antitrypsin, in addition to folding defect, may substantially contribute to its polymerization, a process leading to incurable serpinopathy. The metastability also impedes structural studies on the polymers. There are no crystal structures of Z monomer or any kind of polymers larger than engineered wild type (WT) trimer. Our understanding of polymerization mechanisms is based onmore » biochemical data using in vitro generated WT oligomers and molecular simulations. Here we applied atomic force microscopy (AFM) to compare topography of monomers, in vitro formed WT oligomers, and Z type polymers isolated from transgenic mouse liver. We found the AFM images of monomers closely resembled an antitrypsin outer shell modeled after the crystal structure. We confirmed that the Z variant demonstrated higher spontaneous propensity to dimerize than WT monomers. We also detected an unexpectedly broad range of different types of polymers with periodicity and topography depending on the applied method of polymerization. Short linear oligomers of unit arrangement similar to the Z polymers were especially abundant in heat-treated WT preparations. Long linear polymers were a prominent and unique component of liver extracts. However, the liver preparations contained also multiple types of oligomers of topographies undistinguishable from those found inWT samples polymerized with heat, low pH or guanidine hydrochloride treatments. In conclusion, we established that AFM is an excellent technique to assess morphological diversity of antitrypsin polymers, which is important for etiology of serpinopathies. These data also support previous, but controversial models of in vivo polymerization showing a surprising diversity of polymer topography. PLOS« less

The Role of the Progressive Ankylosis Protein (ANK) in Adipogenic/Osteogenic Fate Decision of Precursor Cells

PubMed Central

Minashima, Takeshi; Quirno, Martin; Lee, You Jin; Kirsch, Thorsten

2017-01-01

The progressive ankylosis protein (ANK) is a transmembrane protein that transports intracellular pyrophosphate (PPi) to the extracellular milieu. In this study we show increased fatty degeneration of the bone marrow of adult ank/ank mice, which lack a functional ANK protein. In addition, isolated bone marrow stromal cells (BMSCs) isolated from ank/ank mice showed a decreased proliferation rate and osteogenic differentiation potential, and an increased adipogenic differentiation potential compared to BMSCs isolated from wild type (WT) littermates. Wnt signaling pathway PCR array analysis revealed that Wnt ligands, Wnt receptors and Wnt signaling proteins that stimulate osteoblast differentiation were expressed at markedly lower levels in ank/ank BMSCs than in WT BMSCs. Lack of ANK function also resulted in impaired bone fracture healing, as indicated by a smaller callus formed and delayed bone formation in the callus site. Whereas 5 weeks after fracture, the fractured bone in WT mice was further remodeled and restored to original shape, the fractured bone in ank/ank mice was not fully restored and remodeled to original shape. In conclusion, our study provides evidence that ANK plays a critical role in the adipogenic/osteogenic fate decision of adult mesenchymal precursor cells. ANK functions in precursor cells are required for osteogenic differentiation of these cells during adult bone homeostasis and repair, whereas lack of ANK functions favors adipogenic differentiation. PMID:28286238

A Real-Time PCR Assay to Identify and Discriminate Among Wild-Type and Vaccine Strains of Varicella-Zoster Virus and Herpes Simplex Virus in Clinical Specimens, and Comparison With the Clinical Diagnoses

PubMed Central

Harbecke, Ruth; Oxman, Michael N.; Arnold, Beth A.; Ip, Charlotte; Johnson, Gary R.; Levin, Myron J.; Gelb, Lawrence D.; Schmader, Kenneth E.; Straus, Stephen E.; Wang, Hui; Wright, Peter F.; Pachucki, Constance T.; Gershon, Anne A.; Arbeit, Robert D.; Davis, Larry E.; Simberkoff, Michael S.; Weinberg, Adriana; Williams, Heather M.; Cheney, Carol; Petrukhin, Luba; Abraham, Katalin G.; Shaw, Alan; Manoff, Susan; Antonello, Joseph M.; Green, Tina; Wang, Yue; Tan, Charles; Keller, Paul M.

2014-01-01

A real-time PCR assay was developed to identify varicella-zoster virus (VZV) and herpes simplex virus (HSV) DNA in clinical specimens from subjects with suspected herpes zoster (HZ; shingles). Three sets of primers and probes were used in separate PCR reactions to detect and discriminate among wild-type VZV (VZV-WT), Oka vaccine strain VZV (VZV-Oka), and HSV DNA, and the reaction for each virus DNA was multiplexed with primers and probe specific for the human β-globin gene to assess specimen adequacy. Discrimination of all VZV-WT strains, including Japanese isolates and the Oka parent strain, from VZV-Oka was based upon a single nucleotide polymorphism at position 106262 in ORF 62, resulting in preferential amplification by the homologous primer pair. The assay was highly sensitive and specific for the target virus DNA, and no cross-reactions were detected with any other infectious agent. With the PCR assay as the gold standard, the sensitivity of virus culture was 53% for VZV and 77% for HSV. There was 92% agreement between the clinical diagnosis of HZ by the Clinical Evaluation Committee and the PCR assay results. PMID:19475609

A single point mutation in Tomato spotted wilt virus NSs protein is sufficient to overcome Tsw-gene-mediated resistance in pepper.

PubMed

Almási, Asztéria; Nemes, Katalin; Csömör, Zsófia; Tóbiás, István; Palkovics, László; Salánki, Katalin

2017-06-01

The nonstructural protein (NSs) of Tomato spotted wilt virus (TSWV) was previously identified as an avirulence determinant for Tsw-based resistance on pepper. The NSs of wild-type (WT) and resistance-breaking (RB) TSWV strains isolated in Hungary had only two amino acid substitutions (104, 461). We have analysed the ability of the NSs and their point mutant variants to trigger Tsw-mediated hypersensitive responses and RNA silencing suppressor (RSS) activity in patch assays. We identified a single amino acid change at position 104 (T-A) that was responsible for the necrosis induction or loss, while a significant difference was not detected in the RSS activity of the two parental strains. We have successfully complemented the infection of the WT strain on resistant pepper cultivar with the infectious S RNA transcript of the RB strain and the WT-T104A point mutant. Our work provides direct evidence that a single amino acid change can induce an RB phenotype.

PGE2 receptor EP3 inhibits water reabsorption and contributes to polyuria and kidney injury in a streptozotocin-induced mouse model of diabetes.

PubMed

Hassouneh, Ramzi; Nasrallah, Rania; Zimpelmann, Joe; Gutsol, Alex; Eckert, David; Ghossein, Jamie; Burns, Kevin D; Hébert, Richard L

2016-06-01

The first clinical manifestation of diabetes is polyuria. The prostaglandin E2 (PGE2) receptor EP3 antagonises arginine vasopressin (AVP)-mediated water reabsorption and its expression is increased in the diabetic kidney. The purpose of this work was to study the contribution of EP3 to diabetic polyuria and renal injury. Male Ep 3 (-/-) (also known as Ptger3 (-/-)) mice were treated with streptozotocin (STZ) to generate a mouse model of diabetes and renal function was evaluated after 12 weeks. Isolated collecting ducts (CDs) were microperfused to study the contribution of EP3 to AVP-mediated fluid reabsorption. Ep 3 (-/-)-STZ mice exhibited attenuated polyuria and increased urine osmolality compared with wild-type STZ (WT-STZ) mice, suggesting enhanced water reabsorption. Compared with WT-STZ mice, Ep 3 (-/-)-STZ mice also had increased protein expression of aquaporin-1, aquaporin-2, and urea transporter A1, and reduced urinary AVP excretion, but increased medullary V2 receptors. In vitro microperfusion studies indicated that Ep 3 (-/-) and WT-STZ CDs responded to AVP stimulation similarly to those of wild-type mice, with a 60% increase in fluid reabsorption. In WT non-injected and WT-STZ mice, EP3 activation with sulprostone (PGE2 analogue) abrogated AVP-mediated water reabsorption; this effect was absent in mice lacking EP3. A major finding of this work is that Ep 3 (-/-)-STZ mice showed blunted renal cyclooxygenase-2 protein expression, reduced renal hypertrophy, reduced hyperfiltration and reduced albuminuria, as well as diminished tubular dilation and nuclear cysts. Taken together, the data suggest that EP3 contributes to diabetic polyuria by inhibiting expression of aquaporins and that it promotes renal injury during diabetes. EP3 may prove to be a promising target for more selective management of diabetic kidney disease.

Wild-type male offspring of fmr-1+/- mothers exhibit characteristics of the fragile X phenotype.

PubMed

Zupan, Bojana; Toth, Miklos

2008-10-01

Fragile X syndrome is an X-linked disorder caused by the inactivation of the FMR-1 gene with symptoms ranging from impaired cognitive functions to seizures, anxiety, sensory abnormalities, and hyperactivity. Males are more severely affected than heterozygote (H) females, who, as carriers, have a 50% chance of transmitting the mutated allele in each pregnancy. fmr-1 knockout (KO) mice reproduce fragile X symptoms, including hyperactivity, seizures, and abnormal sensory processing. In contrast to the expectation that wild-type (WT) males born to H (fmr-1(+/-)) mothers (H>WT) are behaviorally normal and indistinguishable from WT males born to WT mothers (WT>WT); here, we show that H>WT offspring are more active than WT>WT offspring and that their hyperactivity is similar to male KO mice born to H or KO (fmr-1(-/-)) mothers (H>KO/KO>KO). H>WT mice, however, do not exhibit seizures or abnormal sensory processing. Consistent with their hyperactivity, the effect of the D2 agonist quinpirole is reduced in H>WT as well as in H>KO and KO>KO mice compared to WT>WT offspring, suggesting a diminished feedback inhibition of dopamine release. Our data indicate that some aspects of hyperactivity and associated dopaminergic changes in 'fragile X' mice are a maternal fmr-1 genotype rather than an offspring fmr-1 genotype effect.

Wild-Type Male Offspring of fmr-1+/− Mothers Exhibit Characteristics of the Fragile X Phenotype

PubMed Central

Zupan, Bojana; Toth, Miklos

2009-01-01

Fragile X syndrome is an X-linked disorder caused by the inactivation of the FMR-1 gene with symptoms ranging from impaired cognitive functions to seizures, anxiety, sensory abnormalities, and hyperactivity. Males are more severely affected than heterozygote (H) females, who, as carriers, have a 50% chance of transmitting the mutated allele in each pregnancy. fmr-1 knockout (KO) mice reproduce fragile X symptoms, including hyperactivity, seizures, and abnormal sensory processing. In contrast to the expectation that wild-type (WT) males born to H (fmr-1+/−) mothers (H> WT) are behaviorally normal and indistinguishable from WT males born to WT mothers (WT> WT); here, we show that H> WT offspring are more active than WT> WT offspring and that their hyperactivity is similar to male KO mice born to H or KO (fmr-1−/−) mothers (H> KO/KO> KO). H> WT mice, however, do not exhibit seizures or abnormal sensory processing. Consistent with their hyperactivity, the effect of the D2 agonist quinpirole is reduced in H> WT as well as in H> KO and KO> KO mice compared to WT> WT offspring, suggesting a diminished feedback inhibition of dopamine release. Our data indicate that some aspects of hyperactivity and associated dopaminergic changes in ‘fragile X’ mice are a maternal fmr-1 genotype rather than an offspring fmr-1 genotype effect. PMID:18172434

Aequorea green fluorescent protein analysis by flow cytometry

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

Ropp, J.D.; Cuthbertson, R.A.; Donahue, C.J.

The isolation and expression of the cDNA for the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria has highlighted its potential use as a marker for gene expression in a variety of cell types. The longer wavelength peak (470 nm) of GFP`s bimodal absorption spectrum better matches standard fluorescein filter sets; however, it has a considerably lower amplitude than the major absorption peak at 395. In an effort to increase the sensitivity of GFP with routinely available instrumentation, Heim et al. have generated a GFP mutant (serine-65 to threonine; S65T-GFP) which possesses a single absorption peak centered atmore » 490 nm. We have constructed this mutant in order to determine whether it or wild-type GFP (wt-GFP) afforded greater sensitivity when excited near their respective absorption maxima. Using the conventionally available 488 nm and ultraviolet (UV) laser lines from the argon ion laser as well as the 407 nm line from a krypton ion laser with enhanced violet emission, we were able to closely match the absorption maxima of both the S65T and wild-type forms of Aequorea GFP and analyze differences in fluorescence intensity of transiently transfected 293 cells with flow cytometry. The highest fluorescence signal was observed with 488 nm excitation of S65T-GFP relative to all other laser line/GFP pairs. The wt-GFP fluorescence intensity, in contrast, was significantly higher at 407 nm relative to either 488 nm or UV. These results were consistent with parallel spectrofluorometric analysis of the emission spectrum for wt-GFP and S65T- GFP. The relative contribution of cellular autofluorescence at each wavelength was also investigated and shown to be significantly reduced at 407 nm relative to either UV or 488 nm. 29 refs., 5 figs.« less

Virulence genes and plasmid profiles in Rhodococcus equi isolates from domestic pigs and wild boars (Sus scrofa) in Brazil.

PubMed

Ribeiro, Márcio Garcia; Takai, Shinji; Guazzelli, Alessandro; Lara, Gustavo Henrique Batista; da Silva, Aristeu Vieira; Fernandes, Marta Catarina; Condas, Larissa Anuska Zeni; Siqueira, Amanda Keller; Salerno, Tatiana

2011-12-01

The virulence genes and plasmid profiles of 23 Rhodococcus equi isolates from 258 lymph nodes from domestic pigs (129 nodes with lesions and 129 without lesions) and 120 lymph nodes from slaughtered wild boars (60 nodes with lesions and 60 without) were characterized. R. equi was obtained from 19 lymph nodes of domestic pigs, 17 with, and two without lesions, and from four lymph nodes with lesions, from wild boars. The 23 isolates were tested for the presence of vapA and vapB genes, responsible for the 15-17 and 20 kDa virulence-associated proteins, respectively, by PCR in order to characterize as virulent (VapA), intermediately virulent (VapB) and avirulent. Plasmid DNAs were isolated and analyzed by digestion with restriction endonucleases to estimate size and compare their polymorphisms. Of the 19 domestic pigs strains, seven (36.8%) were avirulent and 12 (63.2%) were intermediately virulent, with the intermediately virulent isolates being plasmid types 8 (8 isolates), 10 (2 isolates), 1 (1 isolate) and 29 (1 isolate). The plasmid type of four strains isolated from wild boars was also intermediately virulent type 8. None of the domestic pigs and wild boar isolates showed the vapA gene. These findings demonstrate a high occurrence of plasmid type 8 in isolates from pigs and wild boars, and the similarity of plasmid types in the domestic pigs, wild boars and human isolates in Brazil. Copyright © 2010 Elsevier Ltd. All rights reserved.

Phytoremediation of arsenic from the contaminated soil using transgenic tobacco plants expressing ACR2 gene of Arabidopsis thaliana.

PubMed

Nahar, Noor; Rahman, Aminur; Nawani, Neelu N; Ghosh, Sibdas; Mandal, Abul

2017-11-01

We have cloned, characterized and transformed the AtACR2 gene (arsenic reductase 2) of Arabidopsis thaliana into the genome of tobacco (Nicotiana tabacum, var Sumsun). Our results revealed that the transgenic tobacco plants are more tolerant to arsenic than the wild type ones. These plants can grow on culture medium containing 200μM arsenate, whereas the wild type can barely survive under this condition. Furthermore, when exposed to 100μM arsenate for 35days the amount of arsenic accumulated in the shoots of transgenic plants was significantly lower (28μg/g d wt.) than that found in the shoots of non-transgenic controls (40μg/g d wt.). However, the arsenic content in the roots of transgenic plants was significantly higher (2400μg/g d. wt.) than that (2100μg/g d. wt.) observed in roots of wild type plants. We have demonstrated that Arabidopsis thaliana AtACR2 gene is a potential candidate for genetic engineering of plants to develop new crop cultivars that can be grown on arsenic contaminated fields to reduce arsenic content of the soil and can become a source of food containing no arsenic or exhibiting substantially reduced amount of this metalloid. Copyright © 2017 Elsevier GmbH. All rights reserved.

Biological cost of fosfomycin resistance in Escherichia coli in a murine model of urinary tract infection.

PubMed

Pourbaix, A; Guérin, F; Lastours, V de; Chau, F; Auzou, M; Boulley, E; Cattoir, V; Fantin, B

2017-12-01

Prevalence of fosfomycin resistance in E. coli clinical isolates from UTIs remains very low. Our hypothesis was that fosfomycin resistance may be associated with a biological cost. Three groups of strains of E. coli belonging to the B2 phylogenetic group were used: clinical wild-type (WT) isolates, clinical multidrug-resistant isolates and in vitro fosfomycin-resistant derivatives from the uropathogen clinical strain E. coli CFT073. In each group fosfomycin-susceptible and -resistant isolates were compared. In vitro, we found a significantly decreased growth rate for fosfomycin-resistant strains as compared with susceptible strains in the WT group. In a murine model of ascending UTI, there was a significant reduction in infection rates with fosfomycin-resistant isolates as compared with susceptible ones, in all 3 study groups, ranging from 28 to 39% (P<0.03). All fosfomycin-susceptible clinical strains were virulent in vivo (13/13), while fosfomycin-resistant clinical strains were either virulent (2/7) or non-virulent (5/7) (P<0.002). This difference was not explained by the number of virulence factors or pathogenicity-associated islands. In conclusion, fosfomycin resistance appears to carry some biological cost in E. coli, which may explain in part the apparent paradox of the low prevalence of fosfomycin resistance despite a high rate of spontaneous mutants. Copyright © 2017 Elsevier GmbH. All rights reserved.

T-type Ca2+ channels regulate the exit of cardiac myocytes from the cell cycle after birth

PubMed Central

Wang, Fang; Gao, Hui; Kubo, Hajime; Fan, Xiaoxuan; Zhang, Hongyu; Berretta, Remus; Chen, Xiongwen; Sharp, Thomas; Starosta, Timothy; Makarewich, Catherine; Li, Ying; Molkentin, Jeffrey D.; Houser, Steven R.

2013-01-01

T-type Ca2+ channels (TTCCs) are expressed in the fetal heart and then disappear from ventricular myocytes after birth. The hypothesis examined in this study was the α1G TTCCs' influence in myocyte maturation and their rapid withdrawal from the cell cycle after birth. Methods Cardiac myocytes were isolated from neonatal and adult wild type (WT), α1G−/− and α1G over expressing (α1GDT) mice. Bromodeoxyuridine (BrdU) uptake, myocyte nucleation, cell cycle analysis, and T-type Ca2+ currents were measured. Results All myocytes were mono-nucleated at birth and 35% of WT myocytes expressed functional TTCCs. Very few neonatal myocytes had functional TTCCs in α1G−/− hearts. By the end of the first week after birth no WT or α1G−/− had functional TTCCs. During the first week after birth about 25% of WT myocytes were BrdU+ and became bi-nucleated. Significantly fewer α1G−/− myocytes became bi-nucleated and fewer of these myocytes were BrdU+. Neonatal α1G−/− myocytes were also smaller than WT. Adult WT and α1G−/− hearts were similar in size, but α1G−/− myocytes were smaller and a greater % were mono-nucleated. α1G over expressing hearts were smaller than WT but their myocytes were larger. Conclusions The studies performed show that loss of functional TTCCs is associated with bi-nucleation and myocyte withdrawal from the cell cycle. Loss of α1G TTCCs slowed the transition from mono- to bi-nucleation and resulted in an adult heart with a greater number of small cardiac myocytes. These results suggest that TTCCs are involved in the regulation of myocyte size and the exit of myocytes from the cell cycle during the first week after birth. PMID:23743021

Isolated lymphoid follicles are not IgA inductive sites for recombinant Salmonella

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

Hashizume, Tomomi; Momoi, Fumiki; Kurita-Ochiai, Tomoko

2007-08-24

In this study, we investigated whether isolated lymphoid follicles (ILF) play a role in the regulation of intestinal IgA antibody (Ab) responses. The transfer of wild type (WT) bone marrow (BM) to lymphotoxin-{alpha}-deficient (LT{alpha}{sup -/-}) mice resulted in the formation of mature ILF containing T cells, B cells, and FDC clusters in the absence of mesenteric lymph nodes and Peyer's patches. Although the ILF restored total IgA Abs in the intestine, antigen (Ag)-specific IgA responses were not induced after oral immunization with recombinant Salmonella expressing fragment C of tetanus toxin. Moreover, Ag-specific cell proliferation was not detected in the ILF.more » Interestingly, no IgA anti-LPS Abs were detected in the fecal extracts of LT{alpha}{sup -/-} mice reconstituted with WT BM. On the basis of these findings, ILF can be presumed to play a role in the production of IgA Abs, but lymphoid nodules are not inductive sites for the regulation of Ag-specific intestinal IgA responses to recombinant Salmonella.« less

Efficient assembly of full-length infectious clone of Brazilian IBDV isolate by homologous recombination in yeast

PubMed Central

Silva, J.V.J.; Arenhart, S.; Santos, H.F.; Almeida-Queiroz, S.R.; Silva, A.N.M.R.; Trevisol, I.M.; Bertani, G.R.; Gil, L.H.V.G.

2014-01-01

The Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens. Advances in molecular virology and vaccines for IBDV have been achieved by viral reverse genetics (VRG). VRG for IBDV has undergone changes over time, however all strategies used to generate particles of IBDV involves multiple rounds of amplification and need of in vitro ligation and restriction sites. The aim of this research was to build the world’s first VRG for IBDV by yeast-based homologous recombination; a more efficient, robust and simple process than cloning by in vitro ligation. The wild type IBDV (Wt-IBDV-Br) was isolated in Brazil and had its genome cloned in pJG-CMV-HDR vector by yeast-based homologous recombination. The clones were transfected into chicken embryo fibroblasts and the recovered virus (IC-IBDV-Br) showed genetic stability and similar phenotype to Wt-IBDV-Br, which were observed by nucleotide sequence, focus size/morphology and replication kinetics, respectively. Thus, IBDV reverse genetics by yeast-based homologous recombination provides tools to IBDV understanding and vaccines/viral vectors development. PMID:25763067

Antimicrobial Susceptibility of Flavobacterium psychrophilum from Chilean Salmon Farms and Their Epidemiological Cut-Off Values Using Agar Dilution and Disk Diffusion Methods.

PubMed

Miranda, Claudio D; Smith, Peter; Rojas, Rodrigo; Contreras-Lynch, Sergio; Vega, J M Alonso

2016-01-01

Flavobacterium psychrophilum is the most important bacterial pathogen for freshwater farmed salmonids in Chile. The aims of this study were to determine the susceptibility to antimicrobials used in fish farming of Chilean isolates and to calculate their epidemiological cut-off (CO WT ) values. A number of 125 Chilean isolates of F. psychrophilum were isolated from reared salmonids presenting clinical symptoms indicative of flavobacteriosis and their identities were confirmed by 16S rRNA polymerase chain reaction. Susceptibility to antibacterials was tested on diluted Mueller-Hinton by using an agar dilution MIC method and a disk diffusion method. The CO WT values calculated by Normalized Resistance Interpretation (NRI) analysis allow isolates to be categorized either as wild-type fully susceptible (WT) or as manifesting reduced susceptibility (NWT). When MIC data was used, NRI analysis calculated a CO WT of ≤0.125, ≤2, and ≤0.5 μg mL -1 for amoxicillin, florfenicol, and oxytetracycline, respectively. For the quinolones, the CO WT were ≤1, ≤0.5, and ≤0.125 μg mL -1 for oxolinic acid, flumequine, and enrofloxacin, respectively. The disk diffusion data sets obtained in this work were extremely diverse and were spread over a wide range. For the quinolones there was a close agreement between the frequencies of NWT isolates calculated using MIC and disk data. For oxolinic acid, flumequine, and enrofloxacin the frequencies were 45, 39, and 38% using MIC data, and 42, 41, and 44%, when disk data were used. There was less agreement with the other antimicrobials, because NWT frequencies obtained using MIC and disk data, respectively, were 24 and 10% for amoxicillin, 8 and 2% for florfenicol, and 70 and 64% for oxytetracycline. Considering that the MIC data was more precise than the disk diffusion data, MIC determination would be the preferred method for susceptibility testing for this species and the NWT frequencies derived from the MIC data sets should be considered as the more authoritative. Despite the high frequency of isolates showing full susceptibility to florfenicol, the significant frequencies of isolates exhibiting reduced susceptibility to oxytetracycline and quinolones may result in treatment failures when these agents are used.

Cellobiohydrolase I enzymes

DOEpatents

Adney, William S; Himmel, Michael E; Decker, Stephen R; Knoshaug, Eric P; Nimlos, Mark R; Crowley, Michael F; Jeoh, Tina

2014-01-28

Provided herein is an isolated Cel7A polypeptide comprising mutations in the catalytic domain of the polypeptide relative to the catalytic domain of a wild type Cel7A polypeptide, wherein the mutations reduce N-linked glycosylation of the isolated polypeptide relative to the wild type polypeptide. Also provided herein is an isolated Cel7A polypeptide comprising increased O-linked glycosylation of the linker domain relative to a linker domain of a wild type Cel7A polypeptide. The increased O-linked glycosylation is a result of the addition of and/or substitution of one or more serine and/or threonine residues to the linker domain relative to the linker domain of the wild type polypeptide. In some embodiments, the isolated Cel7A polypeptide comprising mutations in the catalytic domain of the polypeptide relative to the catalytic domain of a wild type Cel7A polypeptide further comprises increased O-linked glycosylation of the linker domain relative to a linker domain of a wild type Cel7A polypeptide. The mutations in the catalytic domain reduce N-linked glycosylation of the isolated polypeptide relative to the wild type polypeptide. The addition of and/or substitution of one or more serine and/or threonine residues to the linker domain relative to the linker domain of the wild type polypeptide increases O-linked glycosylation of the isolated polypeptide. Further provided are compositions comprising such polypeptides and nucleic acids encoding such polypeptides. Still further provided are methods for making such polypeptides.

Effects of targeted deletion of A1 adenosine receptors on postischemic cardiac function and expression of adenosine receptor subtypes.

PubMed

Morrison, R Ray; Teng, Bunyen; Oldenburg, Peter J; Katwa, Laxmansa C; Schnermann, Jurgen B; Mustafa, S Jamal

2006-10-01

To examine ischemic tolerance in the absence of A(1) adenosine receptors (A(1)ARs), isolated wild-type (WT) and A(1)AR knockout (A(1)KO) murine hearts underwent global ischemia-reperfusion, and injury was measured in terms of functional recovery and efflux of lactate dehydrogenase (LDH). Hearts were analyzed by real-time RT-PCR both at baseline and at intervals during ischemia-reperfusion to determine whether compensatory expression of other adenosine receptor subtypes occurs with either A(1)AR deletion and/or ischemia-reperfusion. A(1)KO hearts had higher baseline coronary flow (CF) and left ventricular developed pressure (LVDP) than WT hearts, whereas heart rate was unchanged by A(1)AR deletion. After 20 min of ischemia, CF was attenuated in A(1)KO compared with WT hearts, and this reduction persisted throughout reperfusion. Final recovery of LVDP was decreased in A(1)KO hearts (54.4 +/- 5.1 vs. WT 81.1 +/- 3.4% preischemic baseline) and correlated with higher diastolic pressure during reperfusion. Postischemic efflux of LDH was greater in A(1)KO compared with WT hearts. Real-time RT-PCR demonstrated the absence of A(1)AR transcript in A(1)KO hearts, and the message for A(2A), A(2B), and A(3) adenosine receptors was similar in uninstrumented A(1)KO and WT hearts. Ischemia-reperfusion increased A(2B) mRNA expression 2.5-fold in both WT and A(1)KO hearts without changing A(1) or A(3) expression. In WT hearts, ischemia transiently doubled A(2A) mRNA, which returned to preischemic level upon reperfusion, a pattern not observed in A(1)KO hearts. Together, these data affirm the cardioprotective role of A(1)ARs and suggest that induced expression of other adenosine receptor subtypes may participate in the response to ischemia-reperfusion in isolated murine hearts.

Curvature induced by amyloplast magnetophoresis in protonemata of the moss Ceratodon purpureus

NASA Technical Reports Server (NTRS)

Kuznetsov, O. A.; Schwuchow, J.; Sack, F. D.; Hasenstein, K. H.

1999-01-01

After gravistimulation of Ceratodon purpureus (Hedw.) Brid. protonemata in the dark, amyloplast sedimentation was followed by upward curvature in the wild-type (WT) and downward curvature in the wwr mutant (wrong way response). We used ponderomotive forces induced by high-gradient magnetic fields (HGMF) to simulate the effect of gravity and displace the presumptive statoliths. The field was applied by placing protonemata either between two permanent magnets at the edge of the gap, close to the edge of a magnetized ferromagnetic wedge, or close to a small (<1 mm) permanent magnet. Continuous application of an HGMF in all three configurations resulted in plastid displacement and induced curvature in tip cells of WT and wwr protonemata. WT cells curved toward the HGMF, and wwr cells curved away from the HGMF, comparable to gravitropism. Plastids isolated from protonemal cultures had densities ranging from 1.24 to 1.38 g cm-3. Plastid density was similar for both genotypes, but the mutant contained larger plastids than the WT. The size difference might explain the stronger response of the wwr protonemata to the HGMF. Our data support the plastid-based theory of gravitropic sensing and suggest that HGMF-induced ponderomotive forces can substitute for gravity.

Global ablation of the mitochondrial calcium uniporter increases glycolysis in cortical neurons subjected to energetic stressors.

PubMed

Nichols, Matthew; Elustondo, Pia A; Warford, Jordan; Thirumaran, Aruloli; Pavlov, Evgeny V; Robertson, George S

2017-08-01

The effects of global mitochondrial calcium (Ca 2+ ) uniporter (MCU) deficiency on hypoxic-ischemic (HI) brain injury, neuronal Ca 2+ handling, bioenergetics and hypoxic preconditioning (HPC) were examined. Forebrain mitochondria isolated from global MCU nulls displayed markedly reduced Ca 2+ uptake and Ca 2+ -induced opening of the membrane permeability transition pore. Despite evidence that these effects should be neuroprotective, global MCU nulls and wild-type (WT) mice suffered comparable HI brain damage. Energetic stress enhanced glycolysis and depressed Complex I activity in global MCU null, relative to WT, cortical neurons. HI reduced forebrain NADH levels more in global MCU nulls than WT mice suggesting that increased glycolytic consumption of NADH suppressed Complex I activity. Compared to WT neurons, pyruvate dehydrogenase (PDH) was hyper-phosphorylated in MCU nulls at several sites that lower the supply of substrates for the tricarboxylic acid cycle. Elevation of cytosolic Ca 2+ with glutamate or ionomycin decreased PDH phosphorylation in MCU null neurons suggesting the use of alternative mitochondrial Ca 2+ transport. Under basal conditions, global MCU nulls showed similar increases of Ca 2+ handling genes in the hippocampus as WT mice subjected to HPC. We propose that long-term adaptations, common to HPC, in global MCU nulls compromise resistance to HI brain injury and disrupt HPC.

Virologic surveillance for wild-type rubella viruses in the Americas.

PubMed

Icenogle, Joseph P; Siqueira, Marilda M; Abernathy, Emily S; Lemos, Xenia R; Fasce, Rodrigo A; Torres, Graciela; Reef, Susan E

2011-09-01

The goal of eliminating rubella from the Americas by 2010 was established in 2003. Subsequently, a systematic nomenclature for wild-type rubella viruses (wtRVs) was established, wtRVs circulating in the region were catalogued, and importations of wtRVs into a number of countries were documented. The geographic distribution of wtRVs of various genotypes in the Americas, interpreted in the context of the global distribution of these viruses, contributed to the documentation of rubella elimination from some countries. Data from virologic surveillance also contributed to the conclusion that viruses of genotype 2B began circulating endemically in the Americas during 2006-2007. Viruses of one genotype (1C), which are restricted to the Americas, will likely disappear completely from the world as they are eliminated from the Americas. Efforts to expand virologic surveillance for wtRVs in the Americas will also provide additional data aiding the elimination of rubella from the region. For example, identification of vaccine virus in specimens from rash and fever cases found during elimination can identify such cases as vaccine associated.

The YAP1/SIX2 axis is required for DDX3-mediated tumor aggressiveness and cetuximab resistance in KRAS-wild-type colorectal cancer

PubMed Central

Wu, De-Wei; Lin, Po-Lin; Wang, Lee; Huang, Chi-Chou; Lee, Huei

2017-01-01

The mechanism underlying tumor aggressiveness and cetuximab (CTX) resistance in KRAS-wild-type (KRAS -WT) colorectal cancer remains obscure. We here provide evidence that DDX3 promoted soft agar growth and invasiveness of KRAS-WT cells, as already confirmed in KRAS-mutated cells. Mechanistically, increased KRAS expression induced ROS production, which elevated HIF-1α and YAP1 expression. Increased HIF-1α persistently promoted DDX3 expression via a KRAS/ROS/HIF-1α feedback loop. DDX3-mediated aggressiveness and CTX resistance were regulated by the YAP1/SIX2 axis in KRAS-WT cells and further confirmed in animal models. Kaplan-Meier and Cox regression analysis indicated that DDX3, KRAS, and YAP1 expression had prognostic value for OS and RFS in KRAS-WT and KRAS-mutated tumors, but SIX2 and YAP1/SIX2 were prognostic value only in KRAS-WT patients. The observation from patients seemed to support the mechanistic action of cell and animal models. We therefore suggest that combining YAP1 inhibitors with CTX may therefore suppress DDX3-mediated tumor aggressiveness and enhance CTX sensitivity in KRAS-WT colorectal cancer. PMID:28435452

Flow cytometry-assisted rapid isolation of recombinant Plasmodium berghei parasites exemplified by functional analysis of aquaglyceroporin

PubMed Central

Kenthirapalan, Sanketha; Waters, Andrew P.; Matuschewski, Kai; Kooij, Taco W.A.

2012-01-01

The most critical bottleneck in the generation of recombinant Plasmodium berghei parasites is the mandatory in vivo cloning step following successful genetic manipulation. This study describes a new technique for rapid selection of recombinant P. berghei parasites. The method is based on flow cytometry to isolate isogenic parasite lines and represents a major advance for the field, in that it will speed the generation of recombinant parasites as well as cut down on animal use significantly. High expression of GFP during blood infection, a prerequisite for robust separation of transgenic lines by flow cytometry, was achieved. Isogenic recombinant parasite populations were isolated even in the presence of a 100-fold excess of wild-type (WT) parasites. Aquaglyceroporin (AQP) loss-of-function mutants and parasites expressing a tagged AQP were generated to validate this approach. aqp− parasites grow normally within the WT phenotypic range during blood infection of NMRI mice. Similarly, colonization of the insect vector and establishment of an infection after mosquito transmission were unaffected, indicating that AQP is dispensable for life cycle progression in vivo under physiological conditions, refuting its use as a suitable drug target. Tagged AQP localized to perinuclear structures and not the parasite plasma membrane. We suggest that flow-cytometric isolation of isogenic parasites overcomes the major roadblock towards a genome-scale repository of mutant and transgenic malaria parasite lines. PMID:23137753

Overexpression of EsMcsu1 from the halophytic plant Eutrema salsugineum promotes abscisic acid biosynthesis and increases drought resistance in alfalfa (Medicago sativa L.).

PubMed

Zhou, C; Ma, Z Y; Zhu, L; Guo, J S; Zhu, J; Wang, J F

2015-12-17

The stress phytohormone abscisic acid (ABA) plays pivotal roles in plants' adaptive responses to adverse environments. Molybdenum cofactor sulfurases influence aldehyde oxidase activity and ABA biosynthesis. In this study, we isolated a novel EsMcsu1 gene encoding a molybdenum cofactor sulfurase from Eutrema salsugineum. EsMcus1 transcriptional patterns varied between organs, and its expression was significantly upregulated by abiotic stress or ABA treatment. Alfalfa plants that overexpressed EsMcsu1 had a higher ABA content than wild-type (WT) plants under drought stress conditions. Furthermore, levels of reactive oxygen species (ROS), ion leakage, and malondialdehyde were lower in the transgenic plants than in the WT plants after drought treatment, suggesting that the transgenic plants experienced less ROS-mediated damage. However, the expression of several stress-responsive genes, antioxidant enzyme activity, and osmolyte (proline and total soluble sugar) levels in the transgenic plants were higher than those in the WT plants after drought treatment. Therefore, EsMcsu1 overexpression improved drought tolerance in alfalfa plants by activating a series of ABA-mediated stress responses.

Host CD40 Is Essential for DCG Treatment Against Metastatic Lung Cancer.

PubMed

Yamashita, Kimihiro; Hasegawa, Hiroshi; Fujita, Mitsugu; Nishi, Masayasu; Tanaka, Tomoko; Arimoto, Akira; Suzuki, Satoshi; Kamigaki, Takashi; Kakeji, Yoshihiro

2016-07-01

For the application of invariant natural killer T (iNKT) cells in cancer therapy, the CD40-CD40L interaction is indispensable in administering alpha-galactosylceramide (αGalCer). We hypothesized that CD40 plays an important role in dendritic cells (DC) pulsed with αGalCer (DCGs) in the treatment of lung metastases. Wild-type (WT) and CD40(-/-) mice were treated with DCGs isolated from WT or CD40(-/-) mice in a B16F10 lung metastases model and NK and NKT cell activity in lungs and the spleen were examined. DCG treatment improved WT mice survival but CD40(-/-) hosts received no survival benefit. Conversely, attenuation of a therapeutic effect in mice treated with CD40(-/-) DCGs was not observed. The functional activities of NK and NKT cells in DCG-treated CD40(-/-) mice were partially suppressed. Host CD40 is essential for DCG treatment to have a therapeutic effect on B16F10 lung metastases. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Drought stress-induced compositional changes in tolerant transgenic rice and its wild type.

PubMed

Nam, Kyong-Hee; Kim, Do-Young; Shin, Hee Jae; Nam, Ki Jung; An, Joo Hee; Pack, In-Soon; Park, Jung-Ho; Jeong, Soon-Chun; Kim, Ho Bang; Kim, Chang-Gi

2014-06-15

Comparing well-watered versus deficit conditions, we evaluated the chemical composition of grains harvested from wild-type (WT) and drought-tolerant, transgenic rice (Oryza sativa L.). The latter had been developed by inserting AtCYP78A7, which encodes a cytochrome P450 protein. Two transgenic Lines, '10B-5' and '18A-4', and the 'Hwayoung' WT were grown under a rainout shelter. After the harvested grains were polished, their levels of key components, including proximates, amino acids, fatty acids, minerals and vitamins were analysed to determine the effect of watering system and genotype. Drought treatment significantly influenced the levels of some nutritional components in both transgenic and WT grains. In particular, the amounts of lignoceric acid and copper in the WT decreased by 12.6% and 39.5%, respectively, by drought stress, whereas those of copper and potassium in the transgenics rose by 88.1-113.3% and 10.4-11.9%, respectively, under water-deficit conditions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Kinetic modeling of batch fermentation for Populus hydrolysate tolerant mutant and wild type strains of Clostridium thermocellum.

PubMed

Linville, Jessica L; Rodriguez, Miguel; Mielenz, Jonathan R; Cox, Chris D

2013-11-01

The extent of inhibition of two strains of Clostridium thermocellum by a Populus hydrolysate was investigated. A Monod-based model of wild type (WT) and Populus hydrolysate tolerant mutant (PM) strains of the cellulolytic bacterium C. thermocellum was developed to quantify growth kinetics in standard media and the extent of inhibition to a Populus hydrolysate. The PM was characterized by a higher growth rate (μmax=1.223 vs. 0.571 h(-1)) and less inhibition (KI,gen=0.991 vs. 0.757) in 10% v/v Populus hydrolysate compared to the WT. In 17.5% v/v Populus hydrolysate inhibition of PM increased slightly (KI,gen=0.888), whereas the WT was strongly inhibited and did not grow in a reproducible manner. Of the individual inhibitors tested, 4-hydroxybenzoic acid was the most inhibitory, followed by galacturonic acid. The PM did not have a greater ability to detoxify the hydrolysate than the WT. Copyright © 2013 Elsevier Ltd. All rights reserved.

NOX2 Deficiency Protects Against Streptozotocin-Induced β-Cell Destruction and Development of Diabetes in Mice

PubMed Central

Xiang, Fu-Li; Lu, Xiangru; Strutt, Brenda; Hill, David J.; Feng, Qingping

2010-01-01

OBJECTIVE The role of NOX2-containing NADPH oxidase in the development of diabetes is not fully understood. We hypothesized that NOX2 deficiency decreases reactive oxygen species (ROS) production and immune response and protects against streptozotocin (STZ)-induced β-cell destruction and development of diabetes in mice. RESEARCH DESIGN AND METHODS Five groups of mice—wild-type (WT), NOX2−/−, WT treated with apocynin, and WT adoptively transferred with NOX2−/− or WT splenocytes—were treated with multiple-low-dose STZ. Blood glucose and insulin levels were monitored, and an intraperitoneal glucose tolerance test was performed. Isolated WT and NOX2−/− pancreatic islets were treated with cytokines for 48 h. RESULTS Significantly lower blood glucose levels, higher insulin levels, and better glucose tolerance was observed in NOX2−/− mice and in WT mice adoptively transferred with NOX2−/− splenocytes compared with the respective control groups after STZ treatment. Compared with WT, β-cell apoptosis, as determined by TUNEL staining, and insulitis were significantly decreased, whereas β-cell mass was significantly increased in NOX2−/− mice. In response to cytokine stimulation, ROS production was significantly decreased, and insulin secretion was preserved in NOX2−/− compared with WT islets. Furthermore, proinflammatory cytokine release induced by concanavalin A was significantly decreased in NOX2−/− compared with WT splenocytes. CONCLUSIONS NOX2 deficiency decreases β-cell destruction and preserves islet function in STZ-induced diabetes by reducing ROS production, immune response, and β-cell apoptosis. PMID:20627937

A fragment substitution in the promoter of CsHDZIV11/CsGL3 is responsible for fruit spine density in cucumber (Cucumis sativus L.).

PubMed

Zhang, Haiyang; Wang, Lina; Zheng, Shuangshuang; Liu, Zezhou; Wu, Xiaoqin; Gao, Zhihui; Cao, Chenxing; Li, Qiang; Ren, Zhonghai

2016-07-01

The indel in the promoter of CsHDZIV11 co-segregates with fruit spine density and could be used for molecular breeding in cucumber. Fruit spine density is an important quality trait for marketing in cucumber (Cucumis sativus L.). However, the molecular basis of fruit spine density in cucumber remains unclear. In this study, we isolated a mutant, few spines 1 (fs1), from CNS2 (wild type, WT), a North China-type cucumber with a high density of fruit spines. Genetic analysis showed that fs1 was controlled by a single recessive Mendelian factor. Bulked segregant analysis combined with genome resequencing were used for mapping fs1 in the F2 population derived from a cross between the fs1 mutant and WT, and it was located on chromosome 6 through association analysis. To develop more polymorphic markers to locate fs1, another F2 population was constructed from the cross between fs1 and 'Chinese long' 9930. Then, fs1 was narrowed down to a 110.4-kb genomic region containing 25 annotated genes. A fragment substitution was identified in the promoter region of Csa6M514870 between fs1 and WT. This fragment in fs1 was also present in wild cucumber. Csa6M514870 encodes a PDF2-related protein, a homeodomain-leucine zipper IV transcription factor (CsHDZIV11/CsGL3) sharing high identity and similarity with proteins related to trichome formation or epidermal cell differentiation. Quantitative reverse-transcription PCR revealed a higher expression level of CsHDZIV11 in young fruits from fs1 compared to WT. A molecular marker based on this indel co-segregated with the spine density. This work provides a solid foundation not only for understanding the molecular mechanism of fruit spine density, but also for molecular breeding in cucumber.

Curcumin-induced heme oxygenase-1 expression prevents H2O2-induced cell death in wild type and heme oxygenase-2 knockout adipose-derived mesenchymal stem cells.

PubMed

Cremers, Niels A J; Lundvig, Ditte M S; van Dalen, Stephanie C M; Schelbergen, Rik F; van Lent, Peter L E M; Szarek, Walter A; Regan, Raymond F; Carels, Carine E; Wagener, Frank A D T G

2014-10-08

Mesenchymal stem cell (MSC) administration is a promising adjuvant therapy to treat tissue injury. However, MSC survival after administration is often hampered by oxidative stress at the site of injury. Heme oxygenase (HO) generates the cytoprotective effector molecules biliverdin/bilirubin, carbon monoxide (CO) and iron/ferritin by breaking down heme. Since HO-activity mediates anti-apoptotic, anti-inflammatory, and anti-oxidative effects, we hypothesized that modulation of the HO-system affects MSC survival. Adipose-derived MSCs (ASCs) from wild type (WT) and HO-2 knockout (KO) mice were isolated and characterized with respect to ASC marker expression. In order to analyze potential modulatory effects of the HO-system on ASC survival, WT and HO-2 KO ASCs were pre-treated with HO-activity modulators, or downstream effector molecules biliverdin, bilirubin, and CO before co-exposure of ASCs to a toxic dose of H2O2. Surprisingly, sensitivity to H2O2-mediated cell death was similar in WT and HO-2 KO ASCs. However, pre-induction of HO-1 expression using curcumin increased ASC survival after H2O2 exposure in both WT and HO-2 KO ASCs. Simultaneous inhibition of HO-activity resulted in loss of curcumin-mediated protection. Co-treatment with glutathione precursor N-Acetylcysteine promoted ASC survival. However, co-incubation with HO-effector molecules bilirubin and biliverdin did not rescue from H2O2-mediated cell death, whereas co-exposure to CO-releasing molecules-2 (CORM-2) significantly increased cell survival, independently from HO-2 expression. Summarizing, our results show that curcumin protects via an HO-1 dependent mechanism against H2O2-mediated apoptosis, and likely through the generation of CO. HO-1 pre-induction or administration of CORMs may thus form an attractive strategy to improve MSC therapy.

Substance P induces adverse myocardial remodelling via a mechanism involving cardiac mast cells.

PubMed

Meléndez, Giselle C; Li, Jianping; Law, Brittany A; Janicki, Joseph S; Supowit, Scott C; Levick, Scott P

2011-12-01

Substance P and neurokinin A (NKA) are sensory nerve neuropeptides encoded by the TAC1 gene. Substance P is a mast cell secretagogue and mast cells are known to play a role in adverse myocardial remodelling. Therefore, we wondered whether substance P and/or NKA modulates myocardial remodelling via a mast cell-mediated mechanism. Volume overload was induced by aortocaval fistula in TAC1(-/-) mice and their respective wild types. Left ventricular internal diameter of wild-type (WT) fistulas increased by 31.9%; this was prevented in TAC1(-/-) mice (4.2%). Matrix metalloproteinase (MMP) activity was significantly increased in WT fistula mice and was prevented in TAC1(-/-) mice. Myocardial collagen volume fraction was decreased in WT fistula mice; this collagen degradation was not observed in the TAC1(-/-) group. There were no significant differences between any groups in tumour necrosis factor (TNF)-α or cell death. Cardiac mast cells were isolated from rat hearts and stimulated with substance P or NKA. We found that these cells degranulated only to substance P, via the neurokinin-1 receptor. To determine the effect of substance P on mast cells in vivo, volume overload was created in Sprague-Dawley rats treated with the NK-1 receptor antagonist L732138 (5 mg/kg/day) for a period of 3 days. L732138 prevented: (i) increases in cardiac mast cell density; (ii) increased myocardial TNF-α; and (iii) collagen degradation. Our studies suggest that substance P may be important in mediating adverse myocardial remodelling secondary to volume overload by activating cardiac mast cells, leading to increased TNF-α and MMP activation with subsequent degradation of the extracellular matrix.

Substance P induces adverse myocardial remodelling via a mechanism involving cardiac mast cells

PubMed Central

Meléndez, Giselle C.; Li, Jianping; Law, Brittany A.; Janicki, Joseph S.; Supowit, Scott C.; Levick, Scott P.

2011-01-01

Aims Substance P and neurokinin A (NKA) are sensory nerve neuropeptides encoded by the TAC1 gene. Substance P is a mast cell secretagogue and mast cells are known to play a role in adverse myocardial remodelling. Therefore, we wondered whether substance P and/or NKA modulates myocardial remodelling via a mast cell-mediated mechanism. Methods and results Volume overload was induced by aortocaval fistula in TAC1−/− mice and their respective wild types. Left ventricular internal diameter of wild-type (WT) fistulas increased by 31.9%; this was prevented in TAC1−/− mice (4.2%). Matrix metalloproteinase (MMP) activity was significantly increased in WT fistula mice and was prevented in TAC1−/− mice. Myocardial collagen volume fraction was decreased in WT fistula mice; this collagen degradation was not observed in the TAC1−/− group. There were no significant differences between any groups in tumour necrosis factor (TNF)-α or cell death. Cardiac mast cells were isolated from rat hearts and stimulated with substance P or NKA. We found that these cells degranulated only to substance P, via the neurokinin-1 receptor. To determine the effect of substance P on mast cells in vivo, volume overload was created in Sprague-Dawley rats treated with the NK-1 receptor antagonist L732138 (5 mg/kg/day) for a period of 3 days. L732138 prevented: (i) increases in cardiac mast cell density; (ii) increased myocardial TNF-α; and (iii) collagen degradation. Conclusions Our studies suggest that substance P may be important in mediating adverse myocardial remodelling secondary to volume overload by activating cardiac mast cells, leading to increased TNF-α and MMP activation with subsequent degradation of the extracellular matrix. PMID:21908647

The Role of Serotonin in Ventricular Repolarization in Pregnant Mice

PubMed Central

Park, Hyelim; Mun, Dasom; Lee, Seung-Hyun; Kim, Hyoeun; Yun, Nuri; Kim, Hail; Kim, Michael; Pak, Hui-Nam; Lee, Moon-Hyoung

2018-01-01

Purpose The mechanisms underlying repolarization abnormalities during pregnancy are not fully understood. Although maternal serotonin (5-hydroxytryptamine, 5-HT) production is an important determinant for normal fetal development in mice, its role in mothers remains unclear. We evaluated the role of serotonin in ventricular repolarization in mice hearts via 5Htr3 receptor (Htr3a) and investigated the mechanism of QT-prolongation during pregnancy. Materials and Methods We measured current amplitudes and the expression levels of voltage-gated K+ (Kv) channels in freshly-isolated left ventricular myocytes from wild-type non-pregnant (WT-NP), late-pregnant (WT-LP), and non-pregnant Htr3a homozygous knockout mice (Htr3a−/−-NP). Results During pregnancy, serotonin and tryptophan hydroxylase 1, a rate-limiting enzyme for the synthesis of serotonin, were markedly increased in hearts and serum. Serotonin increased Kv current densities concomitant with the shortening of the QT interval in WT-NP mice, but not in WT-LP and Htr3a−/−-NP mice. Ondansetron, an Htr3 antagonist, decreased Kv currents in WT-LP mice, but not in WT-NP mice. Kv4.3 directly interacted with Htr3a, and this binding was facilitated by serotonin. Serotonin increased the trafficking of Kv4.3 channels to the cellular membrane in WT-NP. Conclusion Serotonin increases repolarizing currents by augmenting Kv currents. Elevated serotonin levels during pregnancy counterbalance pregnancy-related QT prolongation by facilitating Htr3-mediated Kv currents. PMID:29436197

The Role of Serotonin in Ventricular Repolarization in Pregnant Mice.

PubMed

Cui, Shanyu; Park, Hyewon; Park, Hyelim; Mun, Dasom; Lee, Seung Hyun; Kim, Hyoeun; Yun, Nuri; Kim, Hail; Kim, Michael; Pak, Hui Nam; Lee, Moon Hyoung; Joung, Boyoung

2018-03-01

The mechanisms underlying repolarization abnormalities during pregnancy are not fully understood. Although maternal serotonin (5-hydroxytryptamine, 5-HT) production is an important determinant for normal fetal development in mice, its role in mothers remains unclear. We evaluated the role of serotonin in ventricular repolarization in mice hearts via 5Htr3 receptor (Htr3a) and investigated the mechanism of QT-prolongation during pregnancy. We measured current amplitudes and the expression levels of voltage-gated K⁺ (Kv) channels in freshly-isolated left ventricular myocytes from wild-type non-pregnant (WT-NP), late-pregnant (WT-LP), and non-pregnant Htr3a homozygous knockout mice (Htr3a(-/-)-NP). During pregnancy, serotonin and tryptophan hydroxylase 1, a rate-limiting enzyme for the synthesis of serotonin, were markedly increased in hearts and serum. Serotonin increased Kv current densities concomitant with the shortening of the QT interval in WT-NP mice, but not in WT-LP and Htr3a(-/-)-NP mice. Ondansetron, an Htr3 antagonist, decreased Kv currents in WT-LP mice, but not in WT-NP mice. Kv4.3 directly interacted with Htr3a, and this binding was facilitated by serotonin. Serotonin increased the trafficking of Kv4.3 channels to the cellular membrane in WT-NP. Serotonin increases repolarizing currents by augmenting Kv currents. Elevated serotonin levels during pregnancy counterbalance pregnancy-related QT prolongation by facilitating Htr3-mediated Kv currents. © Copyright: Yonsei University College of Medicine 2018

Liver Fatty acid binding protein (L-Fabp) modulates murine stellate cell activation and diet induced nonalcoholic fatty liver disease

PubMed Central

Chen, Anping; Tang, Youcai; Davis, Victoria; Hsu, Fong-Fu; Kennedy, Susan M.; Song, Haowei; Turk, John; Brunt, Elizabeth M.; Newberry, Elizabeth P.; Davidson, Nicholas O.

2013-01-01

Activation of hepatic stellate cells (HSCs) is crucial to the development of fibrosis in nonalcoholic fatty liver disease. Quiescent HSCs contain lipid droplets (LDs), whose depletion upon activation induces a fibrogenic gene program. Here we show that liver fatty acid-binding protein (L-Fabp), an abundant cytosolic protein that modulates fatty acid (FA) metabolism in enterocytes and hepatocytes also modulates HSC FA utilization and in turn regulates the fibrogenic program. L-Fabp expression decreased 10-fold following HSC activation, concomitant with depletion of LDs. Primary HSCs isolated from L-FABP−/− mice contain fewer LDs than wild type (WT) HSCs, and exhibit upregulated expression of genes involved in HSC activation. Adenoviral L-Fabp transduction inhibited activation of passaged WT HSCs and increased both the expression of prolipogenic genes and also augmented intracellular lipid accumulation, including triglyceride and FA, predominantly palmitate. Freshly isolated HSCs from L-FABP−/− mice correspondingly exhibited decreased palmitate in the free FA pool. To investigate whether L-FABP deletion promotes HSC activation in vivo, we fed L-FABP−/− and WT mice a high fat diet supplemented with trans-fatty acids and fructose (TFF). TFF-fed L-FABP−/− mice exhibited reduced hepatic steatosis along with decreased LD abundance and size compared to WT mice. In addition, TFF-fed L-FABP−/− mice exhibited decreased hepatic fibrosis, with reduced expression of fibrogenic genes, compared to WT mice. Conclusion L-FABP deletion attenuates both diet-induced hepatic steatosis and fibrogenesis, despite the observation that L-Fabp paradoxically promotes FA and LD accumulation and inhibits HSC activation in vitro. These findings highlight the importance of cell-specific modulation of hepatic lipid metabolism in promoting fibrogenesis in nonalcoholic fatty liver disease. PMID:23401290

Analysis of the oncogene BRAF mutation and the correlation of the expression of wild-type BRAF and CREB1 in endometriosis

PubMed Central

Lv, Xiao; Ma, Yue; Long, Zaiqiu

2018-01-01

B-Raf proto-oncogene, serine/threonine kinase (BRAF) has previously been identified as a candidate target gene in endometriosis. Wild-type and mutated BRAF serve important roles in different diseases. The aim of the present study was to explore BRAF mutation, the mRNA and protein expression of wild-type BRAF (wtBRAF) in endometriosis, and the association between the expression levels of wtBRAF and the predicted transcription factor cAMP responsive element binding protein 1 (CREB1). In the present study, BRAF mutation was detected using Sanger sequencing among 30 ectopic and matched eutopic endometrium samples of patients with endometriosis as well as 25 normal endometrium samples, and no BRAF mutation was detected in exons 11 or 15. A region of ~2,000 bp upstream of the BRAF gene was then screened using NCBI and UCSC databases, and CREB1 was identified as a potential transcription factor of BRAF by analysis with the JASPAR and the TRANSFAC databases. Quantitative polymerase chain reaction was used to analysis the mRNA expression levels of wtBRAF and CREB1, and the corresponding protein expression levels were evaluated using immunohistochemistry and western blot analysis. The results revealed that the mRNA and protein expression levels of wtBRAF and CREB1 were significantly upregulated in the eutopic endometrial tissues of patients with endometriosis compared with normal endometrial tissues (P<0.05) and no significant difference in wtBRAF and CREB1 levels was detected between the ectopic and eutopic endometrium (P>0.05). In addition, correlation analysis revealed that the protein expression of CREB1 was positively correlated with the transcript level and protein expression of wtBRAF. It is reasonable to speculate that CREB1 may activate the transcription of wtBRAF through directly binding to its promoter, increasing BRAF expression and regulating the cell proliferation, migration and invasion of endometriosis. PMID:29286077

Flight performance and teneral energy reserves of two genetically-modified and one wild-type strain of the yellow fever mosquito Aedes aegypti.

PubMed

Bargielowski, Irka; Kaufmann, Christian; Alphey, Luke; Reiter, Paul; Koella, Jacob

2012-12-01

The ability of sterile males to survive, disperse, find, and mate with wild females is key to the success of sterile insect technique (SIT). The Release of Insects carrying a Dominant Lethal (RIDL) system is a genetics-based SIT strategy for Aedes aegypti. We examine two aspects of insect performance, flight potential (dispersal ability) and teneral energy reserves, by comparing wild-type (WT) males with genetically-modified lines carrying the tetracycline-repressible constructs OX513A and OX3604C. Our results show significant differences in the flight capacity of the modified lines. OX513A males bred with tetracycline covered 38% less distance, while OX3604C males reared without tetracycline spent 21% less time in flight than their WT counterparts. Such differences in flight performance should be considered when designing release programs (e.g., by placing release sites sufficiently close together to achieve adequate coverage). All mosquito lines had similar teneral carbohydrate contents, though males of the OX3604C line contained more lipids. The addition of tetracycline to the larval diet did not influence the flight potential of the males; however, it did change the teneral sugar reserves of the WT and the lipid reserves of both the WT and the OX3604C lines.

Refractive index of dark-adapted bacteriorhodopsin and tris(hydroxymethyl)aminomethane buffer between 390 and 880 nm.

PubMed

Heiner, Zsuzsanna; Osvay, Károly

2009-08-10

The refractivity of wild-type bacteriorhodopsin (bR(WT)) suspended in tris(hydroxymethyl)aminomethane (TRIS) buffer has been measured in the spectral range of 390-840 nm by the method of angle of minimal deviation with the use of a hollow glass prism. The refractive indices of pure bR(WT) as well as of TRIS buffer have been determined from the concentration dependent refraction values. Sellmeier-type dispersion equations have been fitted for both the TRIS buffer and pure bR(WT).

The yeast Holliday junction resolvase, CCE1, can restore wild-type mitochondrial DNA to human cells carrying rearranged mitochondrial DNA.

PubMed

Sembongi, Hiroshi; Di Re, Miriam; Bokori-Brown, Monika; Holt, Ian J

2007-10-01

Rearrangements of mitochondrial DNA (mtDNA) are a well-recognized cause of human disease; deletions are more frequent, but duplications are more readily transmitted to offspring. In theory, partial duplications of mtDNA can be resolved to partially deleted and wild-type (WT) molecules, via homologous recombination. Therefore, the yeast CCE1 gene, encoding a Holliday junction resolvase, was introduced into cells carrying partially duplicated or partially triplicated mtDNA. Some cell lines carrying the CCE1 gene had substantial amounts of WT mtDNA suggesting that the enzyme can mediate intramolecular recombination in human mitochondria. However, high levels of expression of CCE1 frequently led to mtDNA loss, and so it is necessary to strictly regulate the expression of CCE1 in human cells to ensure the selection and maintenance of WT mtDNA.

Prevalence and Prognostic Impact of Wilms' Tumor 1 (WT1) Gene, Including SNP rs16754 in Cytogenetically Normal Acute Myeloblastic Leukemia (CN-AML): An Iranian Experience.

PubMed

Toogeh, Gholamreza; Ramzi, Mani; Faranoush, Mohammad; Amirizadeh, Naser; Haghpanah, Sezaneh; Moghadam, Mohammad; Cohan, Nader

2016-03-01

The aim of this study was to evaluate the effect of Wilms' tumor 1 (WT1) gene mutations in adult cytogenetically normal acute myeloblastic leukemia (CN-AML) patients on survival and clinical outcome. A total of 88 untreated Iranian adult patients with CN-AML were selected as a study group. Exons 7 (including the SNP rs16754), 8, and 9 as a WT1 gene hotspot region were evaluated by polymerase chain reaction and direct sequencing for detection of mutations. Response to treatment and clinical outcome including overall survival (OS) and disease-free survival (DFS) were evaluated according to WT1 gene mutational status. WT1 gene mutations were found in 12.5% of patients, most of which were found in exon 7. Complete remission was lower and relapse was higher in patients with WT1 gene mutation compared with WT1 gene wild type patients. OS and DFS was significantly lower in patients with WT1 gene mutation compared with patients with WT1 gene wild type (P < .001). Also, we did not find any significant effects of SNP rs16754 in exon 7 on clinical outcome and survival in patients with CN-AML. WT1 gene mutations are a predictor indicator of a poor prognosis factor in CN-AML patients. It is recommended that WT1 gene mutations be included in the molecular testing panel in order to better diagnose and confirm their prognostic significance for better management and treatment strategy. Copyright © 2016 Elsevier Inc. All rights reserved.

Matrix metalloproteinase-20 mediates dental enamel biomineralization by preventing protein occlusion inside apatite crystals

PubMed Central

Prajapati, Saumya; Tao, Jinhui; Ruan, Qichao; De Yoreo, James J.; Moradian-Oldak, Janet

2015-01-01

Reconstruction of enamel-like materials is a central topic of research in dentistry and material sciences. The importance of precise proteolytic mechanisms in amelogenesis to form a hard tissue with more than 95% mineral content has already been reported. A mutation in the Matrix Metalloproteinase-20 (MMP-20) gene results in hypomineralized enamel that is thin, disorganized and breaks from the underlying dentin. We hypothesized that the absence of MMP-20 during amelogenesis results in the occlusion of amelogenin in the enamel hydroxyapatite crystals. We used spectroscopy and electron microscopy techniques to qualitatively and quantitatively analyze occluded proteins within the isolated enamel crystals from MMP-20 null and Wild type (WT) mice. Our results showed that the isolated enamel crystals of MMP-20 null mice had more organic macromolecules occluded inside them than enamel crystals from the WT. The crystal lattice arrangements of MMP-20 null enamel crystals analyzed by High Resolution Transmission Electron Microscopy (HRTEM) were found to be significantly different from those of the WT. Raman studies indicated that the crystallinity of the MMP-20 null enamel crystals was lower than that of the WT. In conclusion, we present a novel functional mechanism of MMP-20, specifically prevention of unwanted organic material entrapped in the forming enamel crystals, which occurs as the result of precise amelogenin cleavage. MMP-20 action guides the growth morphology of the forming hydroxyapatite crystals and enhances their crystallinity. Elucidating such molecular mechanisms can be applied in the design of novel biomaterials for future clinical applications in dental restoration or repair. PMID:26513418

Matrix metalloproteinase-20 mediates dental enamel biomineralization by preventing protein occlusion inside apatite crystals.

PubMed

Prajapati, Saumya; Tao, Jinhui; Ruan, Qichao; De Yoreo, James J; Moradian-Oldak, Janet

2016-01-01

Reconstruction of enamel-like materials is a central topic of research in dentistry and material sciences. The importance of precise proteolytic mechanisms in amelogenesis to form a hard tissue with more than 95% mineral content has already been reported. A mutation in the Matrix Metalloproteinase-20 (MMP-20) gene results in hypomineralized enamel that is thin, disorganized and breaks from the underlying dentin. We hypothesized that the absence of MMP-20 during amelogenesis results in the occlusion of amelogenin in the enamel hydroxyapatite crystals. We used spectroscopy and electron microscopy techniques to qualitatively and quantitatively analyze occluded proteins within the isolated enamel crystals from MMP-20 null and Wild type (WT) mice. Our results showed that the isolated enamel crystals of MMP-20 null mice had more organic macromolecules occluded inside them than enamel crystals from the WT. The crystal lattice arrangements of MMP-20 null enamel crystals analyzed by High Resolution Transmission Electron Microscopy (HRTEM) were found to be significantly different from those of the WT. Raman studies indicated that the crystallinity of the MMP-20 null enamel crystals was lower than that of the WT. In conclusion, we present a novel functional mechanism of MMP-20, specifically prevention of unwanted organic material entrapped in the forming enamel crystals, which occurs as the result of precise amelogenin cleavage. MMP-20 action guides the growth morphology of the forming hydroxyapatite crystals and enhances their crystallinity. Elucidating such molecular mechanisms can be applied in the design of novel biomaterials for future clinical applications in dental restoration or repair. Copyright © 2015 Elsevier Ltd. All rights reserved.

Adsorption of rare earth ions onto the cell walls of wild-type and lipoteichoic acid-defective strains of Bacillus subtilis.

PubMed

Moriwaki, Hiroshi; Koide, Remi; Yoshikawa, Ritsuko; Warabino, Yuya; Yamamoto, Hiroki

2013-04-01

The aim of this study is to investigate the potential of cell walls of wild-type and lipoteichoic acid-defective strains of Bacillus subtilis 168 to adsorb rare earth ions. Freeze-dried cell powders prepared from both strains were used for the evaluation of adsorption ability for the rare earth ions, namely, La(III), Eu(III), and Tm(III). The rare earth ions were efficiently adsorbed onto powders of both wild-type strain (WT powder) and lipoteichoic acid-defective strain (∆LTA powder) at pH 3. The maximum adsorption capacities for Tm(III) by WT and ∆LTA powders were 43 and 37 mg g(-1), respectively. Removal (in percent) of Tm(III), La(III), and Eu(III) from aqueous solution by WT powder was greater than by ∆LTA powder. These results indicate that rare earth ions are adsorbed to functional groups, such as phosphate and carboxyl groups, of lipoteichoic acid. We observed coagulated ∆LTA powder in the removal of rare earth ions (1-20 mg L(-1)) from aqueous solution. In contrast, sedimentation of WT powder did not occur under the same conditions. This unique feature of ∆LTA powder may be caused by the difference of the distribution between lipoteichoic acid and wall teichoic acid. It appears that ∆LTA powder is useful for removal of rare earth ions by adsorption, because aggregation allows for rapid separation of the adsorbent by filtration.

Enteric oxalate elimination is induced and oxalate is normalized in a mouse model of primary hyperoxaluria following intestinal colonization with Oxalobacter

PubMed Central

Gjymishka, Altin; Salido, Eduardo C.; Allison, Milton J.; Freel, Robert W.

2011-01-01

Oxalobacter colonization of rat intestine was previously shown to promote enteric oxalate secretion and elimination, leading to significant reductions in urinary oxalate excretion (Hatch et al. Kidney Int 69: 691–698, 2006). The main goal of the present study, using a mouse model of primary hyperoxaluria type 1 (PH1), was to test the hypothesis that colonization of the mouse gut by Oxalobacter formigenes could enhance enteric oxalate secretion and effectively reduce the hyperoxaluria associated with this genetic disease. Wild-type (WT) mice and mice deficient in liver alanine-glyoxylate aminotransferase (Agxt) exhibiting hyperoxalemia and hyperoxaluria were used in these studies. We compared the unidirectional and net fluxes of oxalate across isolated, short-circuited large intestine of artificially colonized and noncolonized mice. In addition, plasma and urinary oxalate was determined. Our results demonstrate that the cecum and distal colon contribute significantly to enteric oxalate excretion in Oxalobacter-colonized Agxt and WT mice. In colonized Agxt mice, urinary oxalate excretion was reduced 50% (to within the normal range observed for WT mice). Moreover, plasma oxalate concentrations in Agxt mice were also normalized (reduced 50%). Colonization of WT mice was also associated with marked (up to 95%) reductions in urinary oxalate excretion. We conclude that segment-specific effects of Oxalobacter on intestinal oxalate transport in the PH1 mouse model are associated with a normalization of plasma oxalate and urinary oxalate excretion in otherwise hyperoxalemic and hyperoxaluric animals. PMID:21163900

Caveolin-1 is required for fatty acid translocase (FAT/CD36) localization and function at the plasma membrane of mouse embryonic fibroblasts.

PubMed

Ring, Axel; Le Lay, Soazig; Pohl, Juergen; Verkade, Paul; Stremmel, Wolfgang

2006-04-01

Several lines of evidence suggest that lipid rafts are involved in cellular fatty acid uptake and influence fatty acid translocase (FAT/CD36) function. However, it remains unknown whether caveolae, a specialized raft type, are required for this mechanism. Here, we show that wild-type (WT) mouse embryonic fibroblasts (MEFs) and caveolin-1 knockout (KO) MEFs, which are devoid of caveolae, have comparable overall expression of FAT/CD36 protein but altered subcellular FAT/CD36 localization and function. In WT MEFs, FAT/CD36 was isolated with both lipid raft enriched detergent-resistant membranes (DRMs) and detergent-soluble membranes (DSMs), whereas in cav-1 KO cells it was exclusively associated with DSMs. Subcellular fractionation demonstrated that FAT/CD36 in WT MEFs was localized intracellularly and at the plasma membrane level while in cav-1 KO MEFs it was absent from the plasma membrane. This mistargeting of FAT/CD36 in cav-1 KO cells resulted in reduced fatty acid uptake compared to WT controls. Adenoviral expression of caveolin-1 in KO MEFs induced caveolae formation, redirection of FAT/CD36 to the plasma membrane and rescue of fatty acid uptake. In conclusion, our data provide evidence that caveolin-1 is necessary to target FAT/CD36 to the plasma membrane. Caveolin-1 may influence fatty acid uptake by regulating surface availability of FAT/CD36.

Cyclophilin B induces chemoresistance by degrading wild type p53 via interaction with MDM2 in colorectal cancer.

PubMed

Choi, Tae Gyu; Nguyen, Minh Nam; Kim, Jieun; Jo, Yong Hwa; Jang, Miran; Nguyen, Ngoc Ngo Yen; Yun, Hyeong Rok; Choe, Wonchae; Kang, Insug; Ha, Joohun; Tang, Dean G; Kim, Sung Soo

2018-06-06

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. Chemoresistance is a major problem for effective therapy in CRC. Here, we investigated the mechanism by which peptidylprolyl isomerase B (PPIB; cyclophilin B, CypB) regulates chemoresistance in CRC. We found that CypB is a novel wild type p53 (p53WT)-inducible gene but a negative regulator of p53WT in response to oxaliplatin treatment. Overexpression of CypB shortens the half-life of p53WT and inhibits oxaliplatin-induced apoptosis in CRC cells, whereas knockdown of CypB lengthens the half-life of p53WT and stimulates p53WT dependent apoptosis. CypB interacts directly with MDM2, and enhances MDM2-dependent p53WT ubiquitination and degradation. Furthermore, we firmly validated using bioinformatics analyses that overexpression of CypB is associated with poor prognosis in CRC progression and chemoresistance. Hence, we suggest a novel mechanism of chemoresistance caused by overexpressed CypB, which may help to develop new anti-cancer drugs. We also propose that CypB may be utilized as a predictive biomarker in CRC patients. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Exercise ameliorates endoplasmic reticulum stress-mediated vascular dysfunction in mesenteric arteries in atherosclerosis.

PubMed

Hong, Junyoung; Kim, Kwangchan; Park, Eunkyung; Lee, Jonghae; Markofski, Melissa M; Marrelli, Sean P; Park, Yoonjung

2018-05-21

Endoplasmic reticulum (ER) stress is closely associated with atherosclerosis, but the effects of exercise on ER stress-mediated endothelial dysfunction in atherosclerosis is not yet fully understood. We assessed endothelium-dependent vasodilation in isolated mesenteric arteries from wild type (WT), WT with exercise (WT-EX), ApoE knockout (ApoE KO), and ApoE KO mice with exercise (ApoE KO-EX). Vasodilation to acetylcholine (ACh) was elicited in the presence of inhibitors of ER stress, eNOS, caspase-1, and UCP-2 (Tudca, L-NAME, AC-YVARD-cmk, and Genipin, respectively) and the ER stress inducer (Tunicamycin). Immunofluorescence was used to visualize the expression of CHOP, as an indicator of ER stress, in superior mesenteric arteries (SMA). Dilation to ACh was attenuated in ApoE KO but was improved in ApoE KO-EX. Incubation of Tudca and AC-YVARD-cmk improved ACh-induced vasodilation in ApoE KO. L-NAME, tunicamycin, and Genipin attenuated vasodilation in WT, WT-EX and ApoE KO-EX, but not in ApoE KO. Exercise training reversed the increase in CHOP expression in the endothelium of SMA of ApoE KO mice. We conclude that ER stress plays a significant role in endothelial dysfunction of resistance arteries in atherosclerosis and that exercise attenuates ER stress and regulates its critical downstream signaling pathways including eNOS, UCP-2 and caspase-1.

Curvature Induced by Amyloplast Magnetophoresis in Protonemata of the Moss Ceratodon purpureus1

PubMed Central

Kuznetsov, Oleg A.; Schwuchow, Jochen; Sack, Fred D.; Hasenstein, Karl H.

1999-01-01

After gravistimulation of Ceratodon purpureus (Hedw.) Brid. protonemata in the dark, amyloplast sedimentation was followed by upward curvature in the wild-type (WT) and downward curvature in the wwr mutant (wrong way response). We used ponderomotive forces induced by high-gradient magnetic fields (HGMF) to simulate the effect of gravity and displace the presumptive statoliths. The field was applied by placing protonemata either between two permanent magnets at the edge of the gap, close to the edge of a magnetized ferromagnetic wedge, or close to a small (<1 mm) permanent magnet. Continuous application of an HGMF in all three configurations resulted in plastid displacement and induced curvature in tip cells of WT and wwr protonemata. WT cells curved toward the HGMF, and wwr cells curved away from the HGMF, comparable to gravitropism. Plastids isolated from protonemal cultures had densities ranging from 1.24 to 1.38 g cm−3. Plastid density was similar for both genotypes, but the mutant contained larger plastids than the WT. The size difference might explain the stronger response of the wwr protonemata to the HGMF. Our data support the plastid-based theory of gravitropic sensing and suggest that HGMF-induced ponderomotive forces can substitute for gravity. PMID:9952461

Reactivation of wild-type and mutant p53 by tryptophanolderived oxazoloisoindolinone SLMP53-1, a novel anticancer small-molecule

PubMed Central

Soares, Joana; Raimundo, Liliana; Pereira, Nuno A.L.; Monteiro, Ângelo; Gomes, Sara; Bessa, Cláudia; Pereira, Clara; Queiroz, Glória; Bisio, Alessandra; Fernandes, João; Gomes, Célia; Reis, Flávio; Gonçalves, Jorge; Inga, Alberto; Santos, Maria M.M.; Saraiva, Lucília

2016-01-01

Restoration of the p53 pathway, namely by reactivation of mutant (mut) p53, represents a valuable anticancer strategy. Herein, we report the identification of the enantiopure tryptophanol-derived oxazoloisoindolinone SLMP53-1 as a novel reactivator of wild-type (wt) and mut p53, using a yeast-based screening strategy. SLMP53-1 has a p53-dependent anti-proliferative activity in human wt and mut p53R280K-expressing tumor cells. Additionally, SLMP53-1 enhances p53 transcriptional activity and restores wt-like DNA binding ability to mut p53R280K. In wt/mut p53-expressing tumor cells, SLMP53-1 triggers p53 transcription-dependent and mitochondrial apoptotic pathways involving BAX, and wt/mut p53 mitochondrial translocation. SLMP53-1 inhibits the migration of wt/mut p53-expressing tumor cells, and it shows promising p53-dependent synergistic effects with conventional chemotherapeutics. In xenograft mice models, SLMP53-1 inhibits the growth of wt/mut p53-expressing tumors, but not of p53-null tumors, without apparent toxicity. Collectively, besides the potential use of SLMP53-1 as anticancer drug, the tryptophanol-derived oxazoloisoindolinone scaffold represents a promissing starting point for the development of effective p53-reactivating drugs. PMID:26735173

Improved capacity to evaluate changes in intestinal mucosal surface area using mathematical modeling.

PubMed

Greig, Chasen J; Cowles, Robert A

2017-07-01

Quantification of intestinal mucosal growth typically relies on morphometric parameters, commonly villus height, as a surrogate for presumed changes in mucosal surface area (MSA). We hypothesized that using mathematical modeling based on multiple unique measurements would improve discrimination of the effects of interventions on MSA compared to standard measures. To determine the ability of mathematical modeling to resolve differences in MSA, a mouse model with enhanced serotonin (5HT) signaling known to stimulate mucosal growth was used. 5-HT signaling is potentiated by targeting the serotonin reuptake transporter (SERT) molecule. Selective serotonin reuptake inhibitor-treated wild-type (WT-SSRI), SERT-knockout (SERTKO), and wild-type C57Bl/6 (WT) mice were used. Distal ileal sections were H&E-stained. Villus height (VH), width (VW), crypt width (CW), and bowel diameter were used to calculate surface area enlargement factor (SEF) and MSA. VH alone for SERTKO and SSRI was significantly increased compared to WT, without a difference between SERTKO and WT-SSRI. VW and CW were significantly decreased for both SERTKO and WT-SSRI compared to WT, and VW for WT-SSRI was also decreased compared to SERTKO. These changes increased SEF and MSA for SERTKO and WT-SSRI compared to WT. Additionally, SEF and MSA were significantly increased for WT-SSRI compared to SERTKO. Mathematical modeling provides a valuable tool for differentiating changes in intestinal MSA. This more comprehensive assessment of surface area does not appear to correlate linearly with standard morphometric measures and represents a more comprehensive method for discriminating between therapies aimed at increasing functional intestinal mucosa. © 2017 Wiley Periodicals, Inc.

Neurovirulent pathotype of Newcastle disease virus associated with the reduced capacity of NDV to replicate in vivo and in vitro

USDA-ARS?s Scientific Manuscript database

Reverse genetics was used to create two recombinant Newcastle disease viruses derived from a velogenic viscerotropic NDV strain from China, wild type ZJI (wt-ZJ1). One of the recombinant viruses (rZJ1) was identical to the wild type and the other had the gene for the green fluorescent protein (GFP)...

Coronary vasospasm induced in transgenic mouse with increased phospholipase C-δ1 activity.

PubMed

Shibutani, Shuji; Osanai, Tomohiro; Ashitate, Toshihiro; Sagara, Shigeki; Izumiyama, Kei; Yamamoto, Yuko; Hanada, Kenji; Echizen, Takashi; Tomita, Hirofumi; Fujita, Takeshi; Miwa, Takeshi; Matsubara, Hiroaki; Homma, Yoshimi; Okumura, Ken

2012-02-28

We reported that phospholipase C (PLC)-δ1 activity was enhanced 3-fold in patients with coronary spastic angina. We detected variant PLC-δ1 with replacement of arginine 257 by histidine (R257H) showing increased enzymatic activity. We tested the hypothesis that increased PLC-δ1 activity causes enhanced coronary vasomotility. We generated transgenic (TG) mice with human R257H variant PLC-δ1 in vascular smooth muscle cells. PLC enzymatic activity in the coronary artery was increased by 2.57 and 1.89 times, respectively, in homozygous and heterozygous TG compared with wild-type (WT) mice. ST elevation after ergometrine occurred in 17 of 18 homozygous TG, 6 of 20 heterozygous TG, and 3 of 22 WT mice (P<0.01, homozygous TG versus WT; P<0.05, homozygous TG versus heterozygous TG; P=NS, heterozygous TG versus WT). ST elevation was associated with bradyarrhythmias in homozygous TG mice. Focal coronary artery narrowing was documented with the microvascular filling technique in 3 of 5 homozygous TG mice after ergometrine but not in any of 7 WT mice (P<0.05). In the isolated Langendorff hearts, coronary perfusion pressure was increased after ergometrine in homozygous TG mice (P<0.01) but not in heterozygous TG or WT mice. Coronary perfusion pressure increase after prostaglandin F2α was similar among homozygous TG, heterozygous TG, and WT mice. Cultured rat aortic smooth muscle cells transfected with variant PLC-δ1 showed a higher PLC activity than those with WT PLC-δ1 (P<0.05) and furthermore showed greater intracellular Ca2+ response to acetylcholine in variant than in WT PLC-δ1 (P<0.05). Increased PLC-δ1 activity enhances coronary vasomotility such as that seen in patients with coronary spastic angina.

Taste responses to sweet stimuli in alpha-gustducin knockout and wild-type mice.

PubMed

Danilova, Vicktoria; Damak, Sami; Margolskee, Robert F; Hellekant, Göran

2006-07-01

The importance of alpha-gustducin in sweet taste transduction is based on data obtained with sucrose and the artificial sweetener SC45647. Here we studied the role of alpha-gustducin in sweet taste. We compared the behavioral and electrophysiological responses of alpha-gustducin knockout (KO) and wild-type (WT) mice to 11 different sweeteners, representing carbohydrates, artificial sweeteners, and sweet amino acids. In behavioral experiments, over 48-h preference ratios were measured in two-bottle preference tests. In electrophysiological experiments, integrated responses of chorda tympani (CT) and glossopharyngeal (NG) nerves were recorded. We found that preference ratios of the KO mice were significantly lower than those of WT for acesulfame-K, dulcin, fructose, NC00174, D-phenylalanine, L-proline, D-tryptophan, saccharin, SC45647, sucrose, but not neotame. The nerve responses to all sweeteners, except neotame, were smaller in the KO mice than in the WT mice. The differences between the responses in WT and KO mice were more pronounced in the CT than in the NG. These data indicate that alpha-gustducin participates in the transduction of the sweet taste in general.

Changes in In Vitro Susceptibility Patterns of Aspergillus to Triazoles and Correlation With Aspergillosis Outcome in a Tertiary Care Cancer Center, 1999-2015.

PubMed

Heo, Sang Taek; Tatara, Alexander M; Jiménez-Ortigosa, Cristina; Jiang, Ying; Lewis, Russell E; Tarrand, Jeffrey; Tverdek, Frank; Albert, Nathaniel D; Verweij, Paul E; Meis, Jacques F; Mikos, Antonios G; Perlin, David S; Kontoyiannis, Dimitrios P

2017-07-15

Azole-resistant aspergillosis in high-risk patients with hematological malignancy or hematopoietic stem cell transplantation (HSCT) is a cause of concern. We examined changes over time in triazole minimum inhibitory concentrations (MICs) of 290 sequential Aspergillus isolates recovered from respiratory sources during 1999-2002 (before introduction of the Aspergillus-potent triazoles voriconazole and posaconazole) and 2003-2015 at MD Anderson Cancer Center. We also tested for polymorphisms in ergosterol biosynthetic genes (cyp51A, erg3C, erg1) in the 37 Aspergillus fumigatus isolates isolated from both periods that had non-wild-type (WT) MICs. For the 107 patients with hematologic cancer and/or HSCT with invasive pulmonary aspergillosis, we correlated in vitro susceptibility with 42-day mortality. Non-WT MICs were found in 37 (13%) isolates and was only low level (MIC <8 mg/L) in all isolates. Higher-triazole MICs were more frequent in the second period and were Aspergillus-species specific, and only encountered in A. fumigatus. No polymorphisms in cyp51A, erg3C, erg1 genes were identified. There was no correlation between in vitro MICs with 42-day mortality in patients with invasive pulmonary aspergillosis, irrespective of antifungal treatment. Asian race (odds ratio [OR], 20.9; 95% confidence interval [CI], 2.5-173.5; P = .005) and azole exposure in the prior 3 months (OR, 9.6; 95% CI, 1.9-48.5; P = .006) were associated with azole resistance. Non-WT azole MICs in Aspergillus are increasing and this is associated with prior azole exposure in patients with hematologic cancer or HSCT. However, no correlation of MIC with outcome of aspergillosis was found in our patient cohort. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Surface-Expressed Enolase Contributes to the Pathogenesis of Clinical Isolate SSU of Aeromonas hydrophila▿

PubMed Central

Sha, Jian; Erova, Tatiana E.; Alyea, Rebecca A.; Wang, Shaofei; Olano, Juan P.; Pancholi, Vijay; Chopra, Ashok K.

2009-01-01

In this study, we demonstrated that the surface-expressed enolase from diarrheal isolate SSU of Aeromonas hydrophila bound to human plasminogen and facilitated the latter's tissue-type plasminogen activator-mediated activation to plasmin. The bacterial surface-bound plasmin was more resistant to the action of its specific physiological inhibitor, the antiprotease α2-antiplasmin. We found that immunization of mice with purified recombinant enolase significantly protected the animals against a lethal challenge dose of wild-type (WT) A. hydrophila. Minimal histological changes were noted in organs from mice immunized with enolase and then challenged with WT bacteria compared to severe pathological changes found in the infected and nonimmunized group of animals. This correlated with the smaller bacterial load of WT bacteria in the livers and spleens of enolase-immunized mice than that found in the nonimmunized controls. We also showed that the enolase gene could potentially be important for the viability of A. hydrophila SSU as we could delete the chromosomal copy of the enolase gene only when another copy of the targeted gene was supplied in trans. By site-directed mutagenesis, we altered five lysine residues located at positions 343, 394, 420, 427, and 430 of enolase in A. hydrophila SSU; the mutated forms of enolase were hyperexpressed in Escherichia coli, and the proteins were purified. Our results indicated that lysine residues at positions 420 and 427 of enolase were crucial in plasminogen-binding activity. We also identified a stretch of amino acid residues (252FYDAEKKEY260) in the A. hydrophila SSU enolase involved in plasminogen binding. To our knowledge, this is the first report of the direct involvement of surface-expressed enolase in the pathogenesis of A. hydrophila SSU infections and of any gram-negative bacteria in general. PMID:19270100

Identification, Isolation, and Phylogenetic Analysis of Clostridium perfringens Type A and Type C from Wild Boar ( Sus scrofa ) in the People's Republic of China.

PubMed

Li, Meng; Zhang, Xu; Zhu, Lingwei; Wang, Haifeng; Zhao, Na; Luo, Jing; Wang, Chengmin; Wang, Yutian; Liu, Yanhua; Zhou, Wei; Zhang, Bikai; Guo, Huancheng; He, Hongxuan

2017-07-01

Clostridium perfringens is a Gram-positive, anaerobic, spore-forming bacterium that can induces gas gangrene or enteritis in poultry and humans and many other mammalian species. Here, we report an outbreak of C. perfringens type A and type C coinfection in wild boars ( Sus scrofa ). In February 2016, 10 dead wild boars, including two fresh carcasses, were found in Zhaosu County, Xinjiang Province, People's Republic of China. The two fresh carcasses were included in this study. Two strains of C. perfringens were isolated, identified, genotyped, and phylogenetically analyzed. Based on postmortem examination, bacterium isolation and identification, enterotoxin detection, and auxiliary tests, we made a diagnosis that the wild boar were killed by C. perfringens . Our findings provide the evidence that wild boar can be killed by C. perfringens intoxication. Wild boars are important reservoirs for many zoonotic agents. Therefore, more actions should be taken on the surveillance, prevention, and control of wild pig-borne diseases.

Evaluation of biological safety in vitro and immunogenicity in vivo of recombinant Escherichia coli Shiga toxoids as candidate vaccines in cattle.

PubMed

Kerner, Katharina; Bridger, Philip S; Köpf, Gabriele; Fröhlich, Julia; Barth, Stefanie; Willems, Hermann; Bauerfeind, Rolf; Baljer, Georg; Menge, Christian

2015-04-10

Cattle are the most important reservoir for enterohemorrhagic Escherichia coli (EHEC), a subset of shigatoxigenic E. coli (STEC) capable of causing life-threatening infectious diseases in humans. In cattle, Shiga toxins (Stx) suppress the immune system thereby promoting long-term STEC shedding. First infections of animals at calves' age coincide with the lack of Stx-specific antibodies. We hypothesize that vaccination of calves against Shiga toxins prior to STEC infection may help to prevent the establishment of a persistent type of infection. The objectives of this study were to generate recombinant Shiga toxoids (rStx1mut & rStx2mut) by site-directed mutagenesis and to assess their immunomodulatory, antigenic, and immunogenic properties. Cultures of bovine primary immune cells were used as test systems. In ileal intraepithelial lymphocytes both, recombinant wild type Stx1 (rStx1WT) and rStx2WT significantly induced transcription of IL-4 mRNA. rStx1WT and rStx2WT reduced the expression of Stx-receptor CD77 (syn. Globotriaosylceramide, Gb3) on B and T cells from peripheral blood and of CD14 on monocyte-derived macrophages. At the same concentrations, rStx1mut and rStx2mut exhibited neither of these effects. Antibodies in sera of cattle naturally infected with STEC recognized the rStxmut toxoids equally well as the recombinant wild type toxins. Immunization of calves with rStx1mut plus rStx2mut led to induction of antibodies neutralizing Stx1 and Stx2. While keeping their antigenicity and immunogenicity recombinant Shiga toxoids are devoid of the immunosuppressive properties of the corresponding wild type toxins in cattle and candidate vaccines to mitigate long-term STEC shedding by the reservoir host.

Haemodynamics of lambs grazing perennial ryegrass (Lolium perenne L.) either infected with AR6 novel wild-type endophyte or not infected

USDA-ARS?s Scientific Manuscript database

Coopworth ewe lambs were randomly assigned to 3, 0.10-ha pastures of ‘Extreme’ perennial ryegrass that were infected with the AR6 novel endophyte (AR6; n=5), infected with the wild-type endophyte (WT; n=6), or was endophyte-free (Nil; n=5). Lambs were conditioned to the pastures from 25 Feb. to 16 ...

Thc6 protein, isolated from Trichoderma harzianum, can induce maize defense response against Curvularia lunata.

PubMed

Fan, Lili; Fu, Kehe; Yu, Chuanjin; Li, Yingying; Li, Yaqian; Chen, Jie

2015-05-01

Mutant T66 was isolated from 450 mutants (constructed with Agrobacterium tumefaciens-mediated transformation method) of Trichoderma harzianum. Maize seeds coated with T66 were more susceptible to Curvularia lunata when compared with those coated with wild-type (WT) strain. The disease index of maize treated with T66 and WT were 62.5 and 42.1%, respectively. Further research showed T-DNA has inserted into the ORF of one gene, which resulted in the functional difference between WT and T66. The gene was cloned and named Thc6, which encodes a novel 327 amino acid protein. To investigate its function, we obtained knockout, complementation, and overexpression mutants of Thc6. Challenge inoculation studies suggested that the Thc6 overexpression mutant can reduce the disease index of maize inbred line Huangzao 4 against the leaf spot pathogen (C. lunata). Meanwhile, The Thc6 mutants were found to affect the resistance of maize inbred line Huangzao 4 against C. lunata by enhancing the activation of jasmonate-responsive genes expression. Liquid chromatography-mass spectrometry (LC-MS) data further confirmed that the concentration of jasmonate in the induced maize exhibits a parallel change tendency with the expression level of defense-related genes. Hence, the Thc6 gene could be participated in the induced resistance of maize inbred line Huangzao 4 against C. lunata infection through a jasmonic acid-dependent pathway. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Social isolation stress induces ATF-7 phosphorylation and impairs silencing of the 5-HT 5B receptor gene

PubMed Central

Maekawa, Toshio; Kim, Seungjoon; Nakai, Daisuke; Makino, Chieko; Takagi, Tsuyoshi; Ogura, Hiroo; Yamada, Kazuyuki; Chatton, Bruno; Ishii, Shunsuke

2010-01-01

Many symptoms induced by isolation rearing of rodents may be relevant to neuropsychiatric disorders, including depression. However, identities of transcription factors that regulate gene expression in response to chronic social isolation stress remain elusive. The transcription factor ATF-7 is structurally related to ATF-2, which is activated by various stresses, including inflammatory cytokines. Here, we report that Atf-7-deficient mice exhibit abnormal behaviours and increased 5-HT receptor 5B (Htr5b) mRNA levels in the dorsal raphe nuclei. ATF-7 silences the transcription of Htr5B by directly binding to its 5′-regulatory region, and mediates histone H3-K9 trimethylation via interaction with the ESET histone methyltransferase. Isolation-reared wild-type (WT) mice exhibit abnormal behaviours that resemble those of Atf-7-deficient mice. Upon social isolation stress, ATF-7 in the dorsal raphe nucleus is phosphorylated via p38 and is released from the Htr5b promoter, leading to the upregulation of Htr5b. Thus, ATF-7 may have a critical role in gene expression induced by social isolation stress. PMID:19893493

[Study of negative feedback between wild-type BRAF or RAFV600E and Mps1 in melanoma].

PubMed

Zhang, Ling; He, Chanting; Bi, Yanghui; Liu, Feng; Cui, Heyang; Wang, Juan; Song, Bin; Shi, Ruyi; Yang, Bin; Wang, Fang; Jia, Zhiwu; Zhao, Zhenxiang; Liu, Jing

2015-04-01

To study the effect of Mps1 on BRAFWT/MEK/ERK pathway in the presence of wild type BRAF or BRAFV600E in melanoma. Melanoma cells harboring BRAFWT genotype were transfected either with pBabe-puro-GST-BRAF-WT and/or pBabe-puro-GFP-Mps1-WT or pBabe-puro-GST-BRAFV600E and/or pBabe-puro-GFP-Mps1-WT, followed by Western blot to detect Mps1 and p-ERK expression. The melanoma cells harboring BRAFWT and BRAFV600E genotype were infected with pSUPER-Mps1 retrovirus to knockdown the endogenous Mps1 protein, followed by Western blot to detect Mps1 and p-ERK expression. Meanwhile, melanoma cells harboring BRAFV600E genotype were infected with pBabe-puro-GFP-Mps1 and Western blot was performed to detect Mps1 and p-ERK expression. In melanoma cells harboring BRAFWT genotype and transfected with pBabe-puro-GST-BRAF-WT and pBabe-puro-GFP-Mps1-WT, phospho-ERK levels were notably reduced as compared to either negative control or empty vector. However, cells transfected with pBabe-puro-GST-BRAFV600E and pBabe-puro-GFP-Mps1-WT, phospho-ERK levels did not change significantly compared with either negative control or empty vector. Knockout of Mps1 in BRAF wild-type cell lines led to an increased ERK activity. However, there was no significant change of ERK activity in BRAFV600E cell lines in the absence of Mps1. The expression of p-ERK in BRAFV600E mutant cell lines infected with pBabe-puro-GFP-Mps1-WT did not show any significant difference from either negative control or empty vector. Based on these findings, it suggests that there exists an auto-regulatory negative feedback loop between the Mps1 kinase and BRAFWT/ERK signaling. Oncogenic BRAFV600E abrogates the regulatory negative feedback loop of Mps1 on the MAPK pathway.

Plaque and growth characteristics of different polioviruses isolated from acute flaccid paralysis in Northern Nigeria.

PubMed

Sule, W F; Oyedele, O I; Osei-Kwasi, M; Odoom, J K; Adu, F D

2008-03-01

To determine some virulent trait-related properties of poliovirus isolates from children with acute flaccid paralysis following vaccination with oral polio vaccine (OPV). Six polioviruses earlier characterised into wild, vaccine-derived and OPV-like were studied using the plaque morphology and growth kinetics at supra-optimal temperature. Department of Virology, University of Ibadan, Nigeria. Polio isolates from six children who developed acute flaccid paralysis following vaccinations with various doses of OPV were used. All the children were located in the Northern part of the country where poliovirus is still circulating. The two vaccine-derived polioviruses acquired wild type characteristics. All the six poliovirus isolates developed different forms of plaques ranging from tiny, small and large. The plaque formed could however not be used to identify the different isolates. Growth of the different isolates at supra-optimal temperature showed that the three wild polioviruses grew to a higher titre when compared with the Sabin 2 control. The two vaccine derived isolates behaved like the wild poliovirus while the OPV-like virus acquired an intermediate characteristics between wild and sabin. The wild polioviruses represented in this study are among the last vestiges of the circulating polioviruses found in the world. It is possible that the observed biological properties of wild types 1 and 3 described in the study are typical of the West African polioviruses. These properties will provide useful previews to the final identification of some important clinical isolates especially type 1 which may grow rapidly in cell culture.

Detection of MEF-1 laboratory reference strain of poliovirus type 2 in children with poliomyelitis in India in 2002 & 2003.

PubMed

Deshpande, J M; Nadkarni, S S; Siddiqui, Z A

2003-12-01

Significant progress has been made towards eradication of poliomyelitis in India. Surveillance for acute flaccid paralysis (AFP) has reached high standards. Among the 3 types of polioviruses, type 2 had been eliminated in India and eradicated globally as of October 1999. However, we isolated wild poliovirus type 2 from a small number of polio cases in northern India in 2000 and again during December 2002 to February 2003. Using molecular tools the origin, of the wild type 2 poliovirus was investigated. Polioviruses isolated from stool samples collected from patients with AFP were differentiated as wild virus or Sabin vaccine-like by ELISA and probe hybridization assays. Complete VP1 gene nucleotide sequences of the wild type 2 poliovirus isolates were determined by reverse transcriptase polymerase chain reaction (RT-PCR), followed by cycle sequencing. VP1 nucleotide sequences were compared with those of wild type 2 polioviruses that were indigenous in India in the past as well as prototype/laboratory strains and the GenBank database. Wild poliovirus type 2 was detected in stool samples from 6 patients with AFP in western Uttar Pradesh and 1 in Gujarat. In addition, the virus was isolated from one healthy contact child and from environmental sewage sample in Moradabad where three of these patients were reported. These isolates were identified as genetically closely related to laboratory reference strain MEF-1. Molecular characterization of the isolates confirmed that there was no evidence of extensive person-to-person transmission of the virus in the community. Laboratory reference strain (MEF-1) of poliovirus type 2 caused paralytic poliomyelitis in 10 patients in September 2000 and November 2002 to February 2003. The origin of the virus was some laboratory as yet not identified. This episode highlights the urgent need for stringent containment of wild poliovirus containing materials in the laboratories across the country in order to prevent recurrence of such incidents.

Gravity-dependent differentiation and root coils in Arabidopsis thaliana wild type and phospholipase-A-I knockdown mutant grown on the International Space Station.

PubMed

Scherer, G F E; Pietrzyk, P

2014-01-01

Arabidopsis roots on 45° tilted agar in 1-g grow in wave-like figures. In addition to waves, formation of root coils is observed in several mutants compromised in gravitropism and/or auxin transport. The knockdown mutant ppla-I-1 of patatin-related phospholipase-A-I is delayed in root gravitropism and forms increased numbers of root coils. Three known factors contribute to waving: circumnutation, gravisensing and negative thigmotropism. In microgravity, deprivation of wild type (WT) and mutant roots of gravisensing and thigmotropism and circumnutation (known to slow down in microgravity, and could potentially lead to fewer waves or increased coiling in both WT and mutant). To resolve this, mutant ppla-I-1 and WT were grown in the BIOLAB facility in the International Space Station. In 1-g, roots of both types only showed waving. In the first experiment in microgravity, the mutant after 9 days formed far more coils than in 1-g but the WT also formed several coils. After 24 days in microgravity, in both types the coils were numerous with slightly more in the mutant. In the second experiment, after 9 days in microgravity only the mutant formed coils and the WT grew arcuated roots. Cell file rotation (CFR) on the mutant root surface in microgravity decreased in comparison to WT, and thus was not important for coiling. Several additional developmental responses (hypocotyl elongation, lateral root formation, cotyledon expansion) were found to be gravity-influenced. We tentatively discuss these in the context of disturbances in auxin transport, which are known to decrease through lack of gravity. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

The novel endocannabinoid receptor GPR55 is activated by atypical cannabinoids but does not mediate their vasodilator effects

PubMed Central

Johns, D G; Behm, D J; Walker, D J; Ao, Z; Shapland, E M; Daniels, D A; Riddick, M; Dowell, S; Staton, P C; Green, P; Shabon, U; Bao, W; Aiyar, N; Yue, T-L; Brown, A J; Morrison, A D; Douglas, S A

2007-01-01

Background and purpose: Atypical cannabinoids are thought to cause vasodilatation through an as-yet unidentified ‘CBx' receptor. Recent reports suggest GPR55 is an atypical cannabinoid receptor, making it a candidate for the vasodilator ‘CBx' receptor. The purpose of the present study was to test the hypothesis that human recombinant GPR55 is activated by atypical cannabinoids and mediates vasodilator responses to these agents. Experimental approach: Human recombinant GPR55 was expressed in HEK293T cells and specific GTPγS activity was monitored as an index of receptor activation. In GPR55-deficient and wild-type littermate control mice, in vivo blood pressure measurement and isolated resistance artery myography were used to determine GPR55 dependence of atypical cannabinoid-induced haemodynamic and vasodilator responses. Key results: Atypical cannabinoids O-1602 and abnormal cannabidiol both stimulated GPR55-dependent GTPγS activity (EC50 approximately 2 nM), whereas the CB1 and CB2-selective agonist WIN 55,212-2 showed no effect in GPR55-expressing HEK293T cell membranes. Baseline mean arterial pressure and heart rate were not different between WT and GPR55 KO mice. The blood pressure-lowering response to abnormal cannabidiol was not different between WT and KO mice (WT 20±2%, KO 26±5% change from baseline), nor was the vasodilator response to abnormal cannabidiol in isolated mesenteric arteries (IC50 approximately 3 μ M for WT and KO). The abnormal cannabidiol vasodilator response was antagonized equivalently by O-1918 in both strains. Conclusions: These results demonstrate that while GPR55 is activated by atypical cannabinoids, it does not appear to mediate the vasodilator effects of these agents. PMID:17704827

Cross-activating invariant NKT cells and kupffer cells suppress cholestatic liver injury in a mouse model of biliary obstruction.

PubMed

Duwaerts, Caroline C; Sun, Eric P; Cheng, Chao-Wen; van Rooijen, Nico; Gregory, Stephen H

2013-01-01

Both Kupffer cells and invariant natural killer T (iNKT) cells suppress neutrophil-dependent liver injury in a mouse model of biliary obstruction. We hypothesize that these roles are interdependent and require iNKT cell-Kupffer cell cross-activation. Female, wild-type and iNKT cell-deficient C57Bl/6 mice were injected with magnetic beads 3 days prior to bile duct ligation (BDL) in order to facilitate subsequent Kupffer cell isolation. On day three post-BDL, the animals were euthanized and the livers dissected. Necrosis was scored; Kupffer cells were isolated and cell surface marker expression (flow cytometry), mRNA expression (qtPCR), nitric oxide (NO (.) ) production (Griess reaction), and protein secretion (cytometric bead-array or ELISAs) were determined. To address the potential role of NO (.) in suppressing neutrophil accumulation, a group of WT mice received 1400W, a specific inducible nitric oxide synthase (iNOS) inhibitor, prior to BDL. To clarify the mechanisms underlying Kupffer cell-iNKT cell cross-activation, WT animals were administered anti-IFN-γ or anti-lymphocyte function-associated antigen (LFA)-1 antibody prior to BDL. Compared to their WT counterparts, Kupffer cells obtained from BDL iNKT cell-deficient mice expressed lower iNOS mRNA levels, produced less NO (.) , and secreted more neutrophil chemoattractants. Both iNOS inhibition and IFN-γ neutralization increased neutrophil accumulation in the livers of BDL WT mice. Anti-LFA-1 pre-treatment reduced iNKT cell accumulation in these same animals. These data indicate that the LFA-1-dependent cross-activation of iNKT cells and Kupffer cells inhibits neutrophil accumulation and cholestatic liver injury.

Cross-Activating Invariant NKT Cells and Kupffer Cells Suppress Cholestatic Liver Injury in a Mouse Model of Biliary Obstruction

PubMed Central

Duwaerts, Caroline C.; Sun, Eric P.; Cheng, Chao-Wen; van Rooijen, Nico; Gregory, Stephen H.

2013-01-01

Both Kupffer cells and invariant natural killer T (iNKT) cells suppress neutrophil-dependent liver injury in a mouse model of biliary obstruction. We hypothesize that these roles are interdependent and require iNKT cell-Kupffer cell cross-activation. Female, wild-type and iNKT cell-deficient C57Bl/6 mice were injected with magnetic beads 3 days prior to bile duct ligation (BDL) in order to facilitate subsequent Kupffer cell isolation. On day three post-BDL, the animals were euthanized and the livers dissected. Necrosis was scored; Kupffer cells were isolated and cell surface marker expression (flow cytometry), mRNA expression (qtPCR), nitric oxide (NO.) production (Griess reaction), and protein secretion (cytometric bead-array or ELISAs) were determined. To address the potential role of NO. in suppressing neutrophil accumulation, a group of WT mice received 1400W, a specific inducible nitric oxide synthase (iNOS) inhibitor, prior to BDL. To clarify the mechanisms underlying Kupffer cell-iNKT cell cross-activation, WT animals were administered anti-IFN-γ or anti-lymphocyte function-associated antigen (LFA)-1 antibody prior to BDL. Compared to their WT counterparts, Kupffer cells obtained from BDL iNKT cell-deficient mice expressed lower iNOS mRNA levels, produced less NO., and secreted more neutrophil chemoattractants. Both iNOS inhibition and IFN-γ neutralization increased neutrophil accumulation in the livers of BDL WT mice. Anti-LFA-1 pre-treatment reduced iNKT cell accumulation in these same animals. These data indicate that the LFA-1-dependent cross-activation of iNKT cells and Kupffer cells inhibits neutrophil accumulation and cholestatic liver injury. PMID:24260285

The effect of calorie restriction on the presence of apoptotic ovarian cells in normal wild type mice and low-plasma-IGF-1 Laron dwarf mice

PubMed Central

2013-01-01

Background It is known that caloric restriction extends lifespan and can minimize age-related dysfunction of the reproductive system. We became interested in how caloric restriction influences apoptosis, which is a crucial process that maintains ovarian cell homeostasis. Methods We examined ovarian cells in: 2.5-year-old wild type mice on caloric restriction (CR) or fed ad libitum (AL) and Laron dwarf mice (GHR-KO) at the same ages on CR or fed AL. Apoptosis was assessed by histochemical analysis on paraffin sections of ovarian tissue. Results Morphological and histochemical analysis revealed that CR improved reproductive potential in 2.5-year-old WT littermates and GHR-KO female mice, as indicated by the increased number of ovarian follicles. The level of apoptosis in ovarian tissue was higher in WT mice on a CR diet compared with WT mice on the AL diet. In GHR-KO mice, the level of apoptosis in ovaries was similar for mice on CR and on AL diets and bigger than in WT mice on CR. Conclusions Morphological and histochemical analysis revealed a younger biological age of the ovaries in 2-year-old WT littermates and GHR-KO female mice on CR compared with animals fed AL. PMID:24063422

A comparison of the ultrastructure and composition of fruits' cuticular wax from the wild-type 'Newhall' navel orange (Citrus sinensis [L.] Osbeck cv. Newhall) and its glossy mutant.

PubMed

Liu, De-Chun; Zeng, Qiong; Ji, Qing-Xun; Liu, Chuan-Fu; Liu, Shan-Bei; Liu, Yong

2012-12-01

The altered ultrastructure and composition of cuticular wax from 'glossy Newhall' (MT) fruits lead to its glossy phenotype. A novel mutant derived from the wild-type (WT) 'Newhall' navel orange (Citrus sinensis [L.] Osbeck cv. Newhall), named 'glossy Newhall' (MT), which produced much more glossy fruits that were easily distinguishable from the WT fruits was characterized in this report. The total wax loads of both WT and MT fruits varied considerably during the fruit development. The most abundant wax fraction of WT mature fruits was triterpenoids, followed by aldehydes, alkanes, fatty acids, primary alcohol and cholesterol. The total wax load in MT mature fruits was reduced by 44.2 % compared with WT. Except for the minor wax components of primary alcohol and cholesterol, the amounts of all major wax fractions in MT mature fruits were decreased in varying degrees. The major reduction occurred in aldehydes that decreased 96.4 % and alkanes that decreased 81.9 %, which was consistent with scanning electron micrographs of MT mature fruit surfaces that showed a severe loss of wax crystals. Hence, aldehydes and alkanes were suggested to be required for wax crystal formation in 'Newhall' navel orange fruits.

Desulfurization of Dibenzothiophene and Diesel Oils by a Newly Isolated Gordona Strain, CYKS1

PubMed Central

Rhee, Sung-Keun; Chang, Je Hwan; Chang, Yong Keun; Chang, Ho Nam

1998-01-01

A dibenzothiophene (DBT)-desulfurizing bacterial strain was isolated and identified as Gordona strain CYKS1. Strain CYKS1 was found to transform DBT to 2-hydroxybiphenyl via the 4S pathway and to be able to also use organic sulfur compounds other than DBT as a sole sulfur source. Its desulfurization activity was susceptible to sulfate repression. Active resting cells for desulfurization could be prepared only in the early growth phase. When two types of diesel oils, middle distillate unit feed (MDUF) and light gas oil (LGO) containing various organic sulfur compounds including DBT, were treated with resting cells of strain CYKS1 for 12 h, the total sulfur content significantly decreased, from 0.15% (wt/wt) to 0.06% (wt/wt) for MDUF and from 0.3% (wt/wt) to 0.25% (wt/wt) for LGO. The newly isolated strain CYKS1 is considered to have good potential for application in the biodesulfurization of fossil fuels. PMID:9603863

Survival of irradiated recipient mice after transplantation of bone marrow from young, old and "early aging" mice.

PubMed

Guest, Ian; Ilic, Zoran; Scrable, Heidi; Sell, Stewart

2015-12-01

Bone marrow transplantation is used to examine survival, hematopoietic stem cell function and pathology in recipients of young and old wild type bone marrow derived stem cells (BMDSCs) as well as cells from p53-based models of premature aging. There is no difference in the long term survival of recipients of 8 week-old p53+/m donor cells compared to recipients of 8 week-old wild-type (WT) donor cells (70 weeks) or of recipients of 16-18 weeks-old donor cells from either p53+/m or WT mice. There is shorter survival in recipients of older versus younger WT donor bone marrow, but the difference is only significant when comparing 8 and 18 week-old donors. In the p44-based model, short term survival/engraftment is significantly reduced in recipients of 11 month-old p44 donor cells compared to 4 week-old p44 or wild type donor cells of either age; mid-life survival at 40 weeks is also significantly less in recipients of p44 cells. BMDSCs are readily detectable within recipient bone marrow, lymph node, intestinal villi and liver sinusoids, but not in epithelial derived cells. These results indicate that recipients of young BMDSCs may survive longer than recipients of old bone marrow, but the difference is marginal at best.

Survival of irradiated recipient mice after transplantation of bone marrow from young, old and “early aging” mice

PubMed Central

Guest, Ian; Ilic, Zoran; Sell, Stewart

2015-01-01

Bone marrow transplantation is used to examine survival, hematopoietic stem cell function and pathology in recipients of young and old wild type bone marrow derived stem cells (BMDSCs) as well as cells from p53-based models of premature aging. There is no difference in the long term survival of recipients of 8 week-old p53+/m donor cells compared to recipients of 8 week-old wild-type (WT) donor cells (70 weeks) or of recipients of 16–18 weeks-old donor cells from either p53+/m or WT mice. There is shorter survival in recipients of older versus younger WT donor bone marrow, but the difference is only significant when comparing 8 and 18 week-old donors. In the p44-based model, short term survival/engraftment is significantly reduced in recipients of 11 month-old p44 donor cells compared to 4 week-old p44 or wild type donor cells of either age; mid-life survival at 40 weeks is also significantly less in recipients of p44 cells. BMDSCs are readily detectable within recipient bone marrow, lymph node, intestinal villi and liver sinusoids, but not in epithelial derived cells. These results indicate that recipients of young BMDSCs may survive longer than recipients of old bone marrow, but the difference is marginal at best. PMID:26796640

Comparison of effect of gamma ray irradiation on wild-type and N-terminal mutants of αA-crystallin.

PubMed

Ramkumar, Srinivasagan; Fujii, Noriko; Fujii, Norihiko; Thankappan, Bency; Sakaue, Hiroaki; Ingu, Kim; Natarajaseenivasan, Kalimuthusamy; Anbarasu, Kumarasamy

2014-01-01

To study the comparative structural and functional changes between wild-type (wt) and N-terminal congenital cataract causing αA-crystallin mutants (R12C, R21L, R49C, and R54C) upon exposure to different dosages of gamma rays. Alpha A crystallin N-terminal mutants were created with the site-directed mutagenesis method. The recombinantly overexpressed and purified wt and mutant proteins were used for further studies. A (60)Co source was used to generate gamma rays to irradiate wild and mutant proteins at dosages of 0.5, 1.0, and 2.0 kGy. The biophysical property of the gamma irradiated (GI) and non-gamma irradiated (NGI) αA-crystallin wt and N-terminal mutants were determined. Oligomeric size was determined by size exclusion high-performance liquid chromatography (HPLC), the secondary structure with circular dichroism (CD) spectrometry, conformation of proteins with surface hydrophobicity, and the functional characterization were determined regarding chaperone activity using the alcohol dehydrogenase (ADH) aggregation assay. αA-crystallin N-terminal mutants formed high molecular weight (HMW) cross-linked products as well as aggregates when exposed to GI compared to the NGI wt counterparts. Furthermore, all mutants exhibited changed β-sheet and random coil structure. The GI mutants demonstrated decreased surface hydrophobicity when compared to αA-crystallin wt at 0, 1.0, and 1.5 kGy; however, at 2.0 kGy a drastic increase in hydrophobicity was observed only in the mutant R54C, not the wt. In contrast, chaperone activity toward ADH was gradually elevated at the minimum level in all GI mutants, and significant elevation was observed in the R12C mutant. Our findings suggest that the N-terminal mutants of αA-crystallin are structurally and functionally more sensitive to GI when compared to their NGI counterparts and wt. Protein oxidation as a result of gamma irradiation drives the protein to cross-link and aggregate culminating in cataract formation.

Tenofovir alafenamide demonstrates broad cross-genotype activity against wild-type HBV clinical isolates and maintains susceptibility to drug-resistant HBV isolates in vitro.

PubMed

Liu, Yang; Miller, Michael D; Kitrinos, Kathryn M

2017-03-01

Tenofovir alafenamide (TAF) is a novel prodrug of tenofovir (TFV). This study evaluated the antiviral activity of TAF against wild-type genotype A-H HBV clinical isolates as well as adefovir-resistant, lamivudine-resistant, and entecavir-resistant HBV isolates. Full length HBV genomes or the polymerase/reverse transcriptase (pol/RT) region from treatment-naïve patients infected with HBV genotypes A-H were amplified and cloned into an expression vector under the control of a CMV promoter. In addition, 11 drug resistant HBV constructs were created by site-directed mutagenesis of a full length genotype D construct. Activity of TAF was measured by transfection of each construct into HepG2 cells and assessment of HBV DNA levels following treatment across a range of TAF concentrations. TAF activity in vitro was similar against wild-type genotype A-H HBV clinical isolates. All lamivudine- and entecavir-resistant isolates and 4/5 adefovir-resistant isolates were found to be sensitive to inhibition by TAF in vitro as compared to the wild-type isolate. The adefovir-resistant isolate rtA181V + rtN236T exhibited low-level reduced susceptibility to TAF. TAF is similarly active in vitro against wild-type genotype A-H HBV clinical isolates. The TAF sensitivity results for all drug-resistant isolates are consistent with what has been observed with the parent drug TFV. The in vitro cell-based HBV phenotyping assay results support the use of TAF in treatment of HBV infected subjects with diverse HBV genotypes, in both treatment-naive and treatment-experienced HBV infected patients. Copyright © 2016 Elsevier B.V. All rights reserved.

Cell type of origin as well as genetic alterations contribute to breast cancer phenotypes

PubMed Central

West, William W.; Qiu, Fang; Band, Hamid; Band, Vimla

2015-01-01

Breast cancer is classified into different subtypes that are associated with different patient survival outcomes, underscoring the importance of understanding the role of precursor cell and genetic alterations in determining tumor subtypes. In this study, we evaluated the oncogenic phenotype of two distinct mammary stem/progenitor cell types designated as K5+/K19− or K5+/K19+ upon introduction of identical combinations of oncogenes-mutant H-Ras (mRas) and mutant p53 (mp53), together with either wild-type ErbB2(wtErbB2) or wild-type EGFR (wtEGFR). We examined their tumor forming and metastasis potential, using both in-vitro and in-vivo assays. Both the combinations efficiently transformed K5+/K19− or K5+/K19+ cells. Xenograft tumors formed by these cells were histologically heterogeneous, with variable proportions of luminal, basal-like and claudin-low type components depending on the cell types and oncogene combinations. Notably, K5+/K19− cells transformed with mRas/mp53/wtEGFR combination had a significantly longer latency for primary tumor development than other cell lines but more lung metastasis incidence than same cells expressing mRas/mp53/wtErbB2. K5+/K19+ cells exhibit shorter overall tumor latency, and high metastatic potential than K5+/K19− cells, suggesting that these K19+ progenitors are more susceptible to oncogenesis and metastasis. Our results suggest that both genetic alterations and cell type of origin contribute to oncogenic phenotype of breast tumors. PMID:25940703

Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol[S

PubMed Central

Zhu, Xuewei; Owen, John S.; Wilson, Martha D.; Li, Haitao; Griffiths, Gary L.; Thomas, Michael J.; Hiltbold, Elizabeth M.; Fessler, Michael B.; Parks, John S.

2010-01-01

We previously showed that macrophages from macrophage-specific ATP-binding cassette transporter A1 (ABCA1) knockout (Abca1-M/-M) mice had an enhanced proinflammatory response to the Toll-like receptor (TLR) 4 agonist, lipopolysaccharide (LPS), compared with wild-type (WT) mice. In the present study, we demonstrate a direct association between free cholesterol (FC), lipid raft content, and hyper-responsiveness of macrophages to LPS in WT mice. Abca1-M/-M macrophages were also hyper-responsive to specific agonists to TLR2, TLR7, and TLR9, but not TLR3, compared with WT macrophages. We hypothesized that ABCA1 regulates macrophage responsiveness to TLR agonists by modulation of lipid raft cholesterol and TLR mobilization to lipid rafts. We demonstrated that Abca1-M/-M vs. WT macrophages contained 23% more FC in isolated lipid rafts. Further, mass spectrometric analysis suggested raft phospholipid composition was unchanged. Although cell surface expression of TLR4 was similar between Abca1-M/-M and WT macrophages, significantly more TLR4 was distributed in membrane lipid rafts in Abca1-M/-M macrophages. Abca1-M/-M macrophages also exhibited increased trafficking of the predominantly intracellular TLR9 into lipid rafts in response to TLR9-specific agonist (CpG). Collectively, our data suggest that macrophage ABCA1 dampens inflammation by reducing MyD88-dependent TLRs trafficking to lipid rafts by selective reduction of FC content in lipid rafts. PMID:20650929

Genetic characteristics and antimicrobial resistance of Escherichia coli from Japanese macaques (Macaca fuscata) in rural Japan.

PubMed

Ogawa, Keiko; Yamaguchi, Keiji; Suzuki, Masatsugu; Tsubota, Toshio; Ohya, Kenji; Fukushi, Hideto

2011-04-01

Escherichia coli was isolated from wild and captive Japanese macaques (Macaca fuscata) to investigate the risk of zoonotic infections and the prevalence of antimicrobial-resistant Escherichia coli in the wild macaque population in Shimokita Peninsula, a rural area of Japan. We collected 265 fresh fecal samples from wild macaques and 20 samples from captive macaques in 2005 and 2006 for E. coli isolation. The predominant isolates were characterized by serotyping, virulence gene profiling, plasmid profiling, pulsed-field gel electrophoresis (PFGE), and microbial sensitivity tests. In total, 248 E. coli strains were isolated from 159 fecal samples from wild macaques, and 42 E. coli were isolated from 17 samples from captive macaques. None of the virulence genes eae, stx, elt, and est were detected in any of the isolates. The relatedness between wild- and captive-derived isolates was low by serotyping, PFGE, and plasmid profiling. Serotypes O8:H6, O8:H34, O8:H42, O8:HUT, O103:H27, O103:HNM, and OUT:H27 were found in wild macaque feces; serotypes O157:H42 and O119:H21 were recovered from captive macaques. O-and H-serotypes of the 26 isolates were not typed by commercial typing antisera and were named OUT and HUT, respectively. Twenty-eight isolates had no flagellar antigen, and their H-serotypes were named HNM. Similarity of PFGE patterns between wild-derived isolates and captive-derived isolates was <70%. No plasmid profile was shared between wild-derived and captive-derived isolates. The prevalence of antimicrobial-resistant E. coli was 6.5% (n=62) in wild macaques, and these isolates were resistant to cephalothin. We conclude that wild Japanese macaques in Shimokita Peninsula were unlikely to act as a reservoir of pathogenic E. coli for humans and that antimicrobial-resistant E. coli in wild macaques may be derived from humans.

Pyridine-type alkaloid composition affects bacterial community composition of floral nectar

PubMed Central

Aizenberg-Gershtein, Yana; Izhaki, Ido; Santhanam, Rakesh; Kumar, Pavan; Baldwin, Ian T.; Halpern, Malka

2015-01-01

Pyridine-type alkaloids are most common in Nicotiana species. To study the effect of alkaloid composition on bacterial community composition in floral nectar, we compared the nicotine-rich wild type (WT) N. attenuata, the nicotine biosynthesis-silenced N. attenuata that was rich in anatabine and the anabasine-rich WT N. glauca plants. We found that the composition of these secondary metabolites in the floral nectar drastically affected the bacterial community richness, diversity and composition. Significant differences were found between the bacterial community compositions in the nectar of the three plants with a much greater species richness and diversity in the nectar from the transgenic plant. The highest community composition similarity index was detected between the two wild type plants. The different microbiome composition and diversity, caused by the different pyridine-type alkaloid composition, could modify the nutritional content of the nectar and consequently, may contribute to the change in the nectar consumption and visitation. These may indirectly have an effect on plant fitness. PMID:26122961

Pyridine-type alkaloid composition affects bacterial community composition of floral nectar.

PubMed

Aizenberg-Gershtein, Yana; Izhaki, Ido; Santhanam, Rakesh; Kumar, Pavan; Baldwin, Ian T; Halpern, Malka

2015-06-30

Pyridine-type alkaloids are most common in Nicotiana species. To study the effect of alkaloid composition on bacterial community composition in floral nectar, we compared the nicotine-rich wild type (WT) N. attenuata, the nicotine biosynthesis-silenced N. attenuata that was rich in anatabine and the anabasine-rich WT N. glauca plants. We found that the composition of these secondary metabolites in the floral nectar drastically affected the bacterial community richness, diversity and composition. Significant differences were found between the bacterial community compositions in the nectar of the three plants with a much greater species richness and diversity in the nectar from the transgenic plant. The highest community composition similarity index was detected between the two wild type plants. The different microbiome composition and diversity, caused by the different pyridine-type alkaloid composition, could modify the nutritional content of the nectar and consequently, may contribute to the change in the nectar consumption and visitation. These may indirectly have an effect on plant fitness.

Susceptibility profile and epidemiological cut-off values of Cryptococcus neoformans species complex from Argentina.

PubMed

Córdoba, Susana; Isla, Maria G; Szusz, Wanda; Vivot, Walter; Altamirano, Rodrigo; Davel, Graciela

2016-06-01

Epidemiological cut-off values (ECVs) based on minimal inhibitory concentration (MIC) distribution have been recently proposed for some antifungal drug/Cryptococcus neoformans combinations. However, these ECVs vary according to the species studied, being serotypes and the geographical origin of strains, variables to be considered. The aims were to define the wild-type (WT) population of the C. neoformans species complex (C. neoformans) isolated from patients living in Argentina, and to propose ECVs for six antifungal drugs. A total of 707 unique C. neoformans isolates obtained from HIV patients suffering cryptococcal meningitis were studied. The MIC of amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole and posaconazole was determined according to the EDef 7.2 (EUCAST) reference document. The MIC distribution, MIC50 , MIC90 and ECV for each of these drugs were calculated. The highest ECV, which included ≥95% of the WT population modelled, was observed for flucytosine and fluconazole (32 μg ml(-1) each). For amphotericin B, itraconazole, voriconazole and posaconazole, the ECVs were: 0.5, 0.5, 0.5 and 0.06 μg ml(-1) respectively. The ECVs determined in this study may aid in identifying the C. neoformans strains circulating in Argentina with decreased susceptibility to the antifungal drugs tested. © 2016 Blackwell Verlag GmbH.

Reduction of virion-associated σ1 fibers on oncolytic reovirus variants promotes adaptation toward tumorigenic cells.

PubMed

Mohamed, Adil; Teicher, Carmit; Haefliger, Sarah; Shmulevitz, Maya

2015-04-01

Wild-type mammalian orthoreovirus serotype 3 Dearing (T3wt) is nonpathogenic in humans but preferentially infects and kills cancer cells in culture and demonstrates promising antitumor activity in vivo. Using forward genetics, we previously isolated two variants of reovirus, T3v1 and T3v2, with increased infectivity toward a panel of cancer cell lines and improved in vivo oncolysis in a murine melanoma model relative to that of T3wt. Our current study explored how mutations in T3v1 and T3v2 promote infectivity. Reovirions contain trimers of σ1, the reovirus cell attachment protein, at icosahedral capsid vertices. Quantitative Western blot analysis showed that purified T3v1 and T3v2 virions had ∼ 2- and 4-fold-lower levels of σ1 fiber than did T3wt virions. Importantly, using RNA interference to reduce σ1 levels during T3wt production, we were able to generate wild-type reovirus with reduced levels of σ1 per virion. As σ1 levels were reduced, virion infectivity increased by 2- to 5-fold per cell-bound particle, demonstrating a causal relationship between virion σ1 levels and the infectivity of incoming virions. During infection of tumorigenic L929 cells, T3wt, T3v1, and T3v2 uncoated the outer capsid proteins σ3 and μ1C at similar rates. However, having started with fewer σ1 molecules, a complete loss of σ1 was achieved sooner for T3v1 and T3v2. Distinct from intracellular uncoating, chymotrypsin digestion, as a mimic of natural enteric infection, resulted in more rapid σ3 and μ1C removal, unique disassembly intermediates, and a rapid loss of infectivity for T3v1 and T3v2 compared to T3wt. Optimal infectivity toward natural versus therapeutic niches may therefore require distinct reovirus structures and σ1 levels. Wild-type reovirus is currently in clinical trials as a potential cancer therapy. Our molecular studies on variants of reovirus with enhanced oncolytic activity in vitro and in vivo now show that distinct reovirus structures promote adaptation toward cancer cells and away from conditions that mimic natural routes of infection. Specifically, we found that reovirus particles with fewer molecules of the cell attachment protein σ1 became more infectious toward transformed cells. Reduced σ1 levels conferred a benefit to incoming particles only, resulting in an earlier depletion of σ1 and a higher probability of establishing productive infection. Conversely, reovirus variants with fewer σ1 molecules showed reduced stability and infectivity and distinct disassembly when exposed to conditions that mimic natural intestinal proteolysis. These findings support a model where the mode of infection dictates the precise optimum of reovirus structure and provide a molecular rationale for considering alternative reovirus structures during oncolytic therapy. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The role of the PI3K/mTOR signaling pathway in Staphylococcus epidermidis small colony variants intracellular survival.

PubMed

Magryś, Agnieszka; Bogut, Agnieszka; Kiełbus, Michał; Olender, Alina

2018-04-01

The objective of this study was to analyze how Staphylococcus epidermidis SCV and WT strains manipulate the PI3K/Akt/mTOR signaling pathway. Six S. epidermidis strains with normal phenotype (WT) and six S. epidermidis strains with SCV phenotype were isolated in parallel from six patients with the prosthetic hip joint infections. THP-1 activated cells were incubated with or without PI3K inhibitor-wortmannin or with mTOR inhibitor-rapamycin. Next, macrophages were exposed to S. epidermidis WT and SCV strains. After 4 h incubation, bacterial survival inside macrophages as well as PI3K-mTOR activation was analyzed. SCV strains of S. epidermidis increased the level of Akt phosphorylation, compared to uninfected macrophages and to their parental WT forms. Wild type variants of S. epidermidis phosphorylated Akt at similar or lower levels as control uninfected cells. Next, the induction of mTOR target, phosphorylated ribosomal protein S6, was measured in bacteria-infected macrophages. The level of phosphorylation was significantly reduced when the cells were exposed to WT strains of S. epidermidis. In contrast, the SCV strains activated S6 protein mostly at a level comparable to the control cells. Rapamycin inhibited mTOR activation as the number of p-S6 positive cells decreased in the tested cases. To conclude, the SCV strains activate the PI3K-Akt signaling pathway in opposite to WT strains. This fact however did not influence the increase in the number of live SCV bacteria as compared to the WT strains. Knowing that the PI3K-Akt pathway is involved in proinflammatory cytokines suppression, SCVs seem to use this pathway to reduce the inflammatory response during the infection.

TERT promoter mutations contribute to IDH mutations in predicting differential responses to adjuvant therapies in WHO grade II and III diffuse gliomas

PubMed Central

Ding, Xiao-Jie; Qin, Zhi-Yong; Hong, Christopher S.; Chen, Ling-Chao; Zhang, Xin; Zhao, Fang-Ping; Wang, Yin; Wang, Yang; Zhou, Liang-Fu; Zhuang, Zhengping; Ng, Ho-Keung; Yan, Hai; Yao, Yu; Mao, Ying

2015-01-01

IDH mutations frequently occur in WHO grade II and III diffuse gliomas and have favorable prognosis compared to wild-type tumors. However, whether IDH mutations in WHO grade II and II diffuse gliomas predict enhanced sensitivity to adjuvant radiation (RT) or chemotherapy (CHT) is still being debated. Recent studies have identified recurrent mutations in the promoter region of telomerase reverse transcriptase (TERT) in gliomas. We previously demonstrated that TERT promoter mutations may be promising biomarkers in glioma survival prognostication when combined with IDH mutations. This study analyzed IDH and TERT promoter mutations in 295 WHO grade II and III diffuse gliomas treated with or without adjuvant therapies to explore their impact on the sensitivity of tumors to genotoxic therapies. IDH mutations were found in 216 (73.2%) patients and TERT promoter mutations were found in 112 (38%) patients. In multivariate analysis, IDH mutations (p < 0.001) were independent prognostic factors for PFS and OS in patients receiving genotoxic therapies while TERT promoter mutations were not. In univariate analysis, IDH and TERT promoter mutations were not significant prognostic factors in patients who did not receive genotoxic therapies. Adjuvant RT and CHT were factors independently impacting PFS (RT p = 0.001, CHT p = 0.026) in IDH mutated WHO grade II and III diffuse gliomas but not in IDH wild-type group. Univariate and multivariate analyses demonstrated TERT promoter mutations further stratified IDH wild-type WHO grade II and III diffuse gliomas into two subgroups with different responses to genotoxic therapies. Adjuvant RT and CHT were significant parameters influencing PFS in the IDH wt/TERT mut subgroup (RT p = 0.015, CHT p = 0.015) but not in the IDH wt/TERT wt subgroup. Our data demonstrated that IDH mutated WHO grade II and III diffuse gliomas had better PFS and OS than their IDH wild-type counterparts when genotoxic therapies were administered after surgery. Importantly, we also found that TERT promoter mutations further stratify IDH wild-type WHO grade II and III diffuse gliomas into two subgroups with different responses to adjuvant therapies. Taken together, TERT promoter mutations may predict enhanced sensitivity to genotoxic therapies in IDH wild-type WHO grade II and III diffuse gliomas and may justify intensified treatment in this subgroup. PMID:26314843

Inhibition of NLRP3 inflammasome by thioredoxin-interacting protein in mouse Kupffer cells as a regulatory mechanism for non-alcoholic fatty liver disease development

PubMed Central

He, Kun; Zhu, Xiwen; Liu, Yan; Miao, Chunmu; Wang, Tao; Li, Peizhi; Zhao, Lei; Chen, Yaxi; Gong, Junhua; Cai, Can; Li, Jinzheng; Li, Shengwei; Ruan, Xiong Z.; Gong, Jianping

2017-01-01

NOD-like receptor (NLR) NLRP3 inflammasome activation has been implicated in the progression of non-alcoholic fatty liver disease (NAFLD) from non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH). It has been also shown that palmitic acid (PA) activates NLRP3 inflammasome and promotes interleukin-1β (IL-1β) secretion in Kupffer cells (KCs). However, the specific mechanism of the NLRP3 inflammasome activation is unclear. We studies the molecular mechanisms by investigating the roles of Thioredoxin-interacting protein (TXNIP) and NLRP3 on NAFLD development in patients, high-fat diet (HFD)-induced NAFL and methionine choline deficient (MCD) diet-induced NASH in wild type (WT), TXNIP−/− (thioredoxin-interacting protein) and NLRP3−/− mice, and isolated KCs. We found that the expressions of NLRP3 and TXNIP in human liver tissues were higher in NASH group than in NAFL group. Furthermore, co-immunoprecipitation analyses show that activation of the TXNIP-NLRP3 inflammasome protein complex occurred in KCs of NASH WT mice rather than NAFL WT mice, thus suggesting that the formation and activation of this protein complex is mainly involved in the development of NASH. NLRP3−/− mice exhibited less severe NASH than WT mice in MCD diet model, whereas TXNIP deficiency enhanced NLRP3 inflammasome activation and exacerbated liver injury. PA triggered the activation and co-localization of the NLRP3 inflammasome protein complex in KCs isolated from WT and TXNIP−/− but not NLRP3−/− mice, and most of the complex co-localized with mitochondria of KCs following PA stimulation. Taken together, our novel findings indicate that TXNIP plays a protective and anti-inflammatory role in the development of NAFLD through binding and suppressing NLRP3. PMID:28499273

Over-expression of mitochondrial creatine kinase in the murine heart improves functional recovery and protects against injury following ischaemia-reperfusion.

PubMed

Whittington, Hannah J; Ostrowski, Philip J; McAndrew, Debra J; Cao, Fang; Shaw, Andrew; Eykyn, Thomas R; Lake, Hannah; Tyler, Jack; Schneider, Jurgen E; Neubauer, Stefan; Zervou, Sevasti; Lygate, Craig A

2018-03-02

Mitochondrial creatine kinase (MtCK) couples ATP production via oxidative phosphorylation to phosphocreatine in the cytosol, which acts as a mobile energy store available for regeneration of ATP at times of high demand. We hypothesised that elevating MtCK would be beneficial in ischaemia-reperfusion (I/R) injury. Mice were created overexpressing the sarcomeric MtCK gene with αMHC promoter at the Rosa26 locus (MtCK-OE) and compared with wild-type (WT) littermates. MtCK activity was 27% higher than WT, with no change in other CK isoenzymes or creatine levels. Electron microscopy confirmed normal mitochondrial cell density and mitochondrial localisation of transgenic protein. Respiration in isolated mitochondria was unaltered and metabolomic analysis by 1H-NMR suggests that cellular metabolism was not grossly affected by transgene expression. There were no significant differences in cardiac structure or function under baseline conditions by cine-MRI or LV haemodynamics. In Langendorff-perfused hearts subjected to 20min ischaemia and 30 min reperfusion, MtCK-OE exhibited less ischaemic contracture and improved functional recovery (Rate pressure product 58% above WT; P < 0.001). These hearts had reduced myocardial infarct size, which was confirmed in vivo: 55±4% in WT vs 29±4% in MtCK-OE; P < 0.0001). Isolated cardiomyocytes from MtCK-OE hearts exhibited delayed opening of the mitochondrial permeability transition pore (mPTP) compared to WT, which was confirmed by reduced mitochondrial swelling in response to calcium. There was no detectable change in the structural integrity of the mitochondrial membrane. Modest elevation of MtCK activity in the heart does not adversely affect cellular metabolism, mitochondrial or in vivo cardiac function, but modifies mPTP opening to protect against I/R injury and improve functional recovery. Our findings support MtCK as a prime therapeutic target in myocardial ischaemia.

Isolation and characterization of a pigmentless-conidium mutant of Aspergillus fumigatus with altered conidial surface and reduced virulence.

PubMed Central

Jahn, B; Koch, A; Schmidt, A; Wanner, G; Gehringer, H; Bhakdi, S; Brakhage, A A

1997-01-01

Aspergillus fumigatus is an important pathogen of immunocompromised hosts, causing pneumonia and invasive disseminated disease with high mortality. The factors contributing to the predominance of A. fumigatus as an opportunistic pathogen are largely unknown. Since the survival of conidia in the host is a prerequisite for establishing disease, we have been attempting to identify factors which are associated with conidia and, simultaneously, important for infection. Therefore, an A. fumigatus mutant strain (white [W]) lacking conidial pigmentation was isolated. Scanning electron microscopy revealed that conidia of the W mutant also differed in their surface morphology from those of the wild type (WT). Mutant (W) and WT conidia were compared with respect to their capacities to stimulate an oxidative response in human phagocytes, their intracellular survival in human monocytes, and virulence in a murine animal model. Luminol-dependent chemiluminescence was 10-fold higher when human neutrophils or monocytes were challenged with W conidia compared with WT conidia. Furthermore, mutant conidia were more susceptible to killing by oxidants in vitro and were more efficiently damaged by human monocytes in vitro than WT conidia. In a murine animal model, the W mutant strain showed reduced virulence compared with the WT. A reversion analysis of the W mutant demonstrated that all phenotypes associated with the W mutant, i.e., altered conidial surface, amount of reactive oxygen species release, susceptibility to hydrogen peroxide, and reduced virulence in an murine animal model, coreverted in revertants which had regained the ability to produce green spores. This finding strongly suggests that the A. fumigatus mutant described here carries a single mutation which caused all of the observed phenotypes. Our results suggest that the conidium pigment or a structural feature related to it contributes to fungal resistance against host defense mechanisms in A. fumigatus infections. PMID:9393803

Akt-mediated cardioprotective effects of aldosterone in type 2 diabetic mice.

PubMed

Fazal, Loubina; Azibani, Feriel; Bihry, Nicolas; Coutance, Guillaume; Polidano, Evelyne; Merval, Régine; Vodovar, Nicolas; Launay, Jean-Marie; Delcayre, Claude; Samuel, Jane-Lise

2014-06-01

Studies have shown that aldosterone would have angiogenic effects and therefore would be beneficial in the context of cardiovascular diseases. We thus investigated the potential involvement of aldosterone in triggering a cardiac angiogenic response in the context of type-2 diabetes and the molecular pathways involved. Male 3-wk-old aldosterone synthase (AS)-overexpressing mice and their control wild-type (WT) littermates were fed a standard or high-fat, high-sucrose (HFHS) diet. After 6 mo of diet treatment, mice were euthanized, and cardiac samples were assayed by RT-PCR, immunoblotting, and immunohistology. HFHS diet induced type-2 diabetes in WT (WT-D) and AS (AS-D) mice. VEGFa mRNAs decreased in WT-D (-43%, P<0.05 vs. WT) and increased in AS-D mice (+236%, P< 0.01 vs. WT-D). In WT-D mouse hearts, the proapoptotic p38MAPK was activated (P<0.05 vs. WT and AS-D), whereas Akt activity decreased (-64%, P<0.05 vs. WT). The AS mice, which exhibited a cardiac up-regulation of IGF1-R, showed an increase in Akt phosphorylation when diabetes was induced (P<0.05 vs. WT and AS-D). Contrary to WT-D mice, AS-D mouse hearts did not express inflammatory markers and exhibited a normal capillary density (P<0.05 vs. WT-D). To our knowledge, this is the first study providing new insights into the mechanisms whereby aldosterone prevents diabetes-induced cardiac disorders. © FASEB.

Light-dependent gravitropism and negative phototropism of inflorescence stems in a dominant Aux/IAA mutant of Arabidopsis thaliana, axr2.

PubMed

Sato, Atsuko; Sasaki, Shu; Matsuzaki, Jun; Yamamoto, Kotaro T

2014-09-01

Gravitropism and phototropism of the primary inflorescence stems were examined in a dominant Aux/IAA mutant of Arabidopsis, axr2/iaa7, which did not display either tropism in hypocotyls. axr2-1 stems completely lacked gravitropism in the dark but slowly regained it in light condition. Though wild-type stems showed positive phototropism, axr2 stems displayed negative phototropism with essentially the same light fluence-response curve as the wild type (WT). Application of 1-naphthaleneacetic acid-containing lanolin to the stem tips enhanced the positive phototropism of WT, and reduced the negative phototropism of axr2. Decapitation of stems caused a small negative phototropism in WT, but did not affect the negative phototropism of axr2. p-glycoprotein 1 (pgp1) pgp19 double mutants showed no phototropism, while decapitated double mutants exhibited negative phototropism. Expression of auxin-responsive IAA14/SLR, IAA19/MSG2 and SAUR50 genes was reduced in axr2 and pgp1 pgp19 stems relative to that of WT. These suggest that the phototropic response of stem is proportional to the auxin supply from the shoot apex, and that negative phototropism may be a basal response to unilateral blue-light irradiation when the levels of auxin or auxin signaling are reduced to the minimal level in the primary stems. In contrast, all of these treatments reduced or did not affect gravitropism in wild-type or axr2 stems. Tropic responses of the transgenic lines that expressed axr2-1 protein by the endodermis-specific promoter suggest that AXR2-dependent auxin response in the endodermis plays a more crucial role in gravitropism than in phototropism in stems but no significant roles in either tropism in hypocotyls.

A novel TFF2 splice variant (ΔEX2TFF2) correlates with longer overall survival time in cholangiocarcinoma

PubMed Central

KAMLUA, SURASEE; PATRAKITKOMJORN, SIRIPORN; JEARANAIKOON, PATCHAREE; MENHENIOTT, TREVELYAN R.; GIRAUD, ANDREW S.; LIMPAIBOON, TEMDUANG

2012-01-01

Trefoil factor 2 (TFF2) is a member of trefoil factor family found to be overexpressed in many cancers including cholangiocarcinoma (CCA). The majority of studies have focused on wild-type TFF2 (wtTFF2) expression, but information regarding alternative splicing variants of TFF2 mRNA has not been reported. In this study, we aimed to identify and quantify a novel TFF2 splice variant in cholangiocarcinoma (CCA). Seventy-eight tumors and 15 normal adjacent tissues were quantified for the expression of the TFF2 splice variant relative to wild-type (wt) TFF2 mRNA using quantitative reverse transcriptase polymerase chain reaction (QRT-PCR). The ratio of TFF2 splice variant against wtTFF2 was analyzed for associations with clinical parameters. We found a novel TFF2 splice variant, exon 2 skipping (ΔEX2TFF2), resulting in a stop codon (TAG) at exon 1. The ΔEX2TFF2/wtTFF2 ratio in tumors was significantly higher than in normal tissue (P<0.01). Interestingly, high ΔEX2TFF2/wtTFF2 ratio was significantly associated with good prognosis compared with low ratio (P=0.017). In contrast, the presence of wtTFF2 protein was associated with poor survival of CCA patients (P=0.034). This is the first report of a trefoil factor splice variant and its potential application as a prognostic biomarker in CCA. PMID:22159958

Wild-type APC predicts poor prognosis in microsatellite-stable proximal colon cancer.

PubMed

Jorissen, Robert N; Christie, Michael; Mouradov, Dmitri; Sakthianandeswaren, Anuratha; Li, Shan; Love, Christopher; Xu, Zheng-Zhou; Molloy, Peter L; Jones, Ian T; McLaughlin, Stephen; Ward, Robyn L; Hawkins, Nicholas J; Ruszkiewicz, Andrew R; Moore, James; Burgess, Antony W; Busam, Dana; Zhao, Qi; Strausberg, Robert L; Lipton, Lara; Desai, Jayesh; Gibbs, Peter; Sieber, Oliver M

2015-09-15

APC mutations (APC-mt) occur in ∼70% of colorectal cancers (CRCs), but their relationship to prognosis is unclear. APC prognostic value was evaluated in 746 stage I-IV CRC patients, stratifying for tumour location and microsatellite instability (MSI). Microarrays were used to identify a gene signature that could classify APC mutation status, and classifier ability to predict prognosis was examined in an independent cohort. Wild-type APC microsatellite stable (APC-wt/MSS) tumours from the proximal colon showed poorer overall and recurrence-free survival (OS, RFS) than APC-mt/MSS proximal, APC-wt/MSS distal and APC-mt/MSS distal tumours (OS HR⩾1.79, P⩽0.015; RFS HR⩾1.88, P⩽0.026). APC was a stronger prognostic indicator than BRAF, KRAS, PIK3CA, TP53, CpG island methylator phenotype or chromosomal instability status (P⩽0.036). Microarray analysis similarly revealed poorer survival in MSS proximal cancers with an APC-wt-like signature (P=0.019). APC status did not affect outcomes in MSI tumours. In a validation on 206 patients with proximal colon cancer, APC-wt-like signature MSS cases showed poorer survival than APC-mt-like signature MSS or MSI cases (OS HR⩾2.50, P⩽0.010; RFS HR⩾2.14, P⩽0.025). Poor prognosis APC-wt/MSS proximal tumours exhibited features of the sessile serrated neoplasia pathway (P⩽0.016). APC-wt status is a marker of poor prognosis in MSS proximal colon cancer.

Pemetrexed-carboplatin with intercalated icotinib in the treatment of patient with advanced EGFR wild-type lung adenocarcinoma: A case report.

PubMed

Xu, Tongpeng; Wu, Hao; Jin, Shidai; Min, Huang; Zhang, Zhihong; Shu, Yongqian; Wen, Wei; Guo, Renhua

2017-08-01

Tyrosine kinase inhibitors (TKIs) are known to have greater efficacy in epidermal growth factor receptor (EGFR) mutation nonsmall cell lung cancer (NSCLC). However, about 10% of EGFR wild-type (wt) patients respond to TKIs. Several strategies to increase the efficacy of TKIs in wt NSCLC are the subjects of ongoing investigations. One of them is combining EGFR TKI with intercalated chemotherapy. We describe a patient with EGFR wt NSCLC, who was found with ovarian and lung metastasis, was treated with pemetrexed and intercalated icotinib. In this case, we reported the successful long-term maintenance treatment of a patient with EGFR wt NSCLC with pemetrexed and Icotinib. The patient (40-year-old female) was found with ovarian masses and lung masses. Pathological, immunohistochemical, and amplification refractory mutation system (ARMS) assay examinations of ovarian specimen suggested the expression of metastatic lung adenocarcinoma with wt EGFR. After failure treatment with paclitaxel-carboplatin, the patient received 4 cycles of pemetrexed plus platinum with intercalated icotinib and then remained on pemetrexed and icotinib. A partial response was achieved after the treatment. The patient's condition had remained stable on pemetrexed and icotinib for more than 20 months, with no evidence of progression. To our knowledge, this is the first report using the long-term maintenance treatment with pemetrexed and intercalated icotinib in EGFR wt patient. The therapeutic strategies warrant further exploration in selected populations of NSCLC.

Pemetrexed-carboplatin with intercalated icotinib in the treatment of patient with advanced EGFR wild-type lung adenocarcinoma

PubMed Central

Xu, Tongpeng; Wu, Hao; Jin, Shidai; Min, Huang; Zhang, Zhihong; Shu, Yongqian; Wen, Wei; Guo, Renhua

2017-01-01

Abstract Rationale: Tyrosine kinase inhibitors (TKIs) are known to have greater efficacy in epidermal growth factor receptor (EGFR) mutation nonsmall cell lung cancer (NSCLC). However, about 10% of EGFR wild-type (wt) patients respond to TKIs. Patient concerns: Several strategies to increase the efficacy of TKIs in wt NSCLC are the subjects of ongoing investigations. One of them is combining EGFR TKI with intercalated chemotherapy. Diagnoses: We describe a patient with EGFR wt NSCLC, who was found with ovarian and lung metastasis, was treated with pemetrexed and intercalated icotinib. Interventions: In this case, we reported the successful long-term maintenance treatment of a patient with EGFR wt NSCLC with pemetrexed and Icotinib. The patient (40-year-old female) was found with ovarian masses and lung masses. Pathological, immunohistochemical, and amplification refractory mutation system (ARMS) assay examinations of ovarian specimen suggested the expression of metastatic lung adenocarcinoma with wt EGFR. After failure treatment with paclitaxel-carboplatin, the patient received 4 cycles of pemetrexed plus platinum with intercalated icotinib and then remained on pemetrexed and icotinib. Outcomes: A partial response was achieved after the treatment. The patient's condition had remained stable on pemetrexed and icotinib for more than 20 months, with no evidence of progression. Lessons: To our knowledge, this is the first report using the long-term maintenance treatment with pemetrexed and intercalated icotinib in EGFR wt patient. The therapeutic strategies warrant further exploration in selected populations of NSCLC. PMID:28816950

Wild-type measles virus infection upregulates poliovirus receptor-related 4 and causes apoptosis in brain endothelial cells by induction of tumor necrosis factor-related apoptosis-inducing ligand.

PubMed

Abdullah, Hani'ah; Brankin, Brenda; Brady, Clare; Cosby, Sara Louise

2013-07-01

Small numbers of brain endothelial cells (BECs) are infected in children with neurologic complications of measles virus (MV) infection. This may provide a mechanism for virus entry into the central nervous system, but the mechanisms are unclear. Both in vitro culture systems and animal models are required to elucidate events in the endothelium. We compared the ability of wild-type (WT), vaccine, and rodent-adapted MV strains to infect, replicate, and induce apoptosis in human and murine brain endothelial cells (HBECs and MBECs, respectively). Mice also were infected intracerebrally. All MV stains productively infected HBECs and induced the MV receptor PVRL4. Efficient WT MV production also occurred in MBECs. Extensive monolayer destruction associated with activated caspase 3 staining was observed in HBECs and MBECs, most markedly with WT MV. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), but not Fas ligand, was induced by MV infection. Treatment of MBECs with supernatants from MV-infected MBEC cultures with an anti-TRAIL antibody blocked caspase 3 expression and monolayer destruction. TRAIL was also expressed in the endothelium and other cell types in infected murine brains. This is the first demonstration that infection of low numbers of BECs with WT MV allows efficient virus production, induction of TRAIL, and subsequent widespread apoptosis.

Differences in Establishment of Persistence of Vaccine and Wild Type Rubella Viruses in Fetal Endothelial Cells

PubMed Central

Perelygina, Ludmila; Adebayo, Adebola; Metcalfe, Maureen; Icenogle, Joseph

2015-01-01

Both wild type (WT) and vaccine rubella virus (RV) can pass through the placenta to infect a human fetus, but only wtRV routinely causes pathology. To investigate possible reasons for this, we compared establishment of persistence of wtRV and RA27/3 vaccine strains in fetal endothelial cells. We showed that yields of RA27/3 and wtRV were similar after the first round of replication, but then only vaccine-infected cultures went through a crisis characterized by partial cell loss and gradual decline of virus titer followed by recovery and establishment of persistent cultures with low levels of RA27/3 secretion. We compared various steps of virus replication, but we were unable to identify changes, which might explain the 2-log difference in RA27/3 and wtRV yields in persistently infected cultures. Whole genome sequencing did not reveal selection of virus variants in either the wtRV or RA27/3 cultures. Quantitative single-cell analysis of RV replication by in situ hybridization detected, on average, 1–4 copies of negative-strand RNA and ~50 copies of positive-strand genomic RNA in cells infected with both vaccine and WT viruses. The distinct characteristics of RA27/3 replication were the presence of large amounts of negative-strand RV RNA and RV dsRNA at the beginning of the crisis and the accumulation of high amounts of genomic RNA in a subpopulation of infected cells during crisis and persistence. These results suggest that RA27/3 can persist in fetal endothelial cells, but the characteristics of persistence and mechanisms for the establishment and maintenance of persistence are different from wtRV. PMID:26177032

Spaceflight Influences both Mucosal and Peripheral Cytokine Production in PTN-Tg and Wild Type Mice

PubMed Central

Liu, Yi; Kalmokoff, Martin; Brooks, Stephen P. J.; Green-Johnson, Julia M.

2013-01-01

Spaceflight is associated with several health issues including diminished immune efficiency. Effects of long-term spaceflight on selected immune parameters of wild type (Wt) and transgenic mice over-expressing pleiotrophin under the human bone-specific osteocalcin promoter (PTN-Tg) were examined using the novel Mouse Drawer System (MDS) aboard the International Space Station (ISS) over a 91 day period. Effects of this long duration flight on PTN-Tg and Wt mice were determined in comparison to ground controls and vivarium-housed PTN-Tg and Wt mice. Levels of interleukin-2 (IL-2) and transforming growth factor-beta1 (TGF-β1) were measured in mucosal and systemic tissues of Wt and PTN-Tg mice. Colonic contents were also analyzed to assess potential effects on the gut microbiota, although no firm conclusions could be made due to constraints imposed by the MDS payload and the time of sampling. Spaceflight-associated differences were observed in colonic tissue and systemic lymph node levels of IL-2 and TGF-β1 relative to ground controls. Total colonic TGF-β1 levels were lower in Wt and PTN-Tg flight mice in comparison to ground controls. The Wt flight mouse had lower levels of IL-2 and TGF-β1 compared to the Wt ground control in both the inguinal and brachial lymph nodes, however this pattern was not consistently observed in PTN-Tg mice. Vivarium-housed Wt controls had higher levels of active TGF-β1 and IL-2 in inguinal lymph nodes relative to PTN-Tg mice. The results of this study suggest compartmentalized effects of spaceflight and on immune parameters in mice. PMID:23874826

In silico gene expression analysis reveals glycolysis and acetate anaplerosis in IDH1 wild-type glioma and lactate and glutamate anaplerosis in IDH1-mutated glioma.

PubMed

Khurshed, Mohammed; Molenaar, Remco J; Lenting, Krissie; Leenders, William P; van Noorden, Cornelis J F

2017-07-25

Hotspot mutations in isocitrate dehydrogenase 1 (IDH1) initiate low-grade glioma and secondary glioblastoma and induce a neomorphic activity that converts α-ketoglutarate (α-KG) to the oncometabolite D-2-hydroxyglutarate (D-2-HG). It causes metabolic rewiring that is not fully understood. We investigated the effects of IDH1 mutations (IDH1MUT) on expression of genes that encode for metabolic enzymes by data mining The Cancer Genome Atlas. We analyzed 112 IDH1 wild-type (IDH1WT) versus 399 IDH1MUT low-grade glioma and 157 IDH1WT versus 9 IDH1MUT glioblastoma samples. In both glioma types, IDH1WT was associated with high expression levels of genes encoding enzymes that are involved in glycolysis and acetate anaplerosis, whereas IDH1MUT glioma overexpress genes encoding enzymes that are involved in the oxidative tricarboxylic acid (TCA) cycle. In vitro, we observed that IDH1MUT cancer cells have a higher basal respiration compared to IDH1WT cancer cells and inhibition of the IDH1MUT shifts the metabolism by decreasing oxygen consumption and increasing glycolysis. Our findings indicate that IDH1WT glioma have a typical Warburg phenotype whereas in IDH1MUT glioma the TCA cycle, rather than glycolytic lactate production, is the predominant metabolic pathway. Our data further suggest that the TCA in IDH1MUT glioma is driven by lactate and glutamate anaplerosis to facilitate production of α-KG, and ultimately D-2-HG. This metabolic rewiring may be a basis for novel therapies for IDH1MUT and IDH1WT glioma.

Plant surface wax affects parasitoid's response to host footprints

NASA Astrophysics Data System (ADS)

Rostás, Michael; Ruf, Daniel; Zabka, Vanessa; Hildebrandt, Ulrich

2008-10-01

The plant surface is the substrate upon which herbivorous insects and natural enemies meet and thus represents the stage for interactions between the three trophic levels. Plant surfaces are covered by an epicuticular wax layer which is highly variable depending on species, cultivar or plant part. Differences in wax chemistry may modulate ecological interactions. We explored whether caterpillars of Spodoptera frugiperda, when walking over a plant surface, leave a chemical trail (kairomones) that can be detected by the parasitoid Cotesia marginiventris. Chemistry and micromorphology of cuticular waxes of two barley eceriferum wax mutants ( cer-za.126, cer-yp.949) and wild-type cv. Bonus (wt) were assessed. The plants were then used to investigate potential surface effects on the detectability of caterpillar kairomones. Here we provide evidence that C. marginiventris responds to chemical footprints of its host. Parasitoids were able to detect the kairomone on wild-type plants and on both cer mutants but the response to cer-yp.949 (reduced wax, high aldehyde fraction) was less pronounced. Experiments with caterpillar-treated wt and mutant leaves offered simultaneously, confirmed this observation: no difference in wasp response was found when wt was tested against cer-za.126 (reduced wax, wt-like chemical composition) but wt was significantly more attractive than cer-yp.949. This demonstrates for the first time that the wax layer can modulate the detectability of host kairomones.

Impaired Expression of Uncoupling Protein 2 (UCP2) Causes Defective Post-ischemic Angiogenesis in Mice Deficient in AMP-activated Protein Kinase α Subunits

PubMed Central

Xu, Ming-Jiang; Song, Ping; Shirwany, Najeeb; Liang, Bin; Xing, Junjie; Viollet, Benoit; Wang, Xian; Zhu, Yi; Zou, Ming-Hui

2011-01-01

Objective The aim of the present study was to determine whether mitochondrial uncoupling protein (UCP)-2 is required for AMPK-dependent angiogenesis in ischemia in vivo. Methods and Results Angiogenesis was assayed by monitoring endothelial tube formation (a surrogate for angiogenesis) in human umbilical vein endothelial cells (HUVECs), isolated mouse aortic endothelial cells (MAECs), and pulmomary microvascular endothelial cells (PMECs), or in ischemic thigh adductor muscles from wild-type (WT) mice or mice deficient in either AMPKα1 or AMPKα2. AMPK inhibition with pharmacological inhibitor (compound C) or genetic means (transfection of AMPKα-specific siRNA) significantly lowered the tube formation in HUVECs. Consistently, compared with WT mice, tube formation in MAECs isolated from either AMPKα1−/− or AMPKα2−/− mice, which exhibited oxidative stress and reduced expression of UCP2, were significantly impaired. In addition, adenoviral overexpression of UCP2, but not adenoviruses encoding green florescence protein (GFP), normalized tube formation in MAECs from either AMPKα1−/− or AMPKα2−/− mice. Similarly, supplementation with sodium nitroprusside (SNP), a nitric oxide (NO) donor, restored tube formation. Furthermore, ischemia significantly increased angiogenesis, serine 1177 phosphorylation of endothelial NO synthase (eNOS), and UCP2 in ischemic thigh adductor muscles from WT mice, but not from either AMPKα1−/− or AMPKα2−/− mice. Conclusion We conclude that AMPK-dependent UCP2 expression in endothelial cells promotes angiogenesis in vivo. PMID:21597006

Emergence and maintenance of infectious salmon anaemia virus (ISAV) in Europe: a new hypothesis.

PubMed

Nylund, A; Devold, M; Plarre, H; Isdal, E; Aarseth, M

2003-08-15

The present study describes the use of molecular methods in studying infectious salmon anaemia virus (ISAV), an important pathogen of farmed salmon in Norway, Scotland, the Faeroe Islands, Canada, USA and Chile. The nucleotide sequences of the haemagglutinin gene (HA) from 70 ISAV isolates have been analysed for phylogenetic relationship and the average mutation rate of nucleotide substitutions calculated. The isolates constitute 2 major groups, 1 European and 1 North American group. The isolate from Chile is closely related to the North American isolates. The European isolates can be further divided into 3 separate groups reflecting geographical distribution, time of collection, and transmission connected with farming activity. Based on existing information about infectious salmon anaemia (ISA) and new information emerging from the present study, it is hypothesised that: (1) ISAV is maintained in wild populations of trout and salmon in Europe; (2) it is transmitted between wild hosts mainly during their freshwater spawning phase in rivers; (3) wild salmonids, mainly trout, possibly carry benign wild-type ISAV isolates; (4) a change (mutation) in virulence probably results from deletions of amino acid segments from the highly polymorphic region (HPR) of benign wild-type isolates; (5) ISA emerges in farmed Atlantic salmon when mutated isolates are transmitted from wild salmonids or, following mutation of benign isolates, in farmed salmon after transmission from wild salmonids; (6) farming activity is an important factor in transmission of ISAV between farming sites in addition to transmission of ISAV from wild salmonids to farmed salmon; (7) transmission of ISAV from farmed to wild salmonids probably occurs less frequently than transmission from wild to farmed fish due to lower frequency of susceptible wild individuals; (8) the frequency of new outbreaks of ISA in farmed salmon probably reflects natural variation in the prevalence of ISAV in wild populations of salmonids.

The Usher Syndrome Type IIIB Histidyl-tRNA Synthetase Mutation Confers Temperature Sensitivity.

PubMed

Abbott, Jamie A; Guth, Ethan; Kim, Cindy; Regan, Cathy; Siu, Victoria M; Rupar, C Anthony; Demeler, Borries; Francklyn, Christopher S; Robey-Bond, Susan M

2017-07-18

Histidyl-tRNA synthetase (HARS) is a highly conserved translation factor that plays an essential role in protein synthesis. HARS has been implicated in the human syndromes Charcot-Marie-Tooth (CMT) Type 2W and Type IIIB Usher (USH3B). The USH3B mutation, which encodes a Y454S substitution in HARS, is inherited in an autosomal recessive fashion and associated with childhood deafness, blindness, and episodic hallucinations during acute illness. The biochemical basis of the pathophysiologies linked to USH3B is currently unknown. Here, we present a detailed functional comparison of wild-type (WT) and Y454S HARS enzymes. Kinetic parameters for enzymes and canonical substrates were determined using both steady state and rapid kinetics. Enzyme stability was examined using differential scanning fluorimetry. Finally, enzyme functionality in a primary cell culture was assessed. Our results demonstrate that the Y454S substitution leaves HARS amino acid activation, aminoacylation, and tRNA His binding functions largely intact compared with those of WT HARS, and the mutant enzyme dimerizes like the wild type does. Interestingly, during our investigation, it was revealed that the kinetics of amino acid activation differs from that of the previously characterized bacterial HisRS. Despite the similar kinetics, differential scanning fluorimetry revealed that Y454S is less thermally stable than WT HARS, and cells from Y454S patients grown at elevated temperatures demonstrate diminished levels of protein synthesis compared to those of WT cells. The thermal sensitivity associated with the Y454S mutation represents a biochemical basis for understanding USH3B.

Epigenetic Control of Prostate Cancer Metastasis: Role of Runx2 Phosphorylation

DTIC Science & Technology

2014-04-01

prostate cancer cells. In the third budget year, we achieved the following: a. Generation of retrovirus and lentivirus vectors expressing WT RUNX2 and S301A... retrovirus vectors will be developed that express β-galactosidase (negative control), wild type Runx2, S301A/S319A (non-phosphorylated) or S301E/S310E...constitutively active) Runx2 mutants. As described last year, retrovirus and lentivirus vectors were constructed to stably introduce wild type and mutant

Involvement of the mitochondrial permeability transition pore in chronic ethanol-mediated liver injury in mice

PubMed Central

King, Adrienne L.; Swain, Telisha M.; Mao, Zhengkuan; Udoh, Uduak S.; Oliva, Claudia R.; Betancourt, Angela M.; Griguer, Corrine E.; Crowe, David R.; Lesort, Mathieu

2013-01-01

Chronic ethanol consumption increases sensitivity of the mitochondrial permeability transition (MPT) pore induction in liver. Ca2+ promotes MPT pore opening, and genetic ablation of cyclophilin D (CypD) increases the Ca2+ threshold for the MPT. We used wild-type (WT) and CypD-null (CypD−/−) mice fed a control or an ethanol-containing diet to investigate the role of the MPT in ethanol-mediated liver injury. Ca2+-mediated induction of the MPT and mitochondrial respiration were measured in isolated liver mitochondria. Steatosis was present in WT and CypD−/− mice fed ethanol and accompanied by increased terminal deoxynucleotidyl transferase dUTP-mediated nick-end label-positive nuclei. Autophagy was increased in ethanol-fed WT mice compared with ethanol-fed CypD−/− mice, as reflected by an increase in the ratio of microtubule protein 1 light chain 3B II to microtubule protein 1 light chain 3B I. Higher levels of p62 were measured in CypD−/− than WT mice. Ethanol decreased mitochondrial respiratory control ratios and select complex activities in WT and CypD−/− mice. Ethanol also increased CypD protein in liver of WT mice. Mitochondria from control- and ethanol-fed WT mice were more sensitive to Ca2+-mediated MPT pore induction than mitochondria from their CypD−/− counterparts. Mitochondria from ethanol-fed CypD−/− mice were also more sensitive to Ca2+-induced swelling than mitochondria from control-fed CypD−/− mice but were less sensitive than mitochondria from ethanol-fed WT mice. In summary, CypD deficiency was associated with impaired autophagy and did not prevent ethanol-mediated steatosis. Furthermore, increased MPT sensitivity was observed in mitochondria from ethanol-fed WT and CypD−/− mice. We conclude that chronic ethanol consumption likely lowers the threshold for CypD-regulated and -independent characteristics of the ethanol-mediated MPT pore in liver mitochondria. PMID:24356880

Carriage of Staphylococcus aureus by free-living wild animals in Spain.

PubMed

Porrero, M Concepción; Mentaberre, Gregorio; Sánchez, Sergio; Fernández-Llario, Pedro; Casas-Díaz, Encarna; Mateos, Ana; Vidal, Dolors; Lavín, Santiago; Fernández-Garayzábal, José-Francisco; Domínguez, Lucas

2014-08-01

The presence of methicillin-susceptible Staphylococcus aureus (MSSA) was analyzed in different free-living wild animals to assess the genetic diversity and predominant genotypes on each animal species. Samples were taken from the skin and/or nares, and isolates were characterized by spa typing, multilocus sequence typing (MLST) and antimicrobial susceptibility testing. The proportion of MSSA carriers were 5.00, 22.93, 19.78, and 17.67% in Eurasian griffon vulture, Iberian ibex, red deer, and wild boar, respectively (P = 0.057). A higher proportion of isolates (P = 0.000) were recovered from nasal samples (78.51%) than skin samples (21.49%), but the 9.26% of red deer and 18.25% of wild boar would have been undetected if only nasal samples had been tested. Sixty-three different spa types were identified, including 25 new spa types. The most common were t528 (43.59%) in Iberian ibex, t548 and t11212 (15.79% and 14.04%) in red deer, and t3750 (36.11%) in wild boar. By MLST, 27 STs were detected, of which 12 had not been described previously. The most frequent were ST581 for Iberian ibex (48.72%), ST425 for red deer (29.82%), and ST2328 for wild boar (42.36%). Isolates from Eurasian griffon vulture belong to ST133. Host specificity has been observed for the most frequent spa types and STs (P = 0.000). The highest resistance percentage was found against benzylpenicillin (average, 22.2%), although most of the S. aureus isolates were susceptible to all antimicrobial tested. Basically, MSSA isolates were different from those MRSA isolates previously detected in the same animal species. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Occurrence of Carbapenemase-Producing Enterobacteriaceae Isolates in the Wildlife: First Report of OXA-48 in Wild Boars in Algeria.

PubMed

Bachiri, Taous; Bakour, Sofiane; Lalaoui, Rym; Belkebla, Nadia; Allouache, Meriem; Rolain, Jean Marc; Touati, Abdelaziz

2018-04-01

The aim of the present study was to screen for the presence of carbapenemase-producing Enterobacteriaceae (CPE) isolates from wild boars and Barbary macaques in Algeria. Fecal samples were collected from wild boars (n = 168) and Barbary macaques (n = 212), in Bejaia, Algeria, between September 2014 and April 2016. The isolates were identified and antimicrobial susceptibility was determined. Carbapenem resistance determinants were studied using PCR and sequencing, while clonal relatedness was performed using multilocus sequence typing (MLST). PCR was used to investigate certain virulence genes. Three CPE isolates from three different samples (1.8%) recovered from wild boars were identified as Escherichia coli (two isolates) and Klebsiella pneumoniae (one isolate). These isolates were resistant to amoxicillin, amoxicillin-clavulanate, tobramycin, ertapenem, and meropenem. The results of PCR and sequencing analysis showed that all three isolates produced the OXA-48 enzyme. The MLST showed that the two E. coli isolates were assigned to the same sequence type, ST635, and belonged to phylogroup A, whereas K. pneumoniae strain belonged to ST13. The K. pneumoniae strain was positive for multiple virulence factors, whereas no virulence determinants were found in E. coli isolates. This is the first report of OXA-48-producing Enterobacteriaceae in wild animals from Algeria and Africa.

Influence of fungal endophyte infection on phenolic content and antioxidant activity in grasses: interaction between Lolium perenne and different strains of Neotyphodium lolii.

PubMed

Qawasmeh, Abdelqader; Obied, Hassan K; Raman, Anantanarayanan; Wheatley, Warwick

2012-04-04

Lolium perenne is a major forage and turf grass, which is often naturally infected with a "wild-type" strain (E(WT)) of the fungal endophyte Neotyphodium lolii , establishing a symbiotic relationship. In this study, the impacts of different strains wild type E(WT), AR1 (E(AR1)) and AR37 (E(AR37)), of N. lolii on the phenolic profile, phenolic content, and antioxidant capacity of L. perenne were examined. Samples could be ranked according to their phenol content as follows: E(AR1) > E(AR37) ≥ E(-) > E(WT). Radical-scavenging assays showed the same relative ranking of extracts. Flavonoid glycosides and hydroxycinnamic acids were the most abundant polyphenols in L. perenne extracts. Chlorogenic acid and its derivatives were the major compounds responsible for the antioxidant activity. Infection with N. lolii significantly influenced L. perenne phenolic content and antioxidant activity. In conclusion, changes in phenolic composition were merely quantitative. Endophyte infection can have zero, positive, or negative effect on phenol content depending on the endophyte strain.

A proteomics approach to study synergistic and antagonistic interactions of the fungal-bacterial consortium Fusarium oxysporum wild-type MSA 35.

PubMed

Moretti, Marino; Grunau, Alexander; Minerdi, Daniela; Gehrig, Peter; Roschitzki, Bernd; Eberl, Leo; Garibaldi, Angelo; Gullino, Maria Lodovica; Riedel, Kathrin

2010-09-01

Fusarium oxysporum is an important plant pathogen that causes severe damage of many economically important crop species. Various microorganisms have been shown to inhibit this soil-borne plant pathogen, including non-pathogenic F. oxysporum strains. In this study, F. oxysporum wild-type (WT) MSA 35, a biocontrol multispecies consortium that consists of a fungus and numerous rhizobacteria mainly belonging to gamma-proteobacteria, was analyzed by two complementary metaproteomic approaches (2-DE combined with MALDI-Tof/Tof MS and 1-D PAGE combined with LC-ESI-MS/MS) to identify fungal or bacterial factors potentially involved in antagonistic or synergistic interactions between the consortium members. Moreover, the proteome profiles of F. oxysporum WT MSA 35 and its cured counter-part CU MSA 35 (WT treated with antibiotics) were compared with unravel the bacterial impact on consortium functioning. Our study presents the first proteome mapping of an antagonistic F. oxysporum strain and proposes candidate proteins that might play an important role for the biocontrol activity and the close interrelationship between the fungus and its bacterial partners.

Matrix metalloproteinase 7 restrains Helicobacter pylori-induced gastric inflammation and premalignant lesions in the stomach by altering macrophage polarization.

PubMed

Krakowiak, M S; Noto, J M; Piazuelo, M B; Hardbower, D M; Romero-Gallo, J; Delgado, A; Chaturvedi, R; Correa, P; Wilson, K T; Peek, R M

2015-04-02

Helicobacter pylori is the strongest risk factor for the development of gastric cancer. Although the specific mechanisms by which this pathogen induces carcinogenesis have not been fully elucidated, high-expression interleukin (IL)-1β alleles are associated with increased gastric cancer risk among H. pylori-infected persons. In addition, loss of matrix metalloproteinase 7 (MMP7) increases mucosal inflammation in mouse models of epithelial injury, and we have shown that gastric inflammation is increased in H. pylori-infected MMP7(-/-) C57BL/6 mice. In this report, we define mechanisms that underpin such responses and extend these results into a genetic model of MMP7 deficiency and gastric cancer. Wild-type (WT) or MMP7(-/-) C57BL/6 mice were challenged with broth alone as an uninfected control or the H. pylori strain PMSS1. All H. pylori-challenged mice were successfully colonized. As expected, H. pylori-infected MMP7(-/-) C57BL/6 mice exhibited a significant increase in gastric inflammation compared with uninfected or infected WT C57BL/6 animals. Loss of MMP7 resulted in M1 macrophage polarization within H. pylori-infected stomachs, as assessed by Luminex technology and immunohistochemistry, and macrophages isolated from infected MMP7-deficient mice expressed significantly higher levels of the M1 macrophage marker IL-1β compared with macrophages isolated from WT mice. To extend these findings into a model of gastric cancer, hypergastrinemic WT INS-GAS or MMP7(-/-) INS-GAS mice were challenged with H. pylori strain PMSS1. Consistent with findings in the C57BL/6 model, H. pylori-infected MMP7-deficient INS-GAS mice exhibited a significant increase in gastric inflammation compared with either uninfected or infected WT INS-GAS mice. In addition, the incidence of gastric hyperplasia and dysplasia was significantly increased in H. pylori-infected MMP7(-/-) INS-GAS mice compared with infected WT INS-GAS mice, and loss of MMP7 promoted M1 macrophage polarization. These results suggest that MMP7 exerts a restrictive role on H. pylori-induced gastric injury and the development of premalignant lesions by suppressing M1 macrophage polarization.

Cysteine Inhibits Mercury Methylation by Geobacter sulfurreducens PCA Mutant Δ omcBESTZ

DOE PAGES

Lin, Hui; Lu, Xia; Liang, Liyuan; ...

2015-04-21

For cysteine enhances Hg uptake and methylation by Geobacter sulfurreducens PCA wild type (WT) strain in short-term assays. The prevalence of this enhancement in other strains remains poorly understood. We examined the influence of cysteine concentration on time-dependent Hg(II) reduction, sorption and methylation by PCA-WT and its c-type cytochrome-deficient mutant ( omcBESTZ) in phosphate buffered saline. Without cysteine, the mutant methylated twice as much Hg(II) as the PCA-WT, whereas addition of cysteine inhibited Hg methylation, regardless of the reaction time. PCA-WT, but, exhibited both time-dependent and cysteine concentration-dependent methylation. In 144 hour assay, nearly complete sorption of the Hg(II) bymore » PCA-WT occurred in the presence of 1 mM cysteine, resulting in our highest observed methylmercury production. Moreover, the chemical speciation modeling and experimental data suggest that uncharged Hg(II) species are more readily taken up, and that this uptake is kinetic limiting, thereby affecting Hg methylation by both mutant and WT.« less

The Mukundpura meteorite, a new fall of CM chondrite

NASA Astrophysics Data System (ADS)

Ray, Dwijesh; Shukla, Anil D.

2018-02-01

Mukundpura is a new CM chondrite fell near Jaipur, Rajasthan, India on June 6, 2017 at 5:15 IST. The fall was observed by local villager. According to eyewitness, the meteorite was fragmented into several pieces once the object hit the ground. Based on petrography, mineralogy and bulk composition, Mukundpura is classified as CM2 chondrite. The chondrules are mainly similar to type I (Olivine: Fo99). Olivines are often found associated with pyroxene (Wo10-35En62-87Fs2-7) phenocryst. However, occurrences of forsteritic and fayalitic olivine (Fa58-71) as isolated mineral clast in matrix are not uncommon. Other types of chondrules include porphyritic pyroxene (En86Fs14) and barred olivine (Fa32.7±0.3) clast. Chondrules are commonly rimmed by fine-grained accretionary dust mantles. Phyllosilicates are the most dominant secondary mineral in matrix and largely associated with poorly characterised phases (PCP). FeO/SiO2 and S/SiO2 of PCP are 2.7 and 0.4 respectively. Other phases in matrix generally include calcite (pure CaCO3), Fe-Ni metal and sulphides. Spinel and perovskite occur occasionally as inclusions. The spherical or elliptical shaped metals (within chondrule or in isolated grains) are low-Ni type (kamacite <7.5 wt%) and resembles the solar Ni/Co ratio. However, Ni content in metal rarely exceeds 8.5 wt% (up to 23 wt%, taenite). Pyrrhotite (Fe ∼62 wt%; S ∼38 wt%) and pentlandite (Fe ∼31-33 wt%, Ni ∼28-32 wt%, S ∼33 wt%)) are the common sulphides occur as isolated grains within the matrix, however, the former is the most dominant. The bulk chemical composition of Mukundpura is largely similar to other CM type chondrite (e.g. Paris CM). Based on petrography, we infer a modest aqueous alteration stage for Mukundpura while the effect of thermal metamorphism was negligible.

Distinct modes of adventitious rooting in Arabidopsis thaliana.

PubMed

Correa, L da Rocha; Troleis, J; Mastroberti, A A; Mariath, J E A; Fett-Neto, A G

2012-01-01

The literature describes different rooting protocols for Arabidopsis thaliana as models to study adventitious rooting, and results are generally perceived as comparable. However, there is a lack of investigations focusing on the distinct features, advantages and limitations of each method in the study of adventitious rooting with both wild-type (WT) ecotypes and their respective mutants. This investigation was undertaken to evaluate the adventitious rooting process in three different experimental systems, all using A. thaliana, analysing the same rooting parameters after transient exposure to auxin (indole-3-acetic acid) and control conditions: excised leaves, de-rooted plants and etiolated seedlings. The founding tissues and sites of origin of roots differed depending on the system used, whereas all rooting patterns were of the direct type (i.e., without callus formation). None of the systems had an absolute requirement for exogenous auxin, although rooting was enhanced by this phytohormone, with the exception of de-rooted plants, which had adventitious rooting strongly inhibited by exogenous auxin. Root elongation was much favoured in isolated leaves. Auxin-overproducing mutants could not be used in the detached leaf system due to precocious senescence; in the de-rooted plant system, these mutants had a WT-like rooting response, whereas the expression of the 'rooty' phenotype was only evident in the etiolated seedling system. Adventitious rooting of etiolated WT seedlings in the presence of exogenous auxin was inhibited by exogenous flavonoids, which act as auxin transport inhibitors; surprisingly, the flavonoid-deficient mutant chs had a lower rooting response compared to WT. Although Arabidopsis is an excellent model system to study adventitious rooting, physiological and developmental responses differed significantly, underlining the importance of avoiding data generalisation on rooting responses derived from different experimental systems with this species. © 2011 German Botanical Society and The Royal Botanical Society of the Netherlands.

Secreted protein acidic and rich in cysteine functions in colitis via IL17A regulation in mucosal CD4+ T cells.

PubMed

Tanaka, Makoto; Takagi, Tomohisa; Naito, Yuji; Uchiyama, Kazuhiko; Hotta, Yuma; Toyokawa, Yuki; Ushiroda, Chihiro; Hirai, Yasuko; Aoi, Wataru; Higashimura, Yasuki; Mizushima, Katsura; Okayama, Tetsuya; Katada, Kazuhiro; Kamada, Kazuhiro; Ishikawa, Takeshi; Handa, Osamu; Itoh, Yoshito

2018-03-01

Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycol that regulates cell proliferation, tissue repair, and tumorigenesis. Despite evidence linking SPARC to inflammation, the mechanisms are unclear. Accordingly, the role of SPARC in intestinal inflammation was investigated. Colitis was induced in wild-type (WT) and SPARC knockout (KO) mice using trinitrobenzene sulfonic acid (TNBS). Colons were assessed for damage; leukocyte infiltration; Tnf, Ifng, Il17a, and Il10 mRNA expression; and histology. Cytokine profiling of colonic lamina propria mononuclear cells (LPMCs) was performed by flow cytometry. Naïve CD4 + T cells were isolated from WT and SPARC KO mouse spleens, and the effect of SPARC on Th17 cell differentiation was examined. Recombination activating gene 1 knockout (RAG1 KO) mice reconstituted with T cells from either WT or SPARC KO mice were investigated. Trinitrobenzene sulfonic acid exposure significantly reduced bodyweight and increased mucosal inflammation, leukocyte infiltration, and Il17a mRNA expression in WT relative to SPARC KO mice. The percentage of IL17A-producing CD4 + T cells among LPMCs from KO mice was lower than that in WT mice when both groups were exposed to TNBS. Th17 cell differentiation was suppressed in cells from SPARC KO mice. In the T cell transfer colitis model, RAG1 KO mice receiving T cells from WT mice were more severely affected than those reconstituted with cells from SPARC KO mice. Secreted protein acidic and rich in cysteine accelerates colonic mucosal inflammation via modulation of IL17A-producing CD4 + T cells. SPARC is a potential therapeutic target for conditions involving intestinal inflammation. © 2017 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

Insulin-Stimulated Cardiac Glucose Oxidation Is Increased in High-Fat Diet–Induced Obese Mice Lacking Malonyl CoA Decarboxylase

PubMed Central

Ussher, John R.; Koves, Timothy R.; Jaswal, Jagdip S.; Zhang, Liyan; Ilkayeva, Olga; Dyck, Jason R.B.; Muoio, Deborah M.; Lopaschuk, Gary D.

2009-01-01

OBJECTIVE Whereas an impaired ability to oxidize fatty acids is thought to contribute to intracellular lipid accumulation, insulin resistance, and cardiac dysfunction, high rates of fatty acid oxidation could also impair glucose metabolism and function. We therefore determined the effects of diet-induced obesity (DIO) in wild-type (WT) mice and mice deficient for malonyl CoA decarboxylase (MCD−/−; an enzyme promoting mitochondrial fatty acid oxidation) on insulin-sensitive cardiac glucose oxidation. RESEARCH DESIGN AND METHODS WT and MCD−/− mice were fed a low- or high-fat diet for 12 weeks, and intramyocardial lipid metabolite accumulation was assessed. A parallel feeding study was performed to assess myocardial function and energy metabolism (nanomoles per gram of dry weight per minute) in isolated working hearts (+/– insulin). RESULTS DIO markedly reduced insulin-stimulated glucose oxidation compared with low fat–fed WT mice (167 ± 31 vs. 734 ± 125; P < 0.05). MCD−/− mice subjected to DIO displayed a more robust insulin-stimulated glucose oxidation (554 ± 82 vs. 167 ± 31; P < 0.05) and less incomplete fatty acid oxidation, evidenced by a decrease in long-chain acylcarnitines compared with WT counterparts. MCD−/− mice had long-chain acyl CoAs similar to those of WT mice subjected to DIO but had increased triacylglycerol levels (10.92 ± 3.72 vs. 3.29 ± 0.62 μmol/g wet wt; P < 0.05). CONCLUSIONS DIO does not impair cardiac fatty acid oxidation or function, and there exists disassociation between myocardial lipid accumulation and insulin sensitivity. Our results suggest that MCD deficiency is not detrimental to the heart in obesity. PMID:19478144

Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Aggregation Causes Mitochondrial Dysfunction during Oxidative Stress-induced Cell Death*

PubMed Central

Itakura, Masanori; Kubo, Takeya; Kaneshige, Akihiro; Harada, Naoki; Izawa, Takeshi; Azuma, Yasu-Taka; Kuwamura, Mitsuru; Yamaji, Ryouichi; Takeuchi, Tadayoshi

2017-01-01

Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein that also mediates cell death under oxidative stress. We reported previously that the active-site cysteine (Cys-152) of GAPDH plays an essential role in oxidative stress-induced aggregation of GAPDH associated with cell death, and a C152A-GAPDH mutant rescues nitric oxide (NO)-induced cell death by interfering with the aggregation of wild type (WT)-GAPDH. However, the detailed mechanism underlying GAPDH aggregate-induced cell death remains elusive. Here we report that NO-induced GAPDH aggregation specifically causes mitochondrial dysfunction. First, we observed a correlation between NO-induced GAPDH aggregation and mitochondrial dysfunction, when GAPDH aggregation occurred at mitochondria in SH-SY5Y cells. In isolated mitochondria, aggregates of WT-GAPDH directly induced mitochondrial swelling and depolarization, whereas mixtures containing aggregates of C152A-GAPDH reduced mitochondrial dysfunction. Additionally, treatment with cyclosporin A improved WT-GAPDH aggregate-induced swelling and depolarization. In doxycycline-inducible SH-SY5Y cells, overexpression of WT-GAPDH augmented NO-induced mitochondrial dysfunction and increased mitochondrial GAPDH aggregation, whereas induced overexpression of C152A-GAPDH significantly suppressed mitochondrial impairment. Further, NO-induced cytochrome c release into the cytosol and nuclear translocation of apoptosis-inducing factor from mitochondria were both augmented in cells overexpressing WT-GAPDH but ameliorated in C152A-GAPDH-overexpressing cells. Interestingly, GAPDH aggregates induced necrotic cell death via a permeability transition pore (PTP) opening. The expression of either WT- or C152A-GAPDH did not affect other cell death pathways associated with protein aggregation, such as proteasome inhibition, gene expression induced by endoplasmic reticulum stress, or autophagy. Collectively, these results suggest that NO-induced GAPDH aggregation specifically induces mitochondrial dysfunction via PTP opening, leading to cell death. PMID:28167533

Tumour gene expression predicts response to cetuximab in patients with KRAS wild-type metastatic colorectal cancer.

PubMed

Baker, J B; Dutta, D; Watson, D; Maddala, T; Munneke, B M; Shak, S; Rowinsky, E K; Xu, L-A; Harbison, C T; Clark, E A; Mauro, D J; Khambata-Ford, S

2011-02-01

Although it is accepted that metastatic colorectal cancers (mCRCs) that carry activating mutations in KRAS are unresponsive to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies, a significant fraction of KRAS wild-type (wt) mCRCs are also unresponsive to anti-EGFR therapy. Genes encoding EGFR ligands amphiregulin (AREG) and epiregulin (EREG) are promising gene expression-based markers but have not been incorporated into a test to dichotomise KRAS wt mCRC patients with respect to sensitivity to anti-EGFR treatment. We used RT-PCR to test 110 candidate gene expression markers in primary tumours from 144 KRAS wt mCRC patients who received monotherapy with the anti-EGFR antibody cetuximab. Results were correlated with multiple clinical endpoints: disease control, objective response, and progression-free survival (PFS). Expression of many of the tested candidate genes, including EREG and AREG, strongly associate with all clinical endpoints. Using multivariate analysis with two-layer five-fold cross-validation, we constructed a four-gene predictive classifier. Strikingly, patients below the classifier cutpoint had PFS and disease control rates similar to those of patients with KRAS mutant mCRC. Gene expression appears to identify KRAS wt mCRC patients who receive little benefit from cetuximab. It will be important to test this model in an independent validation study.

Pasteurella multocida isolated from wild birds of North America: a serotype and DNA fingerprint study of isolates from 1978 to 1993

USGS Publications Warehouse

Wilson, M.A.; Duncan, R.M.; Nordholm, G.E.; Berlowski, B.M.

1995-01-01

Serotype and DNA fingerprint methods were used to study Pasteurella multocida isolated from 320 wild birds of North America. Isolates were collected during 1978-93. The HhaI profiles of 314 isolates matched the HhaI profile of somatic reference type 1, strain X-73; somatic type 1 antigen was expressed by 310 isolates, and the serotype of four isolates was undetected. Differentiation of the 314 isolates was observed by digestion of DNA with HpaII. None of the HpaII profiles matched the HpaII profile of X-73 (designated HhaI 001/HpaII 001). Three HpaII profiles were recognized among the somatic type 1 isolates: HpaII 002 (n = 18), HpaII 003 (n = 122), and HpaII 004 (n = 174). Profile HpaII 002 was found among isolates collected during 1979-83. Profile HpaII 003 was identified from isolates collected during 1979-89, with the exception of two isolates in 1992. The HpaII 004 profile was identified from isolates collected during 1983-93. Of the six remaining isolates, four expressed somatic type 4 and had HhaI profiles identical to the somatic type 4 reference strain P-1662 profile (designated HhaI 004); these isolates were differentiated by digestion of DNA with HpaII. One isolate was identified as serotype F:11, and another was serotype A:3,4. In the present study, 314 of 316 (99.4%) isolates from wild birds in the Central, Mississippi, and Pacific flyways during 1978-93, were P. multocida somatic type 1.

Pre-S2 Start Codon Mutation of Hepatitis B Virus Subgenotype B3 Effects on NF-κB Expression and Activation in Huh7 Cell Lines.

PubMed

Siburian, Marlinang Diarta; Suriapranata, Ivet Marita; Wanandi, Septelia Inawati

2018-03-19

A cross-sectional study on hepatitis B patients in Indonesia showed association of pre-S2 start codon mutation (M120 V) with cirrhosis and hepatocellular carcinoma (HCC), which was dissimilar from studies from other populations where pre-S2 deletion mutation was more prevalent. Different mutation patterns were attributed to different hepatitis B virus (HBV) subgenotypes in each population study. HBV surface proteins are reported to induce the activation of NF-κB, a transcriptional factor known to play an important role in the development of liver disease. This study aimed to see the effects of HBs variants in HBV subgenotype B3 on the expression and activation of NF-κB as one of the mechanisms in inducing advanced liver disease. HBV subgenotypes B3, each carrying wild-type (wt) HBs, M120 V, and pre-S2 deletion mutation were isolated from three HCC patients. HBs genes were amplified and cloned into pcDNA3.1 and were transfected using Lipofectamine into a Huh7 cell line. NF-κB activation was measured through IκB-α expression, which is regulated by NF-κB. RNA expressions for HBs, IκB-α, and NF-κB subunit (p50) were evaluated using real-time PCR. M120 V mutant had a significantly higher mRNA level compared with wt and pre-S2 deletion mutant; however, there were no significant differences in HBs protein expressions. The transcription level of p50 was higher in M120 V mutation compared with HBs wild-type and pre-S2 deletion mutant. NF-κB activation was higher in HBs wild-type compared with the two mutant variants. Pre-S2 mutations had no effect on the increment of NF-κB activation. However, M120 V mutation may utilize a different pathway in liver disease progression that involves high expression of NF-κB subunit, p50.

MOXI Is a Mitochondrial Micropeptide That Enhances Fatty Acid β-Oxidation.

PubMed

Makarewich, Catherine A; Baskin, Kedryn K; Munir, Amir Z; Bezprozvannaya, Svetlana; Sharma, Gaurav; Khemtong, Chalermchai; Shah, Akansha M; McAnally, John R; Malloy, Craig R; Szweda, Luke I; Bassel-Duby, Rhonda; Olson, Eric N

2018-06-26

Micropeptide regulator of β-oxidation (MOXI) is a conserved muscle-enriched protein encoded by an RNA transcript misannotated as non-coding. MOXI localizes to the inner mitochondrial membrane where it associates with the mitochondrial trifunctional protein, an enzyme complex that plays a critical role in fatty acid β-oxidation. Isolated heart and skeletal muscle mitochondria from MOXI knockout mice exhibit a diminished ability to metabolize fatty acids, while transgenic MOXI overexpression leads to enhanced β-oxidation. Additionally, hearts from MOXI knockout mice preferentially oxidize carbohydrates over fatty acids in an isolated perfused heart system compared to wild-type (WT) animals. MOXI knockout mice also exhibit a profound reduction in exercise capacity, highlighting the role of MOXI in metabolic control. The functional characterization of MOXI provides insight into the regulation of mitochondrial metabolism and energy homeostasis and underscores the regulatory potential of additional micropeptides that have yet to be identified. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Genetic diversity and antibiotic susceptibility of Staphylococcus aureus isolates from wild boars.

PubMed

Seinige, D; Von Altrock, A; Kehrenberg, C

2017-10-01

We here report the occurrence of S. aureus in wild boars and characterize isolates genotypically and phenotypically in order to get knowledge about the occurrence of clonal lineages and genotypes in free-living wild animals. Forty-one S. aureus isolates obtained from 111 wild boars hunted in Lower Saxony, Germany, were investigated and compared to human and livestock isolates. The S. aureus belonged to multilocus sequence types ST1, ST7, ST30, ST133, ST425, ST804, ST890 and to the new ST3237, ST3238, ST3255 and ST3369. The livestock associated CC398-MRSA lineage, however, was not found. In addition to well-known spa types, the new types t14999, t15000, t15001 and t15002 were detected. Macrorestriction analysis revealed a variety of different SmaI fragment patterns. Most isolates were susceptible to all antimicrobials tested, including methicillin, and resistance was detected only to ampicillin, penicillin and erythromycin. PCR analysis confirmed the presence of staphylococcal enterotoxin genes (seh) in all t127-ST1 isolates. A high degree of genetic diversity was detected with many spa types and clonal lineages previously reported in humans and livestock animals. Copyright © 2017 Elsevier Ltd. All rights reserved.

Method for producing capsular polysaccharides

NASA Technical Reports Server (NTRS)

Richards, Gil F. (Inventor); Kern, Roger G. (Inventor); Petersen, Gene R. (Inventor)

1994-01-01

Structurally altered capsular polysaccharides are produced by mutant bacteria. These polysaccharides are isolated by selecting a wild type bacterial strain and a phage producing degradative enzymes that have substrate specificity for the capsular polysaccharides produced by the wild type bacteria. Phage-resistant mutants producing capsular polysaccharides are selected and the structurally altered capsular polysaccharide is isolated therefrom.

Wild poliovirus circulation among healthy children immunized with oral polio vaccine in Antananarivo, Madagascar.

PubMed

Andrianarivelo, M R; Rabarijaona, L; Boisier, P; Chezzi, C; Zeller, H G

1999-01-01

From July 1995 to December 1996, 3185 stool specimens from healthy children aged 6-59 months attending 6 dispensaries in the Antananarivo area were examined for poliovirus. The children had been routinely immunized according to the Expanded Programme on Immunization (EPI) schedule and received the last dose of oral polio vaccine (OPV) more than 1 month before stool collection. 99.4% of the children were immunized with at least 3 doses of OPV. HEp-2 cell culture revealed virus infections in 192 stools (6.0%), including 9 poliovirus (0.3%) and 183 nonpolio enterovirus isolates (5.7%). Infections occurred throughout the year, but incidence was higher during the hot and rainy season (P=0.01). Using a neutralization test with monoclonal antibodies and PCR-RFLP in two genomic regions coding for the VP1 capsid and RNA polymerase, 4 wild polioviruses (3 type 1 and 1 type 3) and 5 vaccine-related polioviruses (2 Sabin 1-like variants, 1 Sabin 2-like and 2 Sabin 3-like) strains were identified. The wild polioviruses were isolated at the beginning and the end of the dry season. Similar RFLP patterns were observed for the 3 wild type 1 polioviruses. Comparison of partial genomic sequences in the VP1/2 A region of 1 of the wild type 1 isolates with 2 wild type strains isolated in Antananarivo in 1992 and 1993 showed a divergence of at least 10% between the strains, suggesting at least two different pathways of transmission during this period. Our findings demonstrate that immunization with 3 doses of OPV did not prevent intestinal carriage of wild poliovirus strains, and that there is a risk of wild poliovirus transmission to susceptible children in the area. Multiple strategies are required to improve immunization coverage in Madagascar.

Altered methanol embryopathies in embryo culture with mutant catalase-deficient mice and transgenic mice expressing human catalase

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

Miller, Lutfiya; Wells, Peter G., E-mail: pg.wells@utoronto.ca; Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON

2011-04-01

The mechanisms underlying the teratogenicity of methanol (MeOH) in rodents, unlike its acute toxicity in humans, are unclear, but may involve reactive oxygen species (ROS). Embryonic catalase, although expressed at about 5% of maternal activity, may protect the embryo by detoxifying ROS. This hypothesis was investigated in whole embryo culture to remove confounding maternal factors, including metabolism of MeOH by maternal catalase. C57BL/6 (C57) mouse embryos expressing human catalase (hCat) or their wild-type (C57 WT) controls, and C3Ga.Cg-Catb/J acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug = GD 1),more » exposed for 24 h to 4 mg/ml MeOH or vehicle, and evaluated for functional and morphological changes. hCat and C57 WT vehicle-exposed embryos developed normally. MeOH was embryopathic in C57 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed and turning, whereas hCat embryos were protected. Vehicle-exposed aCat mouse embryos had lower yolk sac diameters compared to C3H WT controls, suggesting that endogenous ROS are embryopathic. MeOH was more embryopathic in aCat embryos than WT controls, with reduced anterior neuropore closure and head length only in catalase-deficient embryos. These data suggest that ROS may be involved in the embryopathic mechanism of methanol, and that embryonic catalase activity may be a determinant of teratological risk.« less

Arginase-II Promotes Tumor Necrosis Factor-α Release From Pancreatic Acinar Cells Causing β-Cell Apoptosis in Aging.

PubMed

Xiong, Yuyan; Yepuri, Gautham; Necetin, Sevil; Montani, Jean-Pierre; Ming, Xiu-Fen; Yang, Zhihong

2017-06-01

Aging is associated with glucose intolerance. Arginase-II (Arg-II), the type-II L -arginine-ureahydrolase, is highly expressed in pancreas. However, its role in regulation of pancreatic β-cell function is not known. Here we show that female (not male) mice deficient in Arg-II (Arg-II -/- ) are protected from age-associated glucose intolerance and reveal greater glucose induced-insulin release, larger islet size and β-cell mass, and more proliferative and less apoptotic β-cells compared with the age-matched wild-type (WT) controls. Moreover, Arg-II is mainly expressed in acinar cells and is upregulated with aging, which enhances p38 mitogen-activated protein kinase (p38 MAPK) activation and release of tumor necrosis factor-α (TNF-α). Accordingly, conditioned medium of isolated acinar cells from old WT (not Arg-II -/- ) mice contains higher TNF-α levels than the young mice and stimulates β-cell apoptosis and dysfunction, which are prevented by a neutralizing anti-TNF-α antibody. In acinar cells, our study demonstrates an age-associated Arg-II upregulation, which promotes TNF-α release through p38 MAPK leading to β-cell apoptosis, insufficient insulin secretion, and glucose intolerance in female rather than male mice. © 2017 by the American Diabetes Association.

Protection against Oxygen-Glucose Deprivation/Reperfusion Injury in Cortical Neurons by Combining Omega-3 Polyunsaturated Acid with Lyciumbarbarum Polysaccharide.

PubMed

Shi, Zhe; Wu, Di; Yao, Jian-Ping; Yao, Xiaoli; Huang, Zhijian; Li, Peng; Wan, Jian-Bo; He, Chengwei; Su, Huanxing

2016-01-13

Ischemic stroke, characterized by the disturbance of the blood supply to the brain, is a severe worldwide health threat with high mortality and morbidity. However, there is no effective pharmacotherapy for ischemic injury. Currently, combined treatment is highly recommended for this devastating injury. In the present study, we investigated neuroprotective effects of the combination of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) and Lyciumbarbarum polysaccharide (LBP) on cortical neurons using an in vitro ischemic model. Our study demonstrated that treatment with docosahexaenoic acid (DHA), a major component of the ω-3 PUFAs family, significantly inhibited the increase of intracellular Ca(2+) in cultured wild type (WT) cortical neurons subjected to oxygen-glucose deprivation/reperfusion (OGD/R) injury and promoted their survival compared with the vehicle-treated control. The protective effects were further confirmed in cultured neurons with high endogenous ω-3 PUFAs that were isolated from fat-1 mice, in that a higher survival rate was found in fat-1 neurons compared with wild-type neurons after OGD/R injury. Our study also found that treatment with LBP (50 mg/L) activated Trk-B signaling in cortical neurons and significantly attenuated OGD/R-induced cell apoptosis compared with the control. Notably, both combining LBP treatment with ω-3 PUFAs administration to WT neurons and adding LBP to fat-1 neurons showed enhanced effects on protecting cortical neurons against OGD/R injury via concurrently regulating the intracellular calcium overload and neurotrophic pathway. The results of the study suggest that ω-3 PUFAs and LBP are promising candidates for combined pharmacotherapy for ischemic stroke.

Iron dysregulation combined with aging prevents sepsis-induced apoptosis.

PubMed

Javadi, Pardis; Buchman, Timothy G; Stromberg, Paul E; Turnbull, Isaiah R; Vyas, Dinesh; Hotchkiss, Richard S; Karl, Irene E; Coopersmith, Craig M

2005-09-01

Sepsis, iron loading, and aging cause independent increases in gut epithelial and splenic apoptosis. It is unknown how their combination will affect apoptosis and systemic cytokine levels. Hfe-/- mice (a murine homologue of hemochromatosis) abnormally accumulate iron in their tissues. Aged (24-26 months) or mature (16-18 months) Hfe-/- mice and wild type (WT) littermates were subjected to cecal ligation and puncture (CLP) or sham laparotomy. Intestine, spleen, and blood were harvested 24 h later and assessed for apoptosis and cytokine levels. Gut epithelial and splenic apoptosis were low in both aged septic and sham Hfe-/- mice, regardless of the amount of iron in their diet. Mature septic WT mice had increased apoptosis compared to age-matched sham WT mice. Mature septic Hfe-/- mice had similar levels of intestinal cell death to age-matched septic WT mice but higher levels of splenic apoptosis. Apoptosis was significantly lower in septic aged Hfe-/- mice than septic mature Hfe-/- animals. Interleukin-6 was elevated in septic aged Hfe-/- mice compared to sham mice. Although sepsis, chronic iron dysregulation, and aging each increase gut and splenic apoptosis, their combination yields cell death levels similar to sham animals despite the fact that aged Hfe-/- mice are able to mount an inflammatory response following CLP and mature Hfe-/- mice have elevated sepsis-induced apoptosis. Combining sepsis with two risk factors that ordinarily increase cell death and increase mortality in CLP yields an apoptotic response that could not have been predicted based upon each element in isolation.

Normal development of mice lacking PAXX, the paralogue of XRCC4 and XLF.

PubMed

Gago-Fuentes, Raquel; Xing, Mengtan; Sæterstad, Siri; Sarno, Antonio; Dewan, Alisa; Beck, Carole; Bradamante, Stefano; Bjørås, Magnar; Oksenych, Valentyn

2018-03-01

DNA repair consists of several cellular pathways which recognize and repair damaged DNA. The classical nonhomologous DNA end-joining (NHEJ) pathway repairs double-strand breaks in DNA. It is required for maturation of both B and T lymphocytes by supporting V(D)J recombination as well as B-cell differentiation during class switch recombination (CSR). Inactivation of NHEJ factors Ku70, Ku80, XRCC4, DNA ligase 4, DNA-PKcs, and Artemis impairs V(D)J recombination and blocks lymphocyte development. Paralogue of XRCC4 and XLF (PAXX) is an accessory NHEJ factor that has a significant impact on the repair of DNA lesions induced by ionizing radiation in human, murine, and chicken cells. However, the role of PAXX during development is poorly understood. To determine the physiological role of PAXX, we deleted part of the Paxx promoter and the first two exons in mice. Further, we compared Paxx -knockout mice with wild-type (WT) and NHEJ-deficient controls including Ku80- and Dna-pkcs -null and severe combined immunodeficiency mice. Surprisingly, Paxx -deficient mice were not distinguishable from the WT littermates; they were the same weight and size, fertility status, had normal spleen, thymus, and bone marrow. Paxx -deficient mice had the same number of chromosomal and chromatid breaks as WT mice. Moreover, Paxx -deficient primary B lymphocytes had the same level of CSR as lymphocytes isolated from WT mice. We concluded that PAXX is dispensable for normal mouse development.

Overexpression of Heparin-Binding Epidermal Growth Factor-Like Growth Factor Mediates Liver Fibrosis in Transgenic Mice.

PubMed

Guo, Yongze; Ding, Qian; Chen, Lei; Ji, Chenguang; Hao, Huiyao; Wang, Jia; Qi, Wei; Xie, Xiaoli; Ma, Junji; Li, Aidi; Jiang, Xiaoyu; Li, Xiaotian; Jiang, Huiqing

2017-08-01

The role of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in liver fibrosis is not clear and is sometimes even contradictory. To clarify this role, a HB-EGF transgenic (Tg) mouse model was, for the first time, used to evaluate the functions of HB-EGF in liver fibrosis. For the in vivo study, carbon tetrachloride injection and bile duct ligation treatment were used to induce liver fibrosis in HB-EGF Tg mice and wild-type (WT) mice, respectively. Primary hepatic satellite cells (HSCs) were isolated from HB-EGF Tg and WT mice for the in vitro study. Compared with the WT mice, HB-EGF Tg mice were shown to develop more severe liver fibrosis when treated with carbon tetrachloride or bile duct ligation, with increased matrix metalloproteinases 13 activity and enhanced expression of fibrogenic genes including α-smooth muscle actin and collagen I. HB-EGF gene transfer led to an increase in proliferation and a decrease in apoptosis in primary HSCs. The ERK signaling pathway was more highly activated in primary HSCs from HB-EGF Tg mice than in those from WT mice. Our investigation confirmed the profibrotic effect of HB-EGF on the liver using a Tg mouse model. This result may contribute to the elucidation of HB-EGF as a therapeutic target in liver fibrosis. Copyright © 2017 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.

DoGMP1 from Dendrobium officinale contributes to mannose content of water-soluble polysaccharides and plays a role in salt stress response

PubMed Central

He, Chunmei; Yu, Zhenming; Teixeira da Silva, Jaime A.; Zhang, Jianxia; Liu, Xuncheng; Wang, Xiaojuan; Zhang, Xinhua; Zeng, Songjun; Wu, Kunlin; Tan, Jianwen; Ma, Guohua; Luo, Jianping; Duan, Jun

2017-01-01

GDP-mannose pyrophosphorylase (GMP) catalyzed the formation of GDP-mannose, which serves as a donor for the biosynthesis of mannose-containing polysaccharides. In this study, three GMP genes from Dendrobium officinale (i.e., DoGMPs) were cloned and analyzed. The putative 1000 bp upstream regulatory region of these DoGMPs was isolated and cis-elements were identified, which indicates their possible role in responses to abiotic stresses. The DoGMP1 protein was shown to be localized in the cytoplasm. To further study the function of the DoGMP1 gene, 35S:DoGMP1 transgenic A. thaliana plants with an enhanced expression level of DoGMP1 were generated. Transgenic plants were indistinguishable from wild-type (WT) plants in tissue culture or in soil. However, the mannose content of the extracted water-soluble polysaccharides increased 67%, 96% and 92% in transgenic lines #1, #2 and #3, respectively more than WT levels. Germination percentage of seeds from transgenic lines was higher than WT seeds and the growth of seedlings from transgenic lines was better than WT seedlings under salinity stress (150 mM NaCl). Our results provide genetic evidence for the involvement of GMP genes in the biosynthesis of mannose-containing polysaccharides and the mediation of GMP genes in the response to salt stress during seed germination and seedling growth. PMID:28176760

A R2R3-MYB transcription factor gene in common wheat (namely TaMYBsm1) involved in enhancement of drought tolerance in transgenic Arabidopsis.

PubMed

Li, Meng-Jun; Qiao, Yu; Li, Ya-Qing; Shi, Zhan-Liang; Zhang, Nan; Bi, Cai-Li; Guo, Jin-Kao

2016-11-01

We isolated the TaMYBsm1 genes, encoding R2R3-type MYB proteins in common wheat, aimed to uncover the possible molecular mechanisms related to drought response. The TaMYBsm1 genes, TaMYBsm1-A, TaMYBsm1-B and TaMYBsm1-D, were isolated and analyzed from the common wheat cultivar Shimai 15. Their expression patterns under PEG 6000 and mannitol were monitored by semi-quantitative RT-PCR and β-glucuronidase (Gus) assay. The function of TaMYBsm1-D under drought stress in transgenic Arabidopsis plants was investigated, and the germination rate, water loss rate, as well as the proline and malondialdehyde (MDA) content were compared with that in wild type (WT) plants. The expression of three downstream genes (DREB2A, P5CS1 and RD29A) in TaMYBsm1-D transgenic plants was analyzed. The R2R3-MYB domains of the MYBsm1 proteins were highly conserved in plants. In addition, the TaMYBsm1 proteins were targeted to the nucleus and contained transcriptional activation domains (TADs). Gus assay and semi-quantitative RT-PCR analysis demonstrated that the TaMYBsm1 genes were up-regulated when the wheat was treated by PEG and mannitol. Compared with WT plants, the germination rates were much higher, but the water loss rates were much lower in TaMYBsm1-D overexpression plants. TaMYBsm1-D transgenic plants showed distinct higher proline contents but a lower MDA content than the WT plants. The three downstream genes were highly expressed in TaMYBsm1-D transgenic plants. We concluded from these results that TaMYBsm1 genes play an important role in plant drought stress tolerance through up-regulation of DREB2A, P5CS1 and RD29A. The increase of proline content and decrease of MDA content may also be involved in the drought response.

Phenotypic and genotypic characteristics of Staphylococcus aureus isolates from zoo and wild animals.

PubMed

Feßler, Andrea T; Thomas, Patricia; Mühldorfer, Kristin; Grobbel, Mirjam; Brombach, Julian; Eichhorn, Inga; Monecke, Stefan; Ehricht, Ralf; Schwarz, Stefan

2018-05-01

Antimicrobial resistance of Staphylococcus aureus is a major problem in human and veterinary medicine. The aim of this study was to characterise S. aureus isolates from wild and zoo animals mainly associated with bacterial infections. In total, 23 S. aureus isolates, including nine from wild animals and 14 from zoo animals, were obtained during routine diagnostics. All isolates were subjected to multilocus sequence typing (MLST), spa typing, macrorestriction analysis with subsequent SmaI pulsed-field gelelectrophoresis (PFGE), antimicrobial susceptibility testing and S. aureus-specific DNA-microarray analysis. Resistant isolates were also tested for their respective resistance genes by PCR. Isolates from zoo animals and wildlife showed a high diversity of MLST types, spa types and PFGE patterns. Nineteen different spa types were identified, including three novel types and 16 main macrorestriction patterns. Only few isolates were resistant to members of four classes of antimicrobial agents and harboured the respective resistance genes (β-lactams [blaZ, mecA, mecC], tetracyclines [tet(K), tet(L)] and chloramphenicol [cat pC221 ]) or mutations (fluoroquinolones). The DNA microarray analysis identified one isolate from a zoo animal harbouring the toxic shock syndrome toxin gene tst1. Moreover, several enterotoxin genes were detected in five S. aureus isolates. All isolates were negative for Panton-Valentine leukocidin (PVL) genes, but the animal-associated leukocidin genes lukM/lukF-P83 were found in three isolates from two animals. Copyright © 2018 Elsevier B.V. All rights reserved.

Coagulase-positive Staphylococcus isolated from wildlife: Identification, molecular characterization and evaluation of resistance profiles with focus on a methicillin-resistant strain.

PubMed

Nowakiewicz, Aneta; Ziółkowska, Grażyna; Zięba, Przemysław; Gnat, Sebastian; Wojtanowicz-Markiewicz, Katarzyna; Trościańczyk, Aleksandra

2016-02-01

The aim of the study was molecular analysis of coagulase-positive isolates of Staphylococcus bacteria obtained from wild animals and evaluation of their resistance to antimicrobial agents. A total of 76 rectal swabs were taken from wild animals. The species of the Staphylococcus isolates was determined by MALDI TOF MS, susceptibility to antimicrobials was evaluated by phenotypic and molecular methods, epidemiological analysis (ADSRRS-fingerprinting) was also carried out. MRSA isolate was typed by MLST and spa-typing. The animals tested, were carriers (n=38) of coagulase-positive Staphylococcus (S. aureus, S. pseudintermedius and S. delphini B). Analyzed isolates were resistant to 1 or 2 antimicrobials, which was confirmed by the presence of genes (blaZ, ermA, ermB, msrA, tetK and tetM). A multi-drug resistant and methicillin-resistant isolate of S. aureus was obtained as well (MRSA, ST8, t1635, PVL-positive and ACME-negative). The ADSRRS-fingerprinting method enabled interspecific and intraspecific differentiation of coagulase-positive Staphylococcus isolates, revealing a certain degree of correlation between the species of the isolate, and the degree of similarity between the isolates. The presence of resistance genes in 13% (5/38) of the isolates obtained from wild animals, including one methicillin-resistant isolate, is relatively small in comparison to the degree of colonization by resistant strains in humans, livestock or pets. Nevertheless, due to the possibility of contact between wild animals, domestic animals and humans, transmission of resistant strains is possible, as suggested by our isolation of a MRSA strain typed as ST8 and specific spa type t1635, which had previously been isolated exclusively from humans. Copyright © 2015 Elsevier Ltd. All rights reserved.

Infiltration of Matrix-Non-producers Weakens the Salmonella Biofilm and Impairs Its Antimicrobial Tolerance and Pathogenicity.

PubMed

Srinandan, Chakravarthy S; Elango, Monalisha; Gnanadhas, Divya P; Chakravortty, Dipshikha

2015-01-01

Bacterial biofilms display a collective lifestyle, wherein the cells secrete extracellular polymeric substances (EPS) that helps in adhesion, aggregation, stability, and to protect the bacteria from antimicrobials. We asked whether the EPS could act as a public good for the biofilm and observed that infiltration of cells that do not produce matrix components weakened the biofilm of Salmonella enterica serovar Typhimurium. EPS production was costly for the producing cells, as indicated by a significant reduction in the fitness of wild type (WT) cells during competitive planktonic growth relative to the non-producers. Infiltration frequency of non-producers in the biofilm showed a concomitant decrease in overall productivity. It was apparent in the confocal images that the non-producing cells benefit from the EPS produced by the Wild Type (WT) to stay in the biofilm. The biofilm containing non-producing cells were more significantly susceptible to sodium hypochlorite and ciprofloxacin treatment than the WT biofilm. Biofilm infiltrated with non-producers delayed the pathogenesis, as tested in a murine model. The cell types were spatially assorted, with non-producers being edged out in the biofilm. However, cellulose was found to act as a barrier to keep the non-producers away from the WT microcolony. Our results show that the infiltration of non-cooperating cell types can substantially weaken the biofilm making it vulnerable to antibacterials and delay their pathogenesis. Cellulose, a component of EPS, was shown to play a pivotal role of acting as the main public good, and to edge-out the non-producers away from the cooperating microcolony.

Isolation, subunit composition and interaction of the NDH-1 complexes from Thermosynechococcus elongatus BP-1.

PubMed

Zhang, Pengpeng; Battchikova, Natalia; Paakkarinen, Virpi; Katoh, Hirokazu; Iwai, Masako; Ikeuchi, Masahiko; Pakrasi, Himadri B; Ogawa, Teruo; Aro, Eva-Mari

2005-09-01

NDH (NADH-quinone oxidoreductase)-1 complexes in cyanobacteria have specific functions in respiration and cyclic electron flow as well as in active CO2 uptake. In order to isolate NDH-1 complexes and to study complex-complex interactions, several strains of Thermosynechococcus elongatus were constructed by adding a His-tag (histidine tag) to different subunits of NDH-1. Two strains with His-tag on CupA and NdhL were successfully used to isolate NDH-1 complexes by one-step Ni2+ column chromatography. BN (blue-native)/SDS/PAGE analysis of the proteins eluted from the Ni2+ column revealed the presence of three complexes with molecular masses of about 450, 300 and 190 kDa, which were identified by MS to be NDH-1L, NDH-1M and NDH-1S respectively, previously found in Synechocystis sp. PCC 6803. A larger complex of about 490 kDa was also isolated from the NdhL-His strain. This complex, designated 'NDH-1MS', was composed of NDH-1M and NDH-1S. NDH-1L complex was recovered from WT (wild-type) cells of T. elongatus by Ni2+ column chromatography. NdhF1 subunit present only in NDH-1L has a sequence of -HHDHHSHH- internally, which appears to have an affinity for the Ni2+ column. NDH-1S or NDH-1M was not recovered from WT cells by chromatography of this kind. The BN/SDS/PAGE analysis of membranes solubilized by a low concentration of detergent indicated the presence of abundant NDH-1MS, but not NDH-1M or NDH-1S. These results clearly demonstrated that NDH-1S is associated with NDH-1M in vivo.

Prolyl Endopeptidase (PREP) is Associated With Male Reproductive Functions and Gamete Physiology in Mice.

PubMed

Dotolo, Raffaele; Kim, Jung Dae; Pariante, Paolo; Minucci, Sergio; Diano, Sabrina

2016-03-01

Prolyl endopeptidase (PREP) is a serine protease which has been implicated in many biological processes, such as the maturation and degradation of peptide hormones and neuropeptides, learning and memory, cell proliferation and differentiation, and glucose metabolism. A small number of reports have also suggested PREP participation in both male and female reproduction-associated processes. In the present work, we examined PREP distribution in male germ cells and studied the effects of its knockdown (Prep(gt/gt)) on testis and sperm in adult mice. The protein is expressed and localized in elongating spermatids and luminal spermatozoa of wild type (wt) mice, as well as Sertoli, Leydig, and peritubular cells. PREP is also expressed in the head and midpiece of epididymal spermatozoa, whereas the remaining tail region shows a weaker signal. Furthermore, testis weight, histology of seminiferous tubules, and epididymal sperm parameters were assessed in wt and Prep(gt/gt) mice: wild type testes have larger average tubule and lumen diameter; in addition, lumenal composition of seminiferous tubules is dissimilar between wt and Prep(gt/gt), as the percentage of spermiated tubules is much higher in wt. Finally, total sperm count, sperm motility, and normal morphology are also higher in wt than in Prep(gt/gt). These results show for the first time that the expression of PREP could be necessary for a correct reproductive function, and suggest that the enzyme may play a role in mouse spermatogenesis and sperm physiology. © 2015 Wiley Periodicals, Inc.

Molecular and Functional Characterization of Wheat ARGOS Genes Influencing Plant Growth and Stress Tolerance

PubMed Central

Zhao, Yue; Tian, Xuejun; Li, Yuanyuan; Zhang, Liyuan; Guan, Panfeng; Kou, Xiaoxia; Wang, Xiaobo; Xin, Mingming; Hu, Zhaorong; Yao, Yingyin; Ni, Zhongfu; Sun, Qixin; Peng, Huiru

2017-01-01

Auxin Regulated Gene involved in Organ Size (ARGOS) is significantly and positively associated with organ size and is involved in abiotic stress responses in plants. However, no studies on wheat ARGOS genes have been reported to date. In the present study, three TaARGOS homoeologous genes were isolated and located on chromosomes 4A, 4B, and 4D of bread wheat, all of which are highly conserved in wheat and its wild relatives. Comparisons of gene expression in different tissues demonstrated that the TaARGOSs were mainly expressed in the stem. Furthermore, the TaARGOS transcripts were significantly induced by drought, salinity, and various phytohormones. Transient expression of the TaARGOS-D protein in wheat protoplasts showed that TaARGOS-D localized to the endoplasmic reticulum. Moreover, overexpression of TaARGOS-D in Arabidopsis resulted in an enhanced germination rate, larger rosette diameter, increased rosette leaf area, and higher silique number than in wild-type (WT) plants. The roles of TaARGOS-D in the control of plant growth were further studied via RNA-seq, and it was found that 105 genes were differentially expressed; most of these genes were involved in ‘developmental processes.’ Interestingly, we also found that overexpression of TaARGOS-D in Arabidopsis improved drought and salinity tolerance and insensitivity to ABA relative to that in WT plants. Taken together, these results demonstrate that the TaARGOSs are involved in seed germination, seedling growth, and abiotic stress tolerance. PMID:28228774

Comparative Transcriptome of Wild Type and Selected Strains of the Microalgae Tisochrysis lutea Provides Insights into the Genetic Basis, Lipid Metabolism and the Life Cycle

PubMed Central

Carrier, Gregory; Garnier, Matthieu; Le Cunff, Loïc; Bougaran, Gaël; Probert, Ian; De Vargas, Colomban; Corre, Erwan; Cadoret, Jean-Paul; Saint-Jean, Bruno

2014-01-01

The applied exploitation of microalgae cultures has to date almost exclusively involved the use of wild type strains, deposited over decades in dedicated culture collections. Concomitantly, the concept of improving algae with selection programs for particular specific purposes is slowly emerging. Studying since a decade an economically and ecologically important haptophyte Tisochrysis lutea (Tiso), we took advantage of the availability of wild type (Tiso-Wt) and selected (Tiso-S2M2) strains to conduct a molecular variations study. This endeavour presented substantial challenges: the genome assembly was not yet available, the life cycle unknown and genetic diversity of Tiso-Wt poorly documented. This study brings the first molecular data in order to set up a selection strategy for that microalgae. Following high-throughput Illumina sequencing, transcriptomes of Tiso-Wt and Tiso-S2M2 were de novo assembled and annotated. Genetic diversity between both strains was analyzed and revealed a clear conservation, while a comparison of transcriptomes allowed identification of polymorphisms resulting from the selection program. Of 34,374 transcripts, 291 were differentially expressed and 165 contained positional polymorphisms (SNP, Indel). We focused on lipid over-accumulation of the Tiso-S2M2 strain and 8 candidate genes were identified by combining analysis of positional polymorphism, differential expression levels, selection signature and by study of putative gene function. Moreover, genetic analysis also suggests the existence of a sexual cycle and genetic recombination in Tisochrysis lutea. PMID:24489800

Mass Spectrometry Analysis of Wild-Type and Knock-in Q140/Q140 Huntington's Disease Mouse Brains Reveals Changes in Glycerophospholipids Including Alterations in Phosphatidic Acid and Lyso-Phosphatidic Acid.

PubMed

Vodicka, Petr; Mo, Shunyan; Tousley, Adelaide; Green, Karin M; Sapp, Ellen; Iuliano, Maria; Sadri-Vakili, Ghazaleh; Shaffer, Scott A; Aronin, Neil; DiFiglia, Marian; Kegel-Gleason, Kimberly B

2015-01-01

Huntington's disease (HD) is a neurodegenerative disease caused by a CAG expansion in the HD gene, which encodes the protein Huntingtin. Huntingtin associates with membranes and can interact directly with glycerophospholipids in membranes. We analyzed glycerophospholipid profiles from brains of 11 month old wild-type (WT) and Q140/Q140 HD knock-in mice to assess potential changes in glycerophospholipid metabolism. Polar lipids from cerebellum, cortex, and striatum were extracted and analyzed by liquid chromatography and negative ion electrospray tandem mass spectrometry analysis (LC-MS/MS). Gene products involved in polar lipid metabolism were studied using western blotting, immuno-electron microscopy and qPCR. Significant changes in numerous species of glycerophosphate (phosphatidic acid, PA) were found in striatum, cerebellum and cortex from Q140/Q140 HD mice compared to WT mice at 11 months. Changes in specific species could also be detected for other glycerophospholipids. Increases in species of lyso-PA (LPA) were measured in striatum of Q140/Q140 HD mice compared to WT. Protein levels for c-terminal binding protein 1 (CtBP1), a regulator of PA biosynthesis, were reduced in striatal synaptosomes from HD mice compared to wild-type at 6 and 12 months. Immunoreactivity for CtBP1 was detected on membranes of synaptic vesicles in striatal axon terminals in the globus pallidus. These novel results identify a potential site of molecular pathology caused by mutant Huntingtin that may impart early changes in HD.

Wild rodents and shrews are natural hosts of Staphylococcus aureus.

PubMed

Mrochen, Daniel M; Schulz, Daniel; Fischer, Stefan; Jeske, Kathrin; El Gohary, Heba; Reil, Daniela; Imholt, Christian; Trübe, Patricia; Suchomel, Josef; Tricaud, Emilie; Jacob, Jens; Heroldová, Marta; Bröker, Barbara M; Strommenger, Birgit; Walther, Birgit; Ulrich, Rainer G; Holtfreter, Silva

2017-09-22

Laboratory mice are the most commonly used animal model for Staphylococcus aureus infection studies. We have previously shown that laboratory mice from global vendors are frequently colonized with S. aureus. Laboratory mice originate from wild house mice. Hence, we investigated whether wild rodents, including house mice, as well as shrews are naturally colonized with S. aureus and whether S. aureus adapts to the wild animal host. 295 animals of ten different species were caught in different locations over four years (2012-2015) in Germany, France and the Czech Republic. 45 animals were positive for S. aureus (15.3%). Three animals were co-colonized with two different isolates, resulting in 48 S. aureus isolates in total. Positive animals were found in Germany and the Czech Republic in each studied year. The S. aureus isolates belonged to ten different spa types, which grouped into six lineages (clonal complex (CC) 49, CC88, CC130, CC1956, sequence type (ST) 890, ST3033). CC49 isolates were most abundant (17/48, 35.4%), followed by CC1956 (14/48, 29.2%) and ST890 (9/48, 18.8%). The wild animal isolates lacked certain properties that are common among human isolates, e.g., a phage-encoded immune evasion cluster, superantigen genes on mobile genetic elements and antibiotic resistance genes, which suggests long-term adaptation to the wild animal host. One CC130 isolate contained the mecC gene, implying wild rodents might be both reservoir and vector for methicillin-resistant S. aureus. In conclusion, we demonstrated that wild rodents and shrews are naturally colonized with S. aureus, and that those S. aureus isolates show signs of host adaptation. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

Structurally altered capsular polysaccharides produced by mutant bacteria

NASA Technical Reports Server (NTRS)

Petersen, Gene R. (Inventor); Kern, Roger G. (Inventor); Richards, Gil F. (Inventor)

1995-01-01

Structurally altered capsular polysaccharides are produced by mutant bacteria. These polysaccharides are isolated by selecting a wild type bacterial strain and a phage producing degradative enzymes that have substrate specificity for the capsular polysaccharides produced by the wild type bacteria. Phage-resistant mutants producing capsular polysaccharides are selected and the structurally altered capsular polysaccharide is isolated therefrom.

Exercise Training Prevents Coronary Endothelial Dysfunction in Type 2 Diabetic Mice.

PubMed

Lee, Sewon; Park, Yoonjung; Zhang, Cuihua

2011-10-01

Type 2 diabetes (T2D) is a leading risk factor for cardiovascular diseases including atherosclerosis and coronary heart disease. Exercise training (ET) is thought to have a beneficial effect on these disorders, but the basis for this effect is not fully understood. Because endothelial dysfunction plays a key role in the pathological events leading to cardiovascular complications in T2D, we hypothesized that the effects of ET will be evidenced by improvements in coronary endothelial function. To test this hypothesis, we assessed the effects of ET on vascular function of diabetic (db/db, Lepr(db)) mice by evaluating endothelial function of isolated coronary arterioles of wild-type (WT) and db/db mice with/without ET. Although dilation of vessels to the endothelial-independent vasodilator, sodium nitroprusside was not different between db/db and WT, dilation to the endothelial-dependent agonist, acetylcholine (ACh), was impaired in db/db compared to WT mice. Vasodilation to ACh was restored in db/db with ET and insulin sensitivity was improved in the db/db after ET. Exercise did not change body weight of db/db, but superoxide dismutase (SOD1 and SOD2) and phosphorylated- eNOS protein (Ser1177) expression in heart tissue was up-regulated whereas tumor necrosis factor-alpha (TNF-α) protein level was decreased by ET. Serum level of interleukin-6 (IL-6) was higher in db/db mice but ET decreased IL-6. This suggests that ET may improve endothelial function by increasing nitric oxide bioavailability as well as decreasing chronic inflammation. We suggest this connection may be the basis for the benefit of ET in T2D.

Morphology of ovaries in laron dwarf mice, with low circulating plasma levels of insulin-like growth factor-1 (IGF-1), and in bovine GH-transgenic mice, with high circulating plasma levels of IGF-1

PubMed Central

2012-01-01

Background It is well known that somatotrophic/insulin signaling affects lifespan in experimental animals, and one of the signs of aging is progressive gonadal dysfunction. Methods To study the effects of insulin-like growth factor-1 (IGF-1) plasma level on ovaries, we analyzed ovaries isolated from 2-year-old growth hormone receptor knockout (GHR-KO) Laron dwarf mice, with low circulating plasma levels of IGF-1, and 6-month-old bovine growth hormone transgenic (bGHTg) mice, with high circulating plasma levels of IGF-1. The ages of the Laron dwarf mutants employed in our studies were selected based on their overall survival (up to ~ 4 years for Laron dwarf mice and ~ 1 year for bGHTg mice). Results Morphological analysis of the ovaries of mice that reached ~50% of their maximal life span revealed a lower biological age for the ovaries isolated from 2-year-old Laron dwarf mice than their normal-lifespan wild type littermates. By contrast, the ovarian morphology of increased in size 6 month old bGHTg mice was generally normal. Conclusion Ovaries isolated from 2-year-old Laron dwarf mice exhibit a lower biological age compared with ovaries from normal WT littermates at the same age. At the same time, no morphological features of accelerated aging were found in 0.5-year-old bGHTg mice compared with ovaries from normal the same age-matched WT littermates. PMID:22747742

Characterization of the Ala62Pro polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression

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

Lee, Seung Heon; Kang, Sukmo; Dong, Mi Sook

2015-06-15

Cytochrome P450 (CYP) 1A1 is a heme-containing enzyme involved in detoxification of hydrophobic pollutants. Its Ala62Pro variant has been identified previously. Ala62 is located in α-helix A of CYP1A1. Residues such as Pro and Gly are α-helix breakers. In this study, the Ala62Pro variant was characterized using heterologous expression. E. coli expressing the Ala62Pro variant, and the purified variant protein, had lower CYP (i.e. holoenzyme) contents than their wild-type (WT) equivalents. The CYP variant from E. coli and mammalian cells exhibited lower 7-ethoxyresorufin O-dealkylation (EROD) and benzo[a]pyrene hydroxylation activities than the WT. Enhanced supplementation of a heme precursor during E.more » coli culture did not increase CYP content in E. coli expressing the variant, but did for the WT. As for Ala62Pro, E. coli expressing an Ala62Gly variant had a lower CYP content than the WT counterpart, but substitution of Ala62 with α-helix-compatible residues such as Ser and Val partially recovered the level of CYP produced. Microsomes from mammalian cells expressing Ala62Pro and Ala62Gly variants exhibited lower EROD activities than those expressing the WT or Ala62Val variant. A region harboring α-helix A has interactions with another region containing heme-interacting residues. Site-directed mutagenesis analyses suggest the importance of interactions between the two regions on holoenzyme expression. Together, these findings suggest that the Ala62Pro substitution leads to changes in protein characteristics and function of CYP1A1 via structural disturbance of the region where the residue is located. - Highlights: • Ala62 is located in α-helix A of the carcinogen-metabolizing enzyme CYP1A1. • Pro acts as an α-helix breaker. • A variant protein of CYP1A1, Ala62Pro, had lower heme content than the wild-type. • The variant of CYP1A1 had lower enzyme activities than the wild-type.« less

Sabin and wild type polioviruses from children who presented with acute flaccid paralysis in Nigeria.

PubMed

Adedeji, A O; Okonko, I O; Adu, F D

2012-09-01

Sensitive poliovirus surveillance to detect vaccine-derived-polioviruses will continue to increase in importance. Isolating and identifying poliovirus strains from children of pediatrics age in Nigeria. A total of 120 fecal samples were randomly collected from children under the age of five who presented with acute flaccid paralysis. Samples were tested by tissue culture technique and further characterized by intratypic differentiation testing using ELISA and PCR methods. The study confirmed the presence of 22(18.3%) enteroviral isolates comprising 19(86.4%) polioviruses and 3(13.6%) non-polio enteroviruses. These 19 polioviruses include: Sabin-type poliovirus-1 (15.8%), poliovirus-2 (10.5%), poliovirus-3 (10.5%) and wild-type poliovirus-1 (63.2%) isolates. It showed that poliovirus infection was higher in children ages 6-11 months (18.9%), females (18.4%), northern states (91.0%) with no vaccination record (75.0%). Wild-type poliovirus-1 was isolated from the stool samples of 12(54.6%) children from northern states and in all age groups except 18-23 months. No significant differences (P >0.05) between poliovirus infection and age (18.9% vs. 17.7%; 81.9% vs. 18.2%) and sex (18.3% vs. 18.4%). There was significant differences (P<0.05) between poliovirus infection and location (91.0% vs. 9.0%) and history of polio vaccination (75.0% vs. 0.0%). No wild-type poliovirus was found in those with complete vaccination. This study further confirms the presence of Sabin and wild-type poliovirus among children in Nigeria. The isolation of Sabin strain of poliovirus is advantageous to the polio eradication program as it is capable of inducing natural immunity in susceptible hosts. Transmission of wild-type poliovirus among children with incomplete vaccination poses a serious threat to polio eradication program in Nigeria. Environmental and serological surveillance with larger sample size are important for monitoring poliovirus circulation in Nigeria.

Potent activity of the HIV-1 maturation inhibitor bevirimat in SCID-hu Thy/Liv mice.

PubMed

Stoddart, Cheryl A; Joshi, Pheroze; Sloan, Barbara; Bare, Jennifer C; Smith, Philip C; Allaway, Graham P; Wild, Carl T; Martin, David E

2007-11-28

The HIV-1 maturation inhibitor, 3-O-(3',3'-dimethylsuccinyl) betulinic acid (bevirimat, PA-457) is a promising drug candidate with 10 nM in vitro antiviral activity against multiple wild-type (WT) and drug-resistant HIV-1 isolates. Bevirimat has a novel mechanism of action, specifically inhibiting cleavage of spacer peptide 1 (SP1) from the C-terminus of capsid which results in defective core condensation. Oral administration of bevirimat to HIV-1-infected SCID-hu Thy/Liv mice reduced viral RNA by >2 log(10) and protected immature and mature T cells from virus-mediated depletion. This activity was observed at plasma concentrations that are achievable in humans after oral dosing, and bevirimat was active up to 3 days after inoculation with both WT HIV-1 and an AZT-resistant HIV-1 clinical isolate. Consistent with its mechanism of action, bevirimat caused a dose-dependent inhibition of capsid-SP1 cleavage in HIV-1-infected human thymocytes obtained from these mice. HIV-1 NL4-3 with an alanine-to-valine substitution at the N-terminus of SP1 (SP1/A1V), which is resistant to bevirimat in vitro, was also resistant to bevirimat treatment in the mice, and SP1/AIV had replication and thymocyte kinetics similar to that of WT NL4-3 with no evidence of fitness impairment in in vivo competition assays. Interestingly, protease inhibitor-resistant HIV-1 with impaired capsid-SP1 cleavage was hypersensitive to bevirimat in vitro with a 50% inhibitory concentration 140 times lower than for WT HIV-1. These results support further clinical development of this first-in-class maturation inhibitor and confirm the usefulness of the SCID-hu Thy/Liv model for evaluation of in vivo antiretroviral efficacy, drug resistance, and viral fitness.

In Vivo Role of TLR2 and MyD88 Signaling in Eliciting Innate Immune Responses in Staphylococcal Endophthalmitis

PubMed Central

Talreja, Deepa; Singh, Pawan Kumar; Kumar, Ashok

2015-01-01

Purpose. The purpose of this study was to investigate the protective mechanisms evoked by TLR2 and MyD88 signaling in bacterial endophthalmitis in vivo. Methods. Endophthalmitis was induced in wild-type (WT), TLR2−/−, MyD88−/−, and Cnlp−/− mice by intravitreal injections of a laboratory strain (RN6390) and two endophthalmitis isolates of Staphylococcus aureus. Disease progression was monitored by assessing corneal and vitreous haze, bacterial burden, and retinal tissue damage. Levels of inflammatory cytokines/chemokines were determined using quantitative RT-PCR (qRT-PCR) and ELISA. Flow cytometry was used to assess neutrophil infiltration. Cathelicidin-related antimicrobial peptide (CRAMP) expression was determined by immunostaining and dot blot. Results. Eyes infected with either laboratory or clinical isolates exhibited higher levels of inflammatory mediators at the early stages of infection (≤24 hours) in WT mice than in TLR2−/− or MyD88−/− mice. However, their levels surpassed that of WT mice at the later stages of infection (>48 hours), coinciding with increased bacterial burden and retinal damage. Both TLR2−/− and MyD88−/− retinas produced reduced levels of CRAMP, and its deficiency (Cnlp−/−) rendered the mice susceptible to increased bacterial burden and retinal tissue damage as early as 1 day post infection. Analyses of inflammatory mediators and neutrophil levels in WT versus Cnlp−/− mice showed a trend similar to that observed in TLR2 and MyD88 KO mice. Furthermore, we observed that even a 10-fold lower infective dose of S. aureus was sufficient to cause endophthalmitis in TLR2−/− and MyD88−/− mice. Conclusions. TLR2 and MyD88 signaling plays an important role in protecting the retina from staphylococcal endophthalmitis by production of the antimicrobial peptide CRAMP. PMID:25678692

Heterogeneity of transverse-axial tubule system in mouse atria: Remodeling in atrial-specific Na+-Ca2+ exchanger knockout mice.

PubMed

Yue, Xin; Zhang, Rui; Kim, Brian; Ma, Aiqun; Philipson, Kenneth D; Goldhaber, Joshua I

2017-07-01

Transverse-axial tubules (TATs) are commonly assumed to be sparse or absent in atrial myocytes from small animals. Atrial myocytes from rats, cats and rabbits lack TATs, which results in a characteristic "V"-shaped Ca release pattern in confocal line-scan recordings due to the delayed rise of Ca in the center of the cell. To examine TAT expression in isolated mouse atrial myocytes, we loaded them with the membrane dye Di-4-ANEPPS to label TATs. We found that >80% of atrial myocytes had identifiable TATs. Atria from male mice had a higher TAT density than female mice, and TAT density correlated with cell width. Using the fluorescent Ca indicator Fluo-4-AM and confocal imaging, we found that wild type (WT) mouse atrial myocytes generate near-synchronous Ca transients, in contrast to the "V"-shaped pattern typically reported in other small animals such as rat. In atrial-specific Na-Ca exchanger (NCX) knockout (KO) mice, which develop sinus node dysfunction and atrial hypertrophy with dilation, we found a substantial loss of atrial TATs in isolated atrial myocytes. There was a greater loss of transverse tubules compared to axial tubules, resulting in a dominance of axial tubules. Consistent with the overall loss of TATs, NCX KO atrial myocytes displayed a "V"-shaped Ca transient with slower and reduced central (CT) Ca release and uptake in comparison to subsarcolemmal (SS) Ca release. We compared chemically detubulated (DT) WT cells to KO, and found similar slowing of CT Ca release and uptake. However, SS Ca transients in the WT DT cells had faster uptake kinetics than KO cells, consistent with the presence of NCX and normal sarcolemmal Ca efflux in the WT DT cells. We conclude that the remodeling of NCX KO atrial myocytes is accompanied by a loss of TATs leading to abnormal Ca release and uptake that could impact atrial contractility and rhythm. Copyright © 2017 Elsevier Ltd. All rights reserved.

Epidemiological Cutoff Values for Fluconazole, Itraconazole, Posaconazole, and Voriconazole for Six Candida Species as Determined by the Colorimetric Sensititre YeastOne Method

PubMed Central

Pemán, Javier; Iñiguez, Carmen; Hervás, David; Lopez-Hontangas, Jose L.; Pina-Vaz, Cidalia; Camarena, Juan J.; Campos-Herrero, Isolina; García-García, Inmaculada; García-Tapia, Ana M.; Guna, Remedios; Merino, Paloma; Pérez del Molino, Luisa; Rubio, Carmen; Suárez, Anabel

2013-01-01

In the absence of clinical breakpoints (CBP), epidemiological cutoff values (ECVs) are useful to separate wild-type (WT) isolates (without mechanisms of resistance) from non-WT isolates (those that can harbor some resistance mechanisms), which is the goal of susceptibility tests. Sensititre YeastOne (SYO) is a widely used method to determine susceptibility of Candida spp. to antifungal agents. The CLSI CBP have been established, but not for the SYO method. The ECVs for four azoles, obtained using MIC distributions determined by the SYO method, were calculated via five methods (three statistical methods and based on the MIC50 and modal MIC). Respectively, the median ECVs (in mg/liter) of the five methods for fluconazole, itraconazole, posaconazole, and voriconazole (in parentheses: the percentage of isolates inhibited by MICs equal to or less than the ECVs; the number of isolates tested) were as follows: 2 (94.4%; 944), 0.5 (96.7%; 942), 0.25 (97.6%; 673), and 0.06 (96.7%; 849) for Candida albicans; 4 (86.1%; 642), 0.5 (99.4%; 642), 0.12 (93.9%; 392), and 0.06 (86.9%; 559) for C. parapsilosis; 8 (94.9%; 175), 1 (93.7%; 175), 2 (93.6%; 125), and 0.25 (90.4%; 167) for C. tropicalis; 128 (98.6%; 212), 4 (95.8%; 212), 4 (96.0%; 173), and 2 (98.5; 205) for C. glabrata; 256 (100%; 53), 1 (98.1%; 53), 1 (100%; 33), and 1 (97.9%; 48) for C. krusei; 4 (89.2%; 93), 0.5 (100%; 93), 0.25 (100%; 33), and 0.06 (87.7%; 73) for C. orthopsilosis. All methods included ≥94% of isolates and yielded similar ECVs (within 1 dilution). These ECVs would be suitable for monitoring emergence of isolates with reduced susceptibility by using the SYO method. PMID:23761155

Inhibition of MCU forces extramitochondrial adaptations governing physiological and pathological stress responses in heart

PubMed Central

Rasmussen, Tyler P.; Wu, Yuejin; Joiner, Mei-ling A.; Koval, Olha M.; Wilson, Nicholas R.; Luczak, Elizabeth D.; Wang, Qinchuan; Chen, Biyi; Gao, Zhan; Zhu, Zhiyong; Wagner, Brett A.; Soto, Jamie; McCormick, Michael L.; Kutschke, William; Weiss, Robert M.; Yu, Liping; Boudreau, Ryan L.; Abel, E. Dale; Zhan, Fenghuang; Spitz, Douglas R.; Buettner, Garry R.; Song, Long-Sheng; Zingman, Leonid V.; Anderson, Mark E.

2015-01-01

Myocardial mitochondrial Ca2+ entry enables physiological stress responses but in excess promotes injury and death. However, tissue-specific in vivo systems for testing the role of mitochondrial Ca2+ are lacking. We developed a mouse model with myocardial delimited transgenic expression of a dominant negative (DN) form of the mitochondrial Ca2+ uniporter (MCU). DN-MCU mice lack MCU-mediated mitochondrial Ca2+ entry in myocardium, but, surprisingly, isolated perfused hearts exhibited higher O2 consumption rates (OCR) and impaired pacing induced mechanical performance compared with wild-type (WT) littermate controls. In contrast, OCR in DN-MCU–permeabilized myocardial fibers or isolated mitochondria in low Ca2+ were not increased compared with WT, suggesting that DN-MCU expression increased OCR by enhanced energetic demands related to extramitochondrial Ca2+ homeostasis. Consistent with this, we found that DN-MCU ventricular cardiomyocytes exhibited elevated cytoplasmic [Ca2+] that was partially reversed by ATP dialysis, suggesting that metabolic defects arising from loss of MCU function impaired physiological intracellular Ca2+ homeostasis. Mitochondrial Ca2+ overload is thought to dissipate the inner mitochondrial membrane potential (ΔΨm) and enhance formation of reactive oxygen species (ROS) as a consequence of ischemia-reperfusion injury. Our data show that DN-MCU hearts had preserved ΔΨm and reduced ROS during ischemia reperfusion but were not protected from myocardial death compared with WT. Taken together, our findings show that chronic myocardial MCU inhibition leads to previously unanticipated compensatory changes that affect cytoplasmic Ca2+ homeostasis, reprogram transcription, increase OCR, reduce performance, and prevent anticipated therapeutic responses to ischemia-reperfusion injury. PMID:26153425

Mesenchymal Stromal Cells Expressing Heme Oxygenase-1 Reverse Pulmonary Hypertension

PubMed Central

Liang, Olin D.; Mitsialis, S. Alex; Chang, Mun Seog; Vergadi, Eleni; Lee, Changjin; Aslam, Muhammad; Fernandez-Gonzalez, Angeles; Liu, Xianlan; Baveja, Rajiv; Kourembanas, Stella

2012-01-01

Pulmonary arterial hypertension (PAH) remains a serious disease, and, while current treatments may prolong and improve quality of life, search for novel and effective therapies is warranted. Using genetically-modified mouse lines, we tested the ability of bone marrow-derived stromal cells (MSCs), to treat chronic hypoxia-induced PAH. Recipient mice were exposed for five weeks to normobaric hypoxia (8%–10% O2), MSC preparations were delivered through jugular vein injection and their effect on PAH was assessed after two additional weeks in hypoxia. Donor MSCs derived from wild-type (WT) mice or Heme Oxygenase-1 (HO-1) null mice (Hmox1KO) conferred partial protection from PAH when transplanted into WT or Hmox1KO recipients, whereas treatment with MSCs isolated from transgenic mice harboring a human HO-1 transgene under the control of surfactant protein C promoter (SHO1 line) reversed established disease in WT recipients. SH01-MSC treatment of Hmox1KO animals, which develop right ventricular (RV) infarction under prolonged hypoxia, resulted in normal RV systolic pressure, significant reduction of RV hypertrophy and prevention of RV infarction. Donor MSCs isolated from a bitransgenic mouse line with doxycycline-inducible, lung-specific expression of HO-1 exhibited similar therapeutic efficacy only upon doxycycline treatment of the recipients. In vitro experiments indicate that potential mechanisms of MSC action include modulation of hypoxia-induced lung inflammation and inhibition of smooth muscle cell proliferation. Cumulative, our results demonstrate that MSCs ameliorate chronic hypoxia – induced PAH and their efficacy is highly augmented by lung-specific HO-1 expression in the transplanted cells, suggesting an interplay between HO-1 dependent and HO-1 independent protective pathways. PMID:20957739

Number of nitrate groups determines reactivity and potency of organic nitrates: a proof of concept study in ALDH-2−/− mice

PubMed Central

Wenzel, P; Hink, U; Oelze, M; Seeling, A; Isse, T; Bruns, K; Steinhoff, L; Brandt, M; Kleschyov, A L; Schulz, E; Lange, K; Weiner, H; Lehmann, J; Lackner, K J; Kawamoto, T; Münzel, T; Daiber, A

2007-01-01

Background and purpose: Mitochondrial aldehyde dehydrogenase (ALDH-2) has been shown to provide a pathway for bioactivation of organic nitrates and to be prone to desensitization in response to highly potent, but not to less potent, nitrates. We therefore sought to support the hypothesis that bioactivation by ALDH-2 critically depends on the number of nitrate groups within the nitrovasodilator. Experimental approach: Nitrates with one (PEMN), two (PEDN; GDN), three (PETriN; glyceryl trinitrate, GTN) and four (pentaerithrityl tetranitrate, PETN) nitrate groups were investigated. Vasodilatory potency was measured in isometric tension studies using isolated aortic segments of wild type (WT) and ALDH-2−/− mice. Activity of the cGMP-dependent kinase-I (reflected by levels of phosphorylated VAsodilator Stimulated Phosphoprotein, P-VASP) was quantified by Western blot analysis, mitochondrial dehydrogenase activity by HPLC. Following incubation of isolated mitochondria with PETN, PETriN-chromophore and PEDN, metabolites were quantified using chemiluminescence nitrogen detection and mass spectrometry. Key results: Compared to WT, vasorelaxation in response to PETN, PETriN and GTN was attenuated about 10fold in ALDH-2−/− mice, identical to WT vessels preincubated with inhibitors of ALDH-2. Reduced vasodilator potency correlated with reduced P-VASP formation and diminished biotransformation of the tetranitrate- and trinitrate-compounds. None of these findings were observed for PEDN, GDN and PEMN. Conclusions and implications: Our results support the crucial role of ALDH-2 in bioactivating highly reactive nitrates like GTN, PETN and PETriN. ALDH-2-mediated relaxation by organic nitrates therefore depends mainly on the number of nitrate groups. Less potent nitrates like PEDN, GDN and PEMN are apparently biotransformed by other pathways. PMID:17220910

Relationship between the melanocortin-1 receptor (MC1R) variant R306ter and physiological responses to mechanical or thermal stimuli in Labrador Retriever dogs.

PubMed

Perez, Tania E; Mealey, Katrina L; Burke, Neal S; Grubb, Tamara L; Court, Michael H; Greene, Stephen A

2017-03-01

Variants in the MC1R gene have been associated with red hair color and sensitivity to pain in humans. The study objective was to determine if a relationship exists between MC1R genotype and physiological thermal or mechanical nociceptive thresholds in Labrador Retriever dogs. Prospective experimental study. Thirty-four Labrador Retriever dogs were included in the study following public requests for volunteers. Owner consent was obtained and owners verified that their dog was apparently not experiencing pain and had not been treated for pain during the previous 14 days. The study was approved by the Institutional Animal Care and Use Committee. Nociceptive thresholds were determined from a mean of three thermal and five mechanical replications using commercially available algometers. Each dog was genotyped for the previously described MC1R variant (R306ter). Data were analyzed using one-way anova with post hoc comparisons using Tukey's test (p < 0.05). Thirteen dogs were homozygous wild-type (WT/WT), nine were heterozygous (WT/R306ter), and eight were homozygous variant (R306ter/R306ter) genotype. Four dogs could not be genotyped. A significant difference (p = 0.04) in mechanical nociceptive thresholds was identified between dogs with the WT/WT genotype (12.1±2.1 N) and those with the WT/R306ter genotype (9.2±2.4 N). A difference in mechanical, but not thermal, nociceptive threshold was observed between wild-type and heterozygous MC1R variants. Differences in nociceptive thresholds between homozygous R306ter variants and other genotypes for MC1R were not observed. Compared with the wild-type MC1R genotype, nociceptive sensitivity to mechanical force in dogs with a single variant R306ter allele may be greater. However, in contrast to the reported association between homozygous MC1R variants (associated with red hair color) and nociception in humans, we found no evidence of a similar relationship in dogs with the homozygous variant genotype. Copyright © 2017 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by Elsevier Ltd. All rights reserved.

Loss of Interleukin 1 Receptor Antagonist Enhances Susceptibility to Ebola Virus Infection.

PubMed

Hill-Batorski, Lindsay; Halfmann, Peter; Marzi, Andrea; Lopes, Tiago J S; Neumann, Gabriele; Feldmann, Heinz; Kawaoka, Yoshihiro

2015-10-01

The current outbreak of Ebola virus (EBOV) infection in West Africa is unprecedented, with nearly 26 000 confirmed cases and >10 000 deaths. Comprehensive data on the pathogenesis of EBOV infection are lacking; however, recent studies suggested that fatal EBOV infections are characterized by dysregulation of the innate immune response and a subsequent cytokine storm. Specifically, several studies suggested that hypersecretion of interleukin 1 receptor antagonist (IL-1Ra) correlates with lethal EBOV infections. To examine the significance of IL-1Ra in EBOV infections, we infected mice that lack the gene encoding IL-1Ra, Il1rn (IL-1RN-KO), and mice with wild-type Il1rn (IL-1RN-WT) with a mouse-adapted EBOV (MA-EBOV). Infected IL-1RN-KO mice lost more weight and had a lower survival rate than IL-1RN-WT mice infected with MA-EBOV. In addition, IL-1RN-KO mice infected with wild-type EBOV, which does not cause lethal infection in adult immunocompetent mice, such as C57BL/6 mice, experienced greater weight loss than IL-1RN-WT mice infected with wild-type EBOV. Further studies revealed that the levels of 6 cytokines in spleens-IL-1α, IL-1β, interleukin 12p40, interleukin 17, granulocyte colony-stimulating factor, and regulated on activation, normal T-cell expressed and secreted-were significantly different between IL-1RN-KO mice and IL-1RN-WT mice infected with MA-EBOV. Collectively, our data suggest that IL-1Ra may have a protective effect upon EBOV infection, likely by damping an overactive proinflammatory immune response. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Nfib hemizygous mice are protected from hyperoxic lung injury and death.

PubMed

Kumar, Vasantha H S; Chaker El Khoury, Joseph; Gronostajski, Richard; Wang, Huamei; Nielsen, Lori; Ryan, Rita M

2017-08-01

Nuclear Factor I ( Nfi) genes encode transcription factors essential for the development of organ systems including the lung. Nfib null mice die at birth with immature lungs. Nfib hemizygous mice have reduced lung maturation with decreased survival. We therefore hypothesized that these mice would be more sensitive to lung injury and would have lower survival to hyperoxia. Adult Nfib hemizygous mice and their wild-type (Wt) littermates were exposed to 100% O 2 for 89, 80, 72 and 66 h for survival studies with lung outcome measurements at 66 h. Nfib hemizygous and Wt controls were also studied in RA at 66 h. Cell counts and cytokines were measured in bronchoalveolar lavage (BAL); lung sections examined by histopathology; lung angiogenic and oxidative stress gene expression assessed by real-time PCR Unexpectedly, Nfib hemizygous mice (0/14-0%) had significantly lower mortality compared to Wt mice (10/22-45%) at 80 h of hyperoxia ( P  < 0.003). LD 50 was 80 h in the Wt group versus 89 h in the hemizygous group. There were no differences in BAL cell counts between the groups. Among the cytokines studied, MIP-2 was significantly lower in hemizygous mice exposed to hyperoxia. New vessel formation, edema, congestion, and alveolar hemorrhage were noted on histopathology at 72 and 80 h in wild-type mice. Nfib hemizygous lungs had significant downregulation of genes involved in redox signaling and inflammatory pathways. Adult Nfib hemizygous mice are relatively resistant to hyperoxia compared to wild-type littermates. Mechanisms contributing to this resistance are not clear; however, transcription factors such as Nfib may regulate cell survival and play a role in modulating postnatal lung development. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Prevalence of toxoplasmosis and genetic characterization of Toxoplasma gondii strains isolated in wild birds of prey and their relation with previously isolated strains from Turkey.

PubMed

Karakavuk, Muhammet; Aldemir, Duygu; Mercier, Aurélien; Atalay Şahar, Esra; Can, Hüseyin; Murat, Jean-Benjamin; Döndüren, Ömer; Can, Şengül; Özdemir, Hüseyin Gökhan; Değirmenci Döşkaya, Aysu; Pektaş, Bayram; Dardé, Marie-Laure; Gürüz, Adnan Yüksel; Döşkaya, Mert

2018-01-01

Toxoplasma gondii is a protozoon parasite that causes congenital toxoplasmosis, as well as other serious clinical presentations, in immune compromised humans. Analyses of the prevalence and genotyping of strains from the definitive host and intermediate hosts will help to understanding the circulation of the different strains and elucidating the role of the genotype(s) in human toxoplasmosis. Turkey has a specific geographic location bridging Africa, Europe, and Asia. We hypothesized that T. gondii strains may have been transferred to Turkey from these continents via migratory birds or vice versa. The present study aimed to assess the prevalence of toxoplasmosis in wild birds of prey of İzmir and Manisa provinces as well as genetically characterize T. gondii strains from these wild birds to show the relation between bird strains and neighboring stray cats as well as human strains previously isolated in Turkey. Tissues obtained from 48 wild birds were investigated for the presence of T. gondii DNA and then bioassayed in mouse. Isolated strains were genotyped using 15 microsatellite markers. The prevalence of T. gondii DNA was found to be 89.6% (n: 43/48) in wild birds. Out of 43 positive samples, a total of 14 strains were genotyped by 15 microsatellite markers. Among them, eight were type II, three were type III and three were mixture of genotypes (two type II/II and one was II/III). These are the first data that showed the presence of T. gondii and types II and III genotypes in wild birds of Turkey. Moreover, Africa 1 was not detected. In addition, cluster analysis showed that T. gondii strains within type II and III lineage have close relation with strains previously isolated from stray cats in İzmir. Further studies are required to isolate more strains from human cases, other intermediate hosts, and water sources to reveal this relation.

Prevalence of toxoplasmosis and genetic characterization of Toxoplasma gondii strains isolated in wild birds of prey and their relation with previously isolated strains from Turkey

PubMed Central

Karakavuk, Muhammet; Aldemir, Duygu; Mercier, Aurélien; Atalay Şahar, Esra; Can, Hüseyin; Murat, Jean-Benjamin; Döndüren, Ömer; Can, Şengül; Özdemir, Hüseyin Gökhan; Değirmenci Döşkaya, Aysu; Pektaş, Bayram; Dardé, Marie-Laure; Gürüz, Adnan Yüksel

2018-01-01

Toxoplasma gondii is a protozoon parasite that causes congenital toxoplasmosis, as well as other serious clinical presentations, in immune compromised humans. Analyses of the prevalence and genotyping of strains from the definitive host and intermediate hosts will help to understanding the circulation of the different strains and elucidating the role of the genotype(s) in human toxoplasmosis. Turkey has a specific geographic location bridging Africa, Europe, and Asia. We hypothesized that T. gondii strains may have been transferred to Turkey from these continents via migratory birds or vice versa. The present study aimed to assess the prevalence of toxoplasmosis in wild birds of prey of İzmir and Manisa provinces as well as genetically characterize T. gondii strains from these wild birds to show the relation between bird strains and neighboring stray cats as well as human strains previously isolated in Turkey. Tissues obtained from 48 wild birds were investigated for the presence of T. gondii DNA and then bioassayed in mouse. Isolated strains were genotyped using 15 microsatellite markers. The prevalence of T. gondii DNA was found to be 89.6% (n: 43/48) in wild birds. Out of 43 positive samples, a total of 14 strains were genotyped by 15 microsatellite markers. Among them, eight were type II, three were type III and three were mixture of genotypes (two type II/II and one was II/III). These are the first data that showed the presence of T. gondii and types II and III genotypes in wild birds of Turkey. Moreover, Africa 1 was not detected. In addition, cluster analysis showed that T. gondii strains within type II and III lineage have close relation with strains previously isolated from stray cats in İzmir. Further studies are required to isolate more strains from human cases, other intermediate hosts, and water sources to reveal this relation. PMID:29668747

Clonal diversity of extended-spectrum beta-lactamase producing Escherichia coli isolates in fecal samples of wild animals.

PubMed

Cristóvão, Filipe; Alonso, Carla Andrea; Igrejas, Gilberto; Sousa, Margarida; Silva, Vanessa; Pereira, José Eduardo; Lozano, Carmen; Cortés-Cortés, Gerardo; Torres, Carmen; Poeta, Patrícia

2017-03-01

The clonal diversity of extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli isolates from nine different species of wild animals from distinct regions of Portugal and Spain and their content in replicon plasmids were analyzed. Among the initial 53 ESBL-producing E. coli isolates that were studied (from previous studies), 28 were selected, corresponding to different animal origins with distinct ESBL types and pulsed-field gel electrophoresis (PFGE) patterns. These 28 isolates produced different ESBLs ascribed to the following families: CTX-M, SHV and TEM. The isolates were classified into three phylogenetic groups: B1 (n = 11), A (n = 10) and D (n = 7). The seven E. coli of phylogroup D were then typed by multilocus sequence typing and ascribed to four distinct sequence types: ST117, ST115, ST2001 and ST69. The clonal diversity and relationship between isolates was studied by PFGE. Lastly, the plasmids were analyzed according to their incompatibility group using the PCR-based-replicon-typing scheme. A great diversity of replicon types was identified, with up to five per isolate. Most of the CTX-M-1 and SHV-12 producing E. coli isolates carried IncI1 or IncN replicons. The diversity of ESBL-producing E. coli isolates in wild animals, which can be disseminated in the environment, emphasizes the environmental and health problems that we face nowadays. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Multi-locus sequence typing of Salmonella enterica serovar Typhimurium isolates from wild birds in northern England suggests host-adapted strain.

PubMed

Hughes, L A; Wigley, P; Bennett, M; Chantrey, J; Williams, N

2010-10-01

Recent studies have suggested that Salmonella Typhimurium strains associated with mortality in UK garden birds are significantly different from strains that cause disease in humans and livestock and that wild bird strains may be host adapted. However, without further genomic characterization of these strains, it is not possible to determine whether they are host adapted. The aim of this study was to characterize a representative sample of Salm. Typhimurium strains detected in wild garden birds using multi-locus sequence typing (MLST)to investigate evolutionary relationships between them. Multi-locus sequence typing was performed on nine Salm. Typhimurium strains isolated from wild garden birds. Two sequence types were identified, the most common of which was ST568. Examination of the public Salmonella enterica MLST database revealed that only three other ST568 isolates had been cultured from a human in Scotland. Two further isolates of Salm. Typhimurium were determined to be ST19. Results of MLST analysis suggest that there is a predominant strain of Salm. Typhimurium circulating among garden bird populations in the United Kingdom, which is rarely detected in other species, supporting the hypothesis that this strain is host adapted. Host-pathogen evolution is often assumed to lead to pathogens becoming less virulent to avoid the death of their host; however, infection with ST568 led to high mortality rates among the wild birds examined, which were all found dead at wild bird-feeding stations. We hypothesize that by attracting unnaturally high densities of birds, wild bird-feeding stations may facilitate the transmission of ST568 between wild birds, therefore reducing the evolutionary cost of this pathogen killing its host, resulting in a host-adapted strain with increased virulence.

Comparative transcriptomic analysis of silkwormBmovo-1 and wild type silkworm ovary

PubMed Central

Xue, Renyu; Hu, Xiaolong; Zhu, Liyuan; Cao, Guangli; Huang, Moli; Xue, Gaoxu; Song, Zuowei; Lu, Jiayu; Chen, Xueying; Gong, Chengliang

2015-01-01

The detailed molecular mechanism of Bmovo-1 regulation of ovary size is unclear. To uncover the mechanism of Bmovo-1 regulation of ovarian development and oogenesis using RNA-Seq, we compared the transcriptomes of wild type (WT) and Bmovo-1-overexpressing silkworm (silkworm+Bmovo-1) ovaries. Using a pair-end Illumina Solexa sequencing strategy, 5,296,942 total reads were obtained from silkworm+Bmovo-1 ovaries and 6,306,078 from WT ovaries. The average read length was about 100 bp. Clean read ratios were 98.79% for silkworm+Bmovo-1 and 98.87% for WT silkworm ovaries. Comparative transcriptome analysis showed 123 upregulated and 111 downregulated genes in silkworm+Bmovo-1 ovaries. These differentially expressed genes were enriched in the extracellular and extracellular spaces and involved in metabolism, genetic information processing, environmental information processing, cellular processes and organismal systems. Bmovo-1 overexpression in silkworm ovaries might promote anabolism for ovarian development and oogenesis and oocyte proliferation and transport of nutrients to ovaries by altering nutrient partitioning, which would support ovary development. Excessive consumption of nutrients for ovary development alters nutrient partitioning and deters silk protein synthesis. PMID:26643037

Biofilm Production and Antibiofilm Activity of Echinocandins and Liposomal Amphotericin B in Echinocandin-Resistant Yeast Species

PubMed Central

Marcos-Zambrano, Laura Judith; Gómez-Perosanz, Marta; Escribano, Pilar; Zaragoza, Oscar; Bouza, Emilio

2016-01-01

The echinocandins and liposomal amphotericin B are active against biofilm produced by echinocandin-susceptible Candida strains. However, few data have been reported on the production of biofilm by echinocandin-resistant isolates and their antifungal susceptibility. We studied the production of biofilm by fks mutant Candida strains and intrinsically echinocandin-resistant non-Candida isolates and the susceptibility of both entities to liposomal amphotericin B and echinocandins. We analyzed the production of biofilm by isolates from patients with fungemia (fks mutant Candida, n = 5; intrinsically echinocandin-resistant non-Candida, n = 12; and Candida wild type, n = 10). Biofilm formation was measured to classify strains according to biomass (crystal violet assay) and metabolic activity (XTT reduction assay). Preformed biofilms were tested against liposomal amphotericin B, caspofungin, micafungin, and anidulafungin. The sessile MIC was defined as the antifungal concentration yielding a 50% or 80% reduction in the metabolic activity of the biofilm compared to that of the growth control (SMIC50 and SMIC80, respectively). fks mutant Candida isolates formed biofilms in a fashion similar to that of Candida wild-type strains. The echinocandins had the highest activity against biofilms formed by wild-type Candida isolates, followed by fks mutant Candida isolates and non-Candida isolates. Liposomal amphotericin B had the highest activity against fks mutant Candida biofilms. The formation of biofilm by echinocandin-resistant strains was similar to that of wild-type strains, although resistance to echinocandins remained high. PMID:27021323

Mini-dystrophin restores L-type calcium currents in skeletal muscle of transgenic mdx mice

PubMed Central

Friedrich, O; Both, M; Gillis, J M; Chamberlain, J S; Fink, RHA

2004-01-01

L-type calcium currents (iCa) were recorded using the two-microelectrode voltage-clamp technique in single short toe muscle fibres of three different mouse strains: (i) C57/SV129 wild-type mice (wt); (ii) mdx mice (an animal model for Duchenne muscular dystrophy; and (iii) transgenically engineered mini-dystrophin (MinD)-expressing mdx mice. The activation and inactivation properties of iCa were examined in 2- to 18-month-old animals. Ca2+ current densities at 0 mV in mdx fibres increased with age, but were always significantly smaller compared to age-matched wild-type fibres. Time-to-peak (TTP) of iCa was prolonged in mdx fibres compared to wt fibres. MinD fibres always showed similar TTP and current amplitudes compared to age-matched wt fibres. In all three genotypes, the voltage-dependent inactivation and deactivation of iCa were similar. Intracellular resting calcium concentration ([Ca2+]i) and the distribution of dihydropyridine binding sites were also not different in young animals of all three genotypes, whereas iCa was markedly reduced in mdx fibres. We conclude, that dystrophin influences L-type Ca2+ channels via a direct or indirect linkage which may be disrupted in mdx mice and may be crucial for proper excitation–contraction coupling initiating Ca2+ release from the sarcoplasmic reticulum. This linkage seems to be fully restored in the presence of mini-dystrophin. PMID:14594987

ACE as a Mechanosensor to Shear Stress Influences the Control of Its Own Regulation via Phosphorylation of Cytoplasmic Ser1270

PubMed Central

Barauna, Valerio Garrone; Campos, Luciene Cristina Gastalho; Miyakawa, Ayumi Aurea; Krieger, Jose Eduardo

2011-01-01

Objectives We tested whether angiotensin converting enzyme (ACE) and phosphorylation of Ser1270 are involved in shear-stress (SS)-induced downregulation of the enzyme. Methods and Results Western blotting analysis showed that SS (18 h, 15 dyn/cm2) decreases ACE expression and phosphorylation as well as p-JNK inhibition in human primary endothelial cells (EC). CHO cells expressing wild-type ACE (wt-ACE) also displayed SS-induced decrease in ACE and p-JNK. Moreover, SS decreased ACE promoter activity in wt-ACE, but had no effect in wild type CHO or CHO expressing ACE without either the extra- or the intracellular domains, and decreased less in CHO expressing a mutated ACE at Ser1270 compared to wt-ACE (13 vs. 40%, respectively). The JNK inhibitor (SP600125, 18 h), in absence of SS, also decreased ACE promoter activity in wt-ACE. Finally, SS-induced inhibition of ACE expression and phosphorylation in EC was counteracted by simultaneous exposure to an ACE inhibitor. Conclusions ACE displays a key role on its own downregulation in response to SS. This response requires both the extra- and the intracellular domains and ACE Ser1270, consistent with the idea that the extracellular domain behaves as a mechanosensor while the cytoplasmic domain elicits the downstream intracellular signaling by phosphorylation on Ser1270. PMID:21901117

Time Courses of Cortical Glucose Metabolism and Microglial Activity Across the Life Span of Wild-Type Mice: A PET Study.

PubMed

Brendel, Matthias; Focke, Carola; Blume, Tanja; Peters, Finn; Deussing, Maximilian; Probst, Federico; Jaworska, Anna; Overhoff, Felix; Albert, Nathalie; Lindner, Simon; von Ungern-Sternberg, Barbara; Bartenstein, Peter; Haass, Christian; Kleinberger, Gernot; Herms, Jochen; Rominger, Axel

2017-12-01

Contrary to findings in the human brain, 18 F-FDG PET shows cerebral hypermetabolism of aged wild-type (WT) mice relative to younger animals, supposedly due to microglial activation. Therefore, we used dual-tracer small-animal PET to examine directly the link between neuroinflammation and hypermetabolism in aged mice. Methods: WT mice (5-20 mo) were investigated in a cross-sectional design using 18 F-FDG ( n = 43) and translocator protein (TSPO) ( 18 F-GE180; n = 58) small-animal PET, with volume-of-interest and voxelwise analyses. Biochemical analysis of plasma cytokine levels and immunohistochemical confirmation of microglial activity were also performed. Results: Age-dependent cortical hypermetabolism in WT mice relative to young animals aged 5 mo peaked at 14.5 mo (+16%, P < 0.001) and declined to baseline at 20 mo. Similarly, cortical TSPO binding increased to a maximum at 14.5 mo (+15%, P < 0.001) and remained high to 20 mo, resulting in an overall correlation between 18 F-FDG uptake and TSPO binding (R = 0.69, P < 0.005). Biochemical and immunohistochemical analyses confirmed the TSPO small-animal PET findings. Conclusion: Age-dependent neuroinflammation is associated with the controversial observation of cerebral hypermetabolism in aging WT mice. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Mesophyll conductance decreases in the wild type but not in an ABA-deficient mutant (aba1) of Nicotiana plumbaginifolia under drought conditions.

PubMed

Mizokami, Yusuke; Noguchi, Ko; Kojima, Mikiko; Sakakibara, Hitoshi; Terashima, Ichiro

2015-03-01

Under drought conditions, leaf photosynthesis is limited by the supply of CO2 . Drought induces production of abscisic acid (ABA), and ABA decreases stomatal conductance (gs ). Previous papers reported that the drought stress also causes the decrease in mesophyll conductance (gm ). However, the relationships between ABA content and gm are unclear. We investigated the responses of gm to the leaf ABA content [(ABA)L ] using an ABA-deficient mutant, aba1, and the wild type (WT) of Nicotiana plumbaginifolia. We also measured leaf water potential (ΨL ) because leaf hydraulics may be related to gm . Under drought conditions, gm decreased with the increase in (ABA)L in WT, whereas both (ABA)L and gm were unchanged by the drought treatment in aba1. Exogenously applied ABA decreased gm in both WT and aba1 in a dose-dependent manner. ΨL in WT was decreased by the drought treatment to -0.7 MPa, whereas ΨL in aba1 was around -0.8 MPa even under the well-watered conditions and unchanged by the drought treatment. From these results, we conclude that the increase in (ABA)L is crucial for the decrease in gm under drought conditions. We discuss possible relationships between the decrease in gm and changes in the leaf hydraulics. © 2014 John Wiley & Sons Ltd.

Differential regulation of insulin-like growth factor-I receptor gene expression by wild type and mutant androgen receptor in prostate cancer cells.

PubMed

Schayek, Hagit; Seti, Hila; Greenberg, Norman M; Sun, Shihua; Werner, Haim; Plymate, Stephen R

2010-07-29

The progression of prostate cancer from an organ-confined, androgen-sensitive disease to a metastatic one is associated with dysregulation of androgen receptor (AR)-regulated target genes and with a decrease in insulin-like growth factor-I receptor (IGF-IR) expression. To investigate the differential effects of wild type (wt) and mutant AR on IGF-IR levels we employed a series of isogenic prostate-derived cell lines and human xenografts. We show that basal and phosphorylated IGF-IR levels progressively decreased as prostate cancer cells became more tumorigenic and metastatic. In addition, we show that wt, but not mutant, AR along with dihydrotestosterone treatment increased IGF-IR promoter activity and endogenous IGF-IR levels. ChIP analysis show enhanced AR binding to the IGF-IR promoter in AR-overexpressing cells. Finally, wt AR-overexpressing cells display an enhanced proliferation rate. In summary, we provide evidence that activated wt AR enhances IGF-IR transcription in prostate cancer cells via a mechanism that involves AR binding to the IGF-IR promoter. AR mutations alter the ability of the mutated protein to regulate IGF-IR expression. Our results suggest that prostate cancer progression is associated with a decrease in IGF-IR expression that could be the result of impaired ability of AR to stimulate IGF-IR gene expression. 2010 Elsevier Ireland Ltd. All rights reserved.

Differential regulation of insulin-like growth factor-I receptor gene expression by wild type and mutant androgen receptor in prostate cancer cells

PubMed Central

Schayek, Hagit; Seti, Hila; Greenberg, Norman M.; Sun, Shihua; Werner, Haim; Plymate, Stephen R.

2010-01-01

The progression of prostate cancer from an organ-confined, androgen-sensitive disease to a metastatic one is associated with dysregulation of androgen receptor (AR)-regulated target genes and with a decrease in insulin-like growth factor-I receptor (IGF-IR) expression. To investigate the differential effects of wild type (wt) and mutant AR on IGF-IR levels we employed a series of isogenic prostate-derived cell lines and human xenografts. We show that basal and phosphorylated IGF-IR levels progressively decreased as prostate cancer cells became more tumorigenic and metastatic. In addition, we show that wt, but not mutant, AR along with dihydrotestosterone treatment increased IGF-IR promoter activity and endogenous IGF-IR levels. ChIP analysis show enhanced AR binding to the IGF-IR promoter in AR-overexpressing cells. Finally, wt AR-overexpressing cells display an enhanced proliferation rate. In summary, we provide evidence that activated wt AR enhances IGF-IR transcription in prostate cancer cells via a mechanism that involves AR binding to the IGF-IR promoter. AR mutations alter the ability of the mutated protein to regulate IGF-IR expression. Our results suggest that prostate cancer progression is associated with a decrease in IGF-IR expression that could be the result of impaired ability of AR to stimulate IGF-IR gene expression. PMID:20417685

De novo establishment of wild-type song culture in the zebra finch.

PubMed

Fehér, Olga; Wang, Haibin; Saar, Sigal; Mitra, Partha P; Tchernichovski, Ofer

2009-05-28

Culture is typically viewed as consisting of traits inherited epigenetically, through social learning. However, cultural diversity has species-typical constraints, presumably of genetic origin. A celebrated, if contentious, example is whether a universal grammar constrains syntactic diversity in human languages. Oscine songbirds exhibit song learning and provide biologically tractable models of culture: members of a species show individual variation in song and geographically separated groups have local song dialects. Different species exhibit distinct song cultures, suggestive of genetic constraints. Without such constraints, innovations and copying errors should cause unbounded variation over multiple generations or geographical distance, contrary to observations. Here we report an experiment designed to determine whether wild-type song culture might emerge over multiple generations in an isolated colony founded by isolates, and, if so, how this might happen and what type of social environment is required. Zebra finch isolates, unexposed to singing males during development, produce song with characteristics that differ from the wild-type song found in laboratory or natural colonies. In tutoring lineages starting from isolate founders, we quantified alterations in song across tutoring generations in two social environments: tutor-pupil pairs in sound-isolated chambers and an isolated semi-natural colony. In both settings, juveniles imitated the isolate tutors but changed certain characteristics of the songs. These alterations accumulated over learning generations. Consequently, songs evolved towards the wild-type in three to four generations. Thus, species-typical song culture can appear de novo. Our study has parallels with language change and evolution. In analogy to models in quantitative genetics, we model song culture as a multigenerational phenotype partly encoded genetically in an isolate founding population, influenced by environmental variables and taking multiple generations to emerge.

Na+,K+-pump stimulation improves contractility in isolated muscles of mice with hyperkalemic periodic paralysis

PubMed Central

Nielsen, Ole Bækgaard; Clausen, Johannes D.; Pedersen, Thomas Holm; Hayward, Lawrence J.

2011-01-01

In patients with hyperkalemic periodic paralysis (HyperKPP), attacks of muscle weakness or paralysis are triggered by K+ ingestion or rest after exercise. Force can be restored by muscle work or treatment with β2-adrenoceptor agonists. A missense substitution corresponding to a mutation in the skeletal muscle voltage-gated Na+ channel (Nav1.4, Met1592Val) causing human HyperKPP was targeted into the mouse SCN4A gene (mutants). In soleus muscles prepared from these mutant mice, twitch, tetanic force, and endurance were markedly reduced compared with soleus from wild type (WT), reflecting impaired excitability. In mutant soleus, contractility was considerably more sensitive than WT soleus to inhibition by elevated [K+]o. In resting mutant soleus, tetrodotoxin (TTX)-suppressible 22Na uptake and [Na+]i were increased by 470 and 58%, respectively, and membrane potential was depolarized (by 16 mV, P < 0.0001) and repolarized by TTX. Na+,K+ pump–mediated 86Rb uptake was 83% larger than in WT. Salbutamol stimulated 86Rb uptake and reduced [Na+]i both in mutant and WT soleus. Stimulating Na+,K+ pumps with salbutamol restored force in mutant soleus and extensor digitorum longus (EDL). Increasing [Na+]i with monensin also restored force in soleus. In soleus, EDL, and tibialis anterior muscles of mutant mice, the content of Na+,K+ pumps was 28, 62, and 33% higher than in WT, respectively, possibly reflecting the stimulating effect of elevated [Na+]i on the synthesis of Na+,K+ pumps. The results confirm that the functional disorders of skeletal muscles in HyperKPP are secondary to increased Na+ influx and show that contractility can be restored by acute stimulation of the Na+,K+ pumps. Calcitonin gene-related peptide (CGRP) restored force in mutant soleus but caused no detectable increase in 86Rb uptake. Repeated excitation and capsaicin also restored contractility, possibly because of the release of endogenous CGRP from nerve endings in the isolated muscles. These observations may explain how mild exercise helps locally to prevent severe weakness during an attack of HyperKPP. PMID:21708955

An Analysis of Interactions between Fluorescently-Tagged Mutant and Wild-Type SOD1 in Intracellular Inclusions

PubMed Central

Qualls, David A.; Crosby, Keith; Brown, Hilda; Borchelt, David R.

2013-01-01

Background By mechanisms yet to be discerned, the co-expression of high levels of wild-type human superoxide dismutase 1 (hSOD1) with variants of hSOD1 encoding mutations linked familial amyotrophic lateral sclerosis (fALS) hastens the onset of motor neuron degeneration in transgenic mice. Although it is known that spinal cords of paralyzed mice accumulate detergent insoluble forms of WT hSOD1 along with mutant hSOD1, it has been difficult to determine whether there is co-deposition of the proteins in inclusion structures. Methodology/Principal Findings In the present study, we use cell culture models of mutant SOD1 aggregation, focusing on the A4V, G37R, and G85R variants, to examine interactions between WT-hSOD1 and misfolded mutant SOD1. In these studies, we fuse WT and mutant proteins to either yellow or red fluorescent protein so that the two proteins can be distinguished within inclusions structures. Conclusions/Significance Although the interpretation of the data is not entirely straightforward because we have strong evidence that the nature of the fused fluorophores affects the organization of the inclusions that form, our data are most consistent with the idea that normal dimeric WT-hSOD1 does not readily interact with misfolded forms of mutant hSOD1. We also demonstrate the monomerization of WT-hSOD1 by experimental mutation does induce the protein to aggregate, although such monomerization may enable interactions with misfolded mutant SOD1. Our data suggest that WT-hSOD1 is not prone to become intimately associated with misfolded mutant hSOD1 within intracellular inclusions that can be generated in cultured cells. PMID:24391857

Jasmonic acid accumulation and systemic photosynthetic and electrical changes in locally burned wild type tomato, ABA-deficient sitiens mutants and sitiens pre-treated by ABA.

PubMed

Hlavinka, Jan; Nožková-Hlaváčková, Vladimíra; Floková, Kristýna; Novák, Ondřej; Nauš, Jan

2012-05-01

Burning the terminal leaflet of younger tomato (Lycopersicon esculentum Mill.) leaf caused local and systemic changes in the surface electrical potential (SEP) and gas exchange (GE) parameters. The local and systemic accumulation of endogenous abscisic acid (ABA) and jasmonic acid (JA) was measured 85 min after burning. The experiments were conducted with wild type (WT) plants, ABA-deficient mutant sitiens (SIT) and ABA pre-treated SIT plants (SITA). First changes in SEP were detected within 1.5 min after burning and were followed by a decrease in GE parameters within 3-6 min in WT, SIT and SITA plants. GE and SEP time courses of SIT were different and wave amplitudes of SEP of SIT were lower compared to WT and SITA. ABA content in WT and SITA control plants was similar and substantially higher compared to SIT, JA content was similar among WT, SIT and SITA. While changes in the ABA content in systemic leaves have not been recorded after burning, the systemic JA content was substantially increased in WT and more in SIT and SITA. The results suggest that ABA content governs the systemic reaction of GE and the SEP shape upon local burning. ABA, JA and SEP participate in triggering the GE reaction. The ABA shortage in the SIT in the reaction to burning is partly compensated by an enhanced JA accumulation. This JA compensation is maintained even in SIT endogenously supplied with ABA. A correlation between the systemic JA content and changes in GE parameters or SEP was not found. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Human mitochondrial NDUFS3 protein bearing Leigh syndrome mutation is more prone to aggregation than its wild-type.

PubMed

Jaokar, Tulika M; Patil, Deepak P; Shouche, Yogesh S; Gaikwad, Sushama M; Suresh, C G

2013-12-01

NDUFS3 is an integral subunit of the Q module of the mitochondrial respiratory Complex-I. The combined mutation (T145I + R199W) in the subunit is reported to cause optic atrophy and Leigh syndrome accompanied by severe Complex-I deficiency. In the present study, we have cloned and overexpressed the human NDUFS3 subunit and its double mutant in a soluble form in Escherichia coli. The wild-type (w-t) and mutant proteins were purified to homogeneity through a serial two-step chromatographic purification procedure of anion exchange followed by size exclusion chromatography. The integrity and purity of the purified proteins was confirmed by Western blot analysis and MALDI-TOF/TOF. The conformational transitions of the purified subunits were studied through steady state as well as time resolved fluorescence and CD spectroscopy under various denaturing conditions. The mutant protein showed altered polarity around tryptophan residues, changed quenching parameters and also noticeably altered secondary and tertiary structure compared to the w-t protein. Mutant also exhibited a higher tendency than the w-t protein for aggregation which was examined using fluorescent (Thioflavin-T) and spectroscopic (Congo red) dye binding techniques. The pH stability of the w-t and mutant proteins varied at extreme acidic pH and the molten globule like structure of w-t at pH1 was absent in case of the mutant protein. Both the w-t and mutant proteins showed multi-step thermal and Gdn-HCl induced unfolding. Thus, the results provide insight into the alterations of NDUFS3 protein structure caused by the mutations, affecting the overall integrity of the protein and finally leading to disruption of Complex-I assembly. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

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

Nayeb-Hashemi, Hamed; Desai, Anal; Demchev, Valeriy

Fibrinogen like protein-1 (Fgl1) is a predominantly liver expressed protein that has been implicated as both a hepatoprotectant and a hepatocyte mitogen. Fgl1 expression is decreased in hepatocellular carcinoma (HCC) and its loss correlates with a poorly differentiated phenotype. To better elucidate the role of Fgl1 in hepatocarcinogenesis, we treated mice wild type or null for Fgl1 with diethyl nitrosamine and monitored for incidence of hepatocellular cancer. We find that mice lacking Fgl1 develop HCC at more than twice the rate of wild type mice. We show that hepatocellular cancers from Fgl1 null mice are molecularly distinct from those ofmore » the wild type mice. In tumors from Fgl1 null mice there is enhanced activation of Akt and downstream targets of the mammalian target of rapamycin (mTOR). In addition, there is paradoxical up regulation of putative hepatocellular cancer tumor suppressors; tripartite motif-containing protein 35 (Trim35) and tumor necrosis factor super family 10b (Tnfrsf10b). Taken together, these findings suggest that Fgl1 acts as a tumor suppressor in hepatocellular cancer through an Akt dependent mechanism and supports its role as a potential therapeutic target in HCC. - Highlights: • Fgl1 knockout mice (Fgl1KO) are more prone to carcinogen-induced liver cancer compared to wild type (WT) mates. • Tumors from the Fgl1KO are molecularly distinct with enhanced Akt and mTOR activity in comparison with Fgl1WT tumors. • Tumors from the Fgl1KO have enhanced expression of Trim35 and Tnfrsf10b, putative HCC tumor suppressors.« less

Effects of endoplasmic reticulum stressors on maturation and signaling of hemizygous and heterozygous wild-type and mutant forms of KIT.

PubMed

Brahimi-Adouane, Sabrina; Bachet, Jean-Baptiste; Tabone-Eglinger, Séverine; Subra, Frédéric; Capron, Claude; Blay, Jean-Yves; Emile, Jean-François

2013-06-01

Gain of function mutations of KIT are frequent in some human tumors, and are sensible to tyrosine kinase inhibitors. In most tumors, oncogenic mutations are heterozygous, however most in vitro data of KIT activation have been obtained with hemizygous mutation. This study aimed to investigate the maturation and activation of wild-type (WT) and mutant (M) forms of KIT in hemizygous and heterozygous conditions. WT and two types of exon 11 deletions M forms of human KIT were expressed in NIH3T3 cell lines. Membrane expression of KIT was quantified by flow cytometry. Quantification of glycosylated forms of KIT and phosphorylated forms of AKT and ERK were performed by western blot. Simultaneous activation of WT KIT and treatment with endoplasmic reticulum (ER) inhibitors, tunicamycin or brefeldin A induced a complete inhibition of membrane expression of the 145 kDa form of KIT. By contrast activation or ER inhibitors alone, only partly inhibited this form. ER inhibitors also inhibited KIT activation-dependent phosphorylation of AKT and ERK1/2. Brefeldin A induced a complete down regulation of the 145 kDa form in hemizygous M, and induced an intra-cellular accumulation of the 125 kDa form in WT but not in hemizygous M. Heterozygous cells had glycosylation and response to ER inhibitors patterns more similar to WT than to hemizygous M. Phosphorylated AKT was reduced in hemizygous cells in comparison to WT KIT cells and heterozygous cells, and in the presence of brefeldin A in all cell lines. Effects of ER inhibitors are significantly different in hemizygous and heterozygous mutants. Differences in intra-cellular trafficking of KIT forms result in differences in downstream signaling pathways, and activation of PI3K/AKT pathway appears to be tied to the presence of the mature 145 kDa form of KIT at the membrane surface. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Evaluation of Electrical Impedance as a Biomarker of Myostatin Inhibition in Wild Type and Muscular Dystrophy Mice.

PubMed

Sanchez, Benjamin; Li, Jia; Yim, Sung; Pacheck, Adam; Widrick, Jeffrey J; Rutkove, Seward B

2015-01-01

Non-invasive and effort independent biomarkers are needed to better assess the effects of drug therapy on healthy muscle and that affected by muscular dystrophy (mdx). Here we evaluated the use of multi-frequency electrical impedance for this purpose with comparison to force and histological parameters. Eight wild-type (wt) and 10 mdx mice were treated weekly with RAP-031 activin type IIB receptor at a dose of 10 mg kg-1 twice weekly for 16 weeks; the investigators were blinded to treatment and disease status. At the completion of treatment, impedance measurements, in situ force measurements, and histology analyses were performed. As compared to untreated animals, RAP-031 wt and mdx treated mice had greater body mass (18% and 17%, p < 0.001 respectively) and muscle mass (25% p < 0.05 and 22% p < 0.001, respectively). The Cole impedance parameters in treated wt mice, showed a 24% lower central frequency (p < 0.05) and 19% higher resistance ratio (p < 0.05); no significant differences were observed in the mdx mice. These differences were consistent with those seen in maximum isometric force, which was greater in the wt animals (p < 0.05 at > 70 Hz), but not in the mdx animals. In contrast, maximum force normalized by muscle mass was unchanged in the wt animals and lower in the mdx animals by 21% (p < 0.01). Similarly, myofiber size was only non-significantly higher in treated versus untreated animals (8% p = 0.44 and 12% p = 0.31 for wt and mdx animals, respectively). Our findings demonstrate electrical impedance of muscle reproduce the functional and histological changes associated with myostatin pathway inhibition and do not reflect differences in muscle size or volume. This technique deserves further study in both animal and human therapeutic trials.

Dietary quercetin attenuates oxidant-induced endothelial dysfunction and atherosclerosis in apolipoprotein E knockout mice fed a high-fat diet: a critical role for heme oxygenase-1.

PubMed

Shen, Yu; Ward, Natalie C; Hodgson, Jonathan M; Puddey, Ian B; Wang, Yutang; Zhang, Di; Maghzal, Ghassan J; Stocker, Roland; Croft, Kevin D

2013-12-01

Several lines of evidence indicate that quercetin, a polyphenol derived in the diet from fruit and vegetables, contributes to cardiovascular health. We aimed to investigate the effects of dietary quercetin on endothelial function and atherosclerosis in mice fed a high-fat diet. Wild-type C57BL/6 (WT) and apolipoprotein E gene knockout (ApoE(-/-)) mice were fed: (i) a high-fat diet (HFD) or (ii) a HFD supplemented with 0.05% w/w quercetin (HFD+Q), for 14 weeks. Compared with animals fed HFD, HFD+Q attenuated atherosclerosis in ApoE(-/-) mice. Treatment with the HFD+Q significantly improved endothelium-dependent relaxation of aortic rings isolated from WT but not ApoE(-/-) mice and attenuated hypochlorous acid-induced endothelial dysfunction in aortic rings of both WT and ApoE(-/-) mice. Mechanistic studies revealed that HFD+Q significantly improved plasma F2-isoprostanes, 24h urinary nitrite, and endothelial nitric oxide synthase activity, and increased heme oxygenase-1 (HO-1) protein expression in the aortas of both WT and ApoE(-/-) mice (P<0.05). HFD+Q also resulted in small changes in plasma cholesterol (P<0.05 in WT) and plasma triacylglycerols (P<0.05 in ApoE (-/-)mice). In a separate experiment, quercetin did not protect against hypochlorite-induced endothelial dysfunction in arteries obtained from heterozygous HO-1 gene knockout mice with low expression of HO-1 protein. Quercetin protects mice fed a HFD against oxidant-induced endothelial dysfunction and ApoE(-/-) mice against atherosclerosis. These effects are associated with improvements in nitric oxide bioavailability and are critically related to arterial induction of HO-1. © 2013 Elsevier Inc. All rights reserved.

Adiponectin attenuates LPS-induced acute lung injury through suppression of endothelial cell activation1

PubMed Central

Konter, Jason M; Parker, Jennifer L; Baez, Elizabeth; Li, Stephanie Z; Ranscht, Barbara; Denzel, Martin; Little, Frederic F; Nakamura, Kazuto; Ouchi, Noriyuki; Fine, Alan; Walsh, Kenneth; Summer, Ross S

2011-01-01

Adiponectin (APN) is an adipose tissue-derived factor with anti-inflammatory and vascular protective properties whose levels paradoxically decrease with increasing body fat. In this study, APN’s role in the early development of ALI to lipopolysaccharide (LPS) was investigated. Intra-tracheal (i.t.) LPS elicited an exaggerated systemic inflammatory response in APN-deficient (APN−/−) mice compared to wild-type (wt) littermates. Increased lung injury and inflammation were observed in APN−/− mice as early as 4 hours after delivery of LPS. Targeted gene expression profiling performed on immune and endothelial cells isolated from lung digests 4 hours after LPS administration showed increased pro-inflammatory gene expression (e.g. IL-6) only in endothelial cells of APN−/− mice when compared to wt mice. Direct effects on lung endothelium were demonstrated by APN’s ability to inhibit LPS-induced IL-6 production in primary human endothelial cells in culture. Furthermore, T-cadherin-deficient (T-cad−/−) mice that have significantly reduced lung airspace APN but high serum APN levels had pulmonary inflammatory responses after i.t. LPS that were similar to those of wt mice. These findings indicate the importance of serum APN in modulating LPS-induced ALI and suggest that conditions leading to hypoadiponectinemia (e.g. obesity) predispose to development of ALI through exaggerated inflammatory response in pulmonary vascular endothelium. PMID:22156343

Base substitutions at scissile bond sites are sufficient to alter RNA-binding and cleavage activity of RNase III.

PubMed

Kim, Kyungsub; Sim, Se-Hoon; Jeon, Che Ok; Lee, Younghoon; Lee, Kangseok

2011-02-01

RNase III, a double-stranded RNA-specific endoribonuclease, degrades bdm mRNA via cleavage at specific sites. To better understand the mechanism of cleavage site selection by RNase III, we performed a genetic screen for sequences containing mutations at the bdm RNA cleavage sites that resulted in altered mRNA stability using a transcriptional bdm'-'cat fusion construct. While most of the isolated mutants showed the increased bdm'-'cat mRNA stability that resulted from the inability of RNase III to cleave the mutated sequences, one mutant sequence (wt-L) displayed in vivo RNA stability similar to that of the wild-type sequence. In vivo and in vitro analyses of the wt-L RNA substrate showed that it was cut only once on the RNA strand to the 5'-terminus by RNase III, while the binding constant of RNase III to this mutant substrate was moderately increased. A base substitution at the uncleaved RNase III cleavage site in wt-L mutant RNA found in another mutant lowered the RNA-binding affinity by 11-fold and abolished the hydrolysis of scissile bonds by RNase III. Our results show that base substitutions at sites forming the scissile bonds are sufficient to alter RNA cleavage as well as the binding activity of RNase III. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Accumulation of cytolytic CD8{sup +} T cells in B16-melanoma and proliferation of mature T cells in TIS21-knockout mice after T cell receptor stimulation

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

Ryu, Min Sook; Woo, Min-Yeong; Department of Biomedical Sciences, The Graduate School, Ajou University

2014-10-01

In vivo and in vitro effects of TIS21 gene on the mature T cell activation and antitumor activities were explored by employing MO5 melanoma orthograft and splenocytes isolated from the TIS21-knockout (KO) mice. Proliferation and survival of mature T cells were significantly increased in the KO than the wild type (WT) cells, indicating that TIS21 inhibits the rate of mature T cell proliferation and its survival. In MO5 melanoma orthograft model, the KO mice recruited much more CD8{sup +} T cells into the tumors at around day 14 after tumor cell injection along with reduced tumor volumes compared with themore » WT. The increased frequency of granzyme B{sup +} CD8{sup +} T cells in splenocytes of the KO mice compared with the WT may account for antitumor-immunity of TIS21 gene in the melanoma orthograft. In contrast, reduced frequencies of CD107a{sup +} CD8{sup +} T cells in the splenocytes of KO mice may affect the loss of CD8{sup +} T cell infiltration in the orthograft at around day 19. These results indicate that TIS21 exhibits antiproliferative and proapoptotic effects in mature T cells, and differentially affects the frequencies of granzyme B{sup +} CD8{sup +} T-cells and CD107a{sup +} CD8{sup +} T-cells, thus transiently regulating in vivo anti-tumor immunity. - Highlights: • Constitutive expression of TIS21 in splenocytes and upregulation by TCR stimulation. • Proliferation of mature T-cells in spleen of TIS21KO mice after TCR stimulation. • Inhibition of cell death in mature T-cells of TIS21KO mice compared with the wild type. • Inhibition of melanoma growth in TIS21KO mice and CD8{sup +} T cell infiltration in tumor. • Reduction of CD 107{sup +}CD8{sup +} T cells, but increased granzyme B{sup +} CD8{sup +} T cells in TIS21KO mice.« less

Disruption of hypoxia-inducible transcription factor-prolyl hydroxylase domain-1 (PHD-1-/-) attenuates ex vivo myocardial ischemia/reperfusion injury through hypoxia-inducible factor-1α transcription factor and its target genes in mice.

PubMed

Adluri, Ram Sudheer; Thirunavukkarasu, Mahesh; Dunna, Nageswara Rao; Zhan, Lijun; Oriowo, Babatunde; Takeda, Kotaro; Sanchez, Juan A; Otani, Hajime; Maulik, Gautam; Fong, Guo-Hua; Maulik, Nilanjana

2011-10-01

Hypoxia-inducible transcription factor (HIF)-prolyl hydroxylases domain (PHD-1-3) are oxygen sensors that regulate the stability of the HIFs in an oxygen-dependent manner. Suppression of PHD enzymes leads to stabilization of HIFs and offers a potential treatment option for many ischemic disorders, such as peripheral artery occlusive disease, myocardial infarction, and stroke. Here, we show that homozygous disruption of PHD-1 (PHD-1(-/-)) could facilitate HIF-1α-mediated cardioprotection in ischemia/reperfused (I/R) myocardium. Wild-type (WT) and PHD-1(-/-) mice were randomized into WT time-matched control (TMC), PHD-1(-/-) TMC (PHD1TMC), WT I/R, and PHD-1(-/-) I/R (PHD1IR). Isolated hearts from each group were subjected to 30 min of global ischemia followed by 2 h of reperfusion. TMC hearts were perfused for 2 h 30 min without ischemia. Decreased infarct size (35%±0.6% vs. 49%±0.4%) and apoptotic cardiomyocytes (106±13 vs. 233±21 counts/100 high-power field) were observed in PHD1IR compared to wild-type ischemia/reperfusion (WTIR). Protein expression of HIF-1α was significantly increased in PHD1IR compared to WTIR. mRNA expression of β-catenin (1.9-fold), endothelial nitric oxide synthase (1.9-fold), p65 (1.9-fold), and Bcl-2 (2.7-fold) were upregulated in the PHD1IR compared with WTIR, which was studied by real-time quantitative polymerase chain reaction. Further, gel-shift analysis showed increased DNA binding activity of HIF-1α and nuclear factor-kappaB in PHD1IR compared to WTIR. In addition, nuclear translocation of β-catenin was increased in PHD1IR compared with WTIR. These findings indicated that silencing of PHD-1 attenuates myocardial I/R injury probably by enhancing HIF-1α/β-catenin/endothelial nitric oxide synthase/nuclear factor-kappaB and Bcl-2 signaling pathway.

Overexpression of PtABCC1 contributes to mercury tolerance and accumulation in Arabidopsis and poplar.

PubMed

Sun, Liping; Ma, Yifeng; Wang, Huihong; Huang, Weipeng; Wang, Xiaozhu; Han, Li; Sun, Wanmei; Han, Erqin; Wang, Bangjun

2018-03-18

Mercury (Hg) is a highly biotoxic heavy metal that contaminates the environment. Phytoremediation is a green technology for environmental remediation and is used to clean up Hg contaminated soil in recent years. In this study, we isolated an ATP-binding cassette (ABC) transporter gene PtABCC1 from Populus trichocarpa and overexpressed it in Arabidopsis and poplar. The transgenic plants conferred higher Hg tolerance than wild type (WT) plants, and overexpression of PtABCC1 could lead to 26-72% or 7-160% increase of Hg accumulation in Arabidopsis or poplar plants, respectively. These results demonstrated that PtABCC1 plays a crucial role in enhancing tolerance and accumulation to Hg in plants, which provides a promising way for phytoremediation of Hg contamination. Copyright © 2018 Elsevier Inc. All rights reserved.

The type 2 cannabinoid receptor regulates susceptibility to osteoarthritis in mice.

PubMed

Sophocleous, A; Börjesson, A E; Salter, D M; Ralston, S H

2015-09-01

Cannabinoid receptors and their ligands have been implicated in the regulation of various physiological processes but their role in osteoarthritis has not been investigated. The aim of this study was to evaluate the role of the type 2 cannabinoid receptor (Cnr2) in regulating susceptibility to osteoarthritis in mice. We analysed the severity of knee osteoarthritis as assessed by the Osteoarthritis Research Society International (OARSI) scoring system in mice with targeted deletion of Cnr2 (Cnr2(-/-)) and wild type (WT) littermates. Studies were conducted in mice subjected to surgical destabilisation of the medial meniscus (DMM) and in those with spontaneous age-related osteoarthritis (OA). Osteoarthritis was more severe following DMM in the medial compartment of the knee in Cnr2(-/-) compared with WT mice (mean ± sem score = 4.9 ± 0.5 vs 3.6 ± 0.3; P = 0.017). Treatment of WT mice with the CB2-selective agonist HU308 following DMM reduced the severity of OA in the whole joint (HU308 = 8.4 ± 0.2 vs vehicle = 10.4 ± 0.6; P = 0.007). Spontaneous age related osteoarthritis was also more severe in the medial compartment of the knee in 12-month old Cnr2(-/-) mice compared with WT (5.6 ± 0.5 vs 3.5 ± 0.3, P = 0.008). Cultured articular chondrocytes from Cnr2(-/-) mice produced less proteoglycans in vitro than wild type chondrocytes. These studies demonstrate that the Cnr2 pathway plays a role in the pathophysiology of osteoarthritis in mice and shows that pharmacological activation of CB2 has a protective effect. Further studies of the role of cannabinoid receptors in the pathogenesis of osteoarthritis in man are warranted. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Phylogenetic analysis of the haemagglutinin gene of current wild-type canine distemper viruses from South Africa: lineage Africa.

PubMed

Woma, Timothy Y; van Vuuren, Moritz; Bosman, Ana-Mari; Quan, Melvyn; Oosthuizen, Marinda

2010-07-14

There are no reports of CDV isolations in southern Africa, and although CDV is said to have geographically distinct lineages, molecular information of African strains has not yet been documented. Viruses isolated in cell cultures were subjected to reverse transcription-polymerase chain reaction (RT-PCR), and the complete H gene was sequenced and phylogenetically analysed with other strains from GenBank. Phylogenetic comparisons of the complete H gene of CDV isolates from different parts of the world (available in GenBank) with wild-type South African isolates revealed nine clades. All South African isolates form a separate African clade of their own and thus are clearly separated from the American, European, Asian, Arctic and vaccine virus clades. It is likely that only the 'African lineage' of CDV may be circulating in South Africa currently, and the viruses isolated from dogs vaccinated against CDV are not the result of reversion to virulence of vaccine strains, but infection with wild-type strains. (c) 2009 Elsevier B.V. All rights reserved.

Screening of virulence-associated genes as a molecular typing method for characterization of Streptococcus suis isolates recovered from wild boars and pigs.

PubMed

Sánchez del Rey, Verónica; Fernández-Garayzábal, José F; Domínguez, Lucas; Gottschalk, Marcelo; Vela, Ana I

2016-03-01

Streptococcus suis is an important zoonotic pathogen associated with a wide range of diseases in pigs, but has also been isolated from wild animals such as rabbits and wild boars. In the current study, 126 S. suis isolates recovered from pigs (n = 85) and wild boars (n = 41) were tested by polymerase chain reaction (PCR) for the presence of nine virulence-associated genes. S. suis isolates from wild boars were differentiated by the lower detection rates of the epf, sly, mrp, sao and dltA genes (0%, 2.4%, 2.4%, 4.8% and 21.9%, respectively) compared with the isolates from pigs (56.5%, 75.3%, 56.5%, 88.2.0% and 88.2%, respectively). The differences in the content of these virulence-associated genes were statistically significant (P < 0.05). There was a correlation between the variants saoM and saoL and serotypes 2 and 9, respectively (P < 0.05). Isolates were classified into 31 virulence-associated gene profiles (VPs). Ten VPs were detected among wild boar isolates and 22 VPs among pig isolates, with only two VPs common to wild boars and pigs. The predominant VPs among isolates from wild boars (VP1, VP7) were different from those observed in pig isolates (VP16 and VP26). VP16 was detected exclusively in clinical pig isolates of serotype 9 and VP26 was detected in 71.4% of the serotype 2 clinical pig isolates. Further multilocus sequence typing (MLST) analysis showed a significant correlation association between certain VPs and STs (VP16 and VP17 with ST123 and ST125 and VP26 with ST1). In conclusion, the current study showed that combination of virulence-associated gene profiling and MLST analysis may provide more information of the relatedness of the S. suis strains from different animal species that could be useful for epidemiological purposes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Endothelin-1 Mediated Induction of Extracellular Matrix Genes in Strial Marginal Cells Underlies Strial Pathology in Alport Mice

PubMed Central

Meehan, Daniel T.; Delimont, Duane; Dufek, Brianna; Zallocchi, Marisa; Phillips, Grady; Gratton, Michael Anne; Cosgrove, Dominic

2016-01-01

Alport syndrome, a type IV collagen disorder, manifests as glomerular disease associated with hearing loss with thickening of the glomerular and strial capillary basement membranes (SCBMs). We have identified a role for endothelin-1 (ET-1) activation of endothelin A receptors (ETARs) in glomerular pathogenesis. Here we explore whether ET-1 plays a role in strial pathology. Wild type (WT) and Alport mice were treated with the ETAR antagonist, sitaxentan. The stria vascularis was analyzed for SCBM thickness and for extracellular matrix (ECM) proteins. Additional WT and Alport mice were exposed to noise or hypoxia and the stria analyzed for hypoxia-related and ECM genes. A strial marginal cell line cultured under hypoxic conditions, or stimulated with ET-1 was analyzed for expression of hypoxia-related and ECM transcripts. Noise exposure resulted in significantly elevated ABR thresholds in Alport mice relative to wild type littermates. Alport stria showed elevated expression of collagen α1(IV), laminin α2, and laminin α5 proteins relative to WT. SCBM thickening and elevated ECM protein expression was ameliorated by ETAR blockade. Stria from normoxic Alport mice and hypoxic WT mice showed upregulation of hypoxia-related, ECM, and ET-1 transcripts. Both ET-1 stimulation and hypoxia up-regulated ECM transcripts in cultured marginal cells. We conclude that ET-1 mediated activation of ETARs on strial marginal cells results in elevated expression of ECM genes and thickening of the SCBMs in Alport mice. SCBM thickening results in hypoxic stress further elevating ECM and ET-1 gene expression, exacerbating strial pathology. PMID:27553900

Solution structure of lysine-free (K0) ubiquitin

PubMed Central

Huang, Tao; Li, Jess; Byrd, R Andrew

2014-01-01

Lysine-free ubiquitin (K0-Ub) is commonly used to study the ubiquitin-signaling pathway, where it is assumed to have the same structure and function as wild-type ubiquitin (wt-Ub). However, the K0-Ub 15N heteronuclear single quantum correlation NMR spectrum differs significantly from wt-Ub and the melting temperature is depressed by 19°C, raising the question of the structural integrity and equivalence to wt-Ub. The three-dimensional structure of K0-Ub was determined by solution NMR, using chemical shift and residual dipolar coupling data. K0-Ub adopts the same backbone structure as wt-Ub, and all significant chemical shifts can be related to interactions impacted by the K to R mutations. PMID:24591328

Isolation of Clostridium perfringens type A from wild bharals (Pseudois nayaur) following sudden death in Tibet, China.

PubMed

Zhu, Lingwei; Zhou, Wei; Wang, Tiecheng; Xiang, Haiyang; Ji, Xue; Han, Yixiao; Tian, Yuan; Sun, Yang; Liu, Jun; Guo, Xuejun

2017-04-01

Dozens of wild bharals died suddenly in Tibet. Necropsy showed severe congestion and hemorrhage in multiple organs, with large numbers of Gram-positive bacilli. Strains of Clostridium perfringens type A were isolated from the different organs and the intestinal contents. The other possible pathogens were ruled out by PCR. Copyright © 2017 Elsevier Ltd. All rights reserved.

Wild-type MIC distributions for aminoglycoside and cyclic polypeptide antibiotics used for treatment of Mycobacterium tuberculosis infections.

PubMed

Juréen, P; Angeby, K; Sturegård, E; Chryssanthou, E; Giske, C G; Werngren, J; Nordvall, M; Johansson, A; Kahlmeter, G; Hoffner, S; Schön, T

2010-05-01

The aminoglycosides and cyclic polypeptides are essential drugs in the treatment of multidrug-resistant tuberculosis, underscoring the need for accurate and reproducible drug susceptibility testing (DST). The epidemiological cutoff value (ECOFF) separating wild-type susceptible strains from non-wild-type strains is an important but rarely used tool for indicating susceptibility breakpoints against Mycobacterium tuberculosis. In this study, we established wild-type MIC distributions on Middlebrook 7H10 medium for amikacin, kanamycin, streptomycin, capreomycin, and viomycin using 90 consecutive clinical isolates and 21 resistant strains. Overall, the MIC variation between and within runs did not exceed +/-1 MIC dilution step, and validation of MIC values in Bactec 960 MGIT demonstrated good agreement. Tentative ECOFFs defining the wild type were established for all investigated drugs, including amikacin and viomycin, which currently lack susceptibility breakpoints for 7H10. Five out of seven amikacin- and kanamycin-resistant isolates were classified as susceptible to capreomycin according to the current critical concentration (10 mg/liter) but were non-wild type according to the ECOFF (4 mg/liter), suggesting that the critical concentration may be too high. All amikacin- and kanamycin-resistant isolates were clearly below the ECOFF for viomycin, and two of them were below the ECOFF for streptomycin, indicating that these two drugs may be considered for treatment of amikacin-resistant strains. Pharmacodynamic indices (peak serum concentration [Cmax]/MIC) were more favorable for amikacin and viomycin compared to kanamycin and capreomycin. In conclusion, our data emphasize the importance of establishing wild-type MIC distributions for improving the quality of drug susceptibility testing against Mycobacterium tuberculosis.

Wild-Type MIC Distributions for Aminoglycoside and Cyclic Polypeptide Antibiotics Used for Treatment of Mycobacterium tuberculosis Infections▿

PubMed Central

Juréen, P.; Ängeby, K.; Sturegård, E.; Chryssanthou, E.; Giske, C. G.; Werngren, J.; Nordvall, M.; Johansson, A.; Kahlmeter, G.; Hoffner, S.; Schön, T.

2010-01-01

The aminoglycosides and cyclic polypeptides are essential drugs in the treatment of multidrug-resistant tuberculosis, underscoring the need for accurate and reproducible drug susceptibility testing (DST). The epidemiological cutoff value (ECOFF) separating wild-type susceptible strains from non-wild-type strains is an important but rarely used tool for indicating susceptibility breakpoints against Mycobacterium tuberculosis. In this study, we established wild-type MIC distributions on Middlebrook 7H10 medium for amikacin, kanamycin, streptomycin, capreomycin, and viomycin using 90 consecutive clinical isolates and 21 resistant strains. Overall, the MIC variation between and within runs did not exceed ±1 MIC dilution step, and validation of MIC values in Bactec 960 MGIT demonstrated good agreement. Tentative ECOFFs defining the wild type were established for all investigated drugs, including amikacin and viomycin, which currently lack susceptibility breakpoints for 7H10. Five out of seven amikacin- and kanamycin-resistant isolates were classified as susceptible to capreomycin according to the current critical concentration (10 mg/liter) but were non-wild type according to the ECOFF (4 mg/liter), suggesting that the critical concentration may be too high. All amikacin- and kanamycin-resistant isolates were clearly below the ECOFF for viomycin, and two of them were below the ECOFF for streptomycin, indicating that these two drugs may be considered for treatment of amikacin-resistant strains. Pharmacodynamic indices (peak serum concentration [Cmax]/MIC) were more favorable for amikacin and viomycin compared to kanamycin and capreomycin. In conclusion, our data emphasize the importance of establishing wild-type MIC distributions for improving the quality of drug susceptibility testing against Mycobacterium tuberculosis. PMID:20237102

Estrogen Receptor Alpha Binding to ERE is Required for Full Tlr7- and Tlr9-Induced Inflammation

PubMed Central

Cunningham, Melissa A; Wirth, Jena R; Naga, Osama; Eudaly, Jackie; Gilkeson, Gary S

2014-01-01

We previously found that a maximum innate inflammatory response induced by stimulation of Toll-like receptors (TLRs) 3, 7 and 9 requires ERα, but does not require estrogen in multiple cell types from both control and lupus-prone mice. Given the estrogen-independence, we hypothesized that ERα mediates TLR signaling by tethering to, and enhancing, the activity of downstream transcription factors such as NFκB, rather than acting classically by binding EREs on target genes. To investigate the mechanism of ERα impact on TLR signaling, we utilized mice with a knock-in ERα mutant that is unable to bind ERE. After stimulation with TLR ligands, both ex vivo spleen cells and bone marrow-derived dendritic cells (BM-DCs) isolated from mutant ERα (“KIKO”) mice produced significantly less IL-6 compared with cells from wild-type (WT) littermates. These results suggest that ERα modulation of TLR signaling does indeed require ERE binding for its effect on the innate immune response. PMID:25061615

Trafficking-deficient long QT syndrome mutation KCNQ1-T587M confers severe clinical phenotype by impairment of KCNH2 membrane localization: evidence for clinically significant IKr-IKs alpha-subunit interaction.

PubMed

Biliczki, Peter; Girmatsion, Zenawit; Brandes, Ralf P; Harenkamp, Sabine; Pitard, Bruno; Charpentier, Flavien; Hébert, Terence E; Hohnloser, Stefan H; Baró, Isabelle; Nattel, Stanley; Ehrlich, Joachim R

2009-12-01

KCNQ1-T587M is a trafficking-deficient long QT syndrome (LQTS) missense mutation. Affected patients exhibit severe clinical phenotypes that are not explained by the mutant's effects on I(Ks). Previous work showed a KCNH2 and KCNQ1 alpha-subunit interaction that increases KCNH2 membrane localization and function. We hypothesized that failure of trafficking-deficient KCNQ1-T587M to enhance KCNH2 membrane expression could reduce KCNH2 current versus wild-type KCNQ1 (KCNQ1-WT), contributing to the LQTS phenotype of KCNQ1-T587M carriers. Patch-clamp, protein biochemical studies, confocal imaging, and in vivo transfection of guinea pig cardiomyocytes were performed. KCNQ1-T587M failed to generate functional current when coexpressed with KCNE1 and caused haploinsufficiency when coexpressed with KCNQ1-WT/KCNE1. Coexpression of KCNQ1-WT with KCNH2 increased I(KCNH2) versus KCNH2 alone (P <.05). Immunoblots and confocal microscopy indicated increased plasma membrane localization of KCNH2 alpha-subunits in cells cotransfected with KCNQ1-WT plasmid, while total KCNH2 protein synthesis and KCNH2 glycosylation remained unaffected, which suggests a chaperone effect of KCNQ1-WT to enhance the membrane localization of KCNH2. KCNH2 also coimmunoprecipitated with KCNQ1-WT. Although KCNQ1-T587M coprecipitated with KCNH2, the mutant was retained intracellularly and failed to increase KCNH2 membrane localization, abolishing the KCNQ1-WT chaperone function and reducing I(KCNH2) upon coexpression substantially compared with coexpression with KCNQ1-WT (P <.05). In vivo transfection of KCNQ1-T587M in guinea pigs suppressed I(Kr) in isolated cardiomyocytes. The trafficking-deficient LQTS mutation KCNQ1-T587M fails to show the chaperoning function that enhances KCNH2 membrane localization with KCNQ1-WT. This novel mechanism results in reduced I(KCNH2), which would be expected to decrease repolarization reserve and synergize with reduced I(KCNQ1) caused directly by the mutation, potentially explaining the malignant clinical phenotype in affected patients.

Monitoring Rates and Heterogeneity of High-Pressure Germination of Bacillus Spores by Phase-Contrast Microscopy of Individual Spores

DTIC Science & Technology

2014-01-01

wild-type spores but ~15-fold higher deltaTrelease values; v ) germination kinetics of wild-type spores given a ? 30 sec 140 MPa HP pulse followed by...15-fold longer than those for wild-type spores, but the two types of spores exhibited similar average Tlag values; and ( v ) the germination of wild-type...committed spores, as it does for nutrient-committed spores (14)? ( v ) Can these HP-com- mitted spores be isolated under conditions that do not allow

Capmatinib, Ceritinib, Regorafenib, or Entrectinib in Treating Patients With BRAF/NRAS Wild-Type Stage III-IV Melanoma

ClinicalTrials.gov

2017-12-20

ALK Fusion Protein Expression; BRAF wt Allele; Invasive Skin Melanoma; MET Fusion Gene Positive; NRAS wt Allele; NTRK1 Fusion Positive; NTRK2 Fusion Positive; NTRK3 Fusion Positive; RET Fusion Positive; ROS1 Fusion Positive; Stage III Cutaneous Melanoma AJCC v7; Stage IIIA Cutaneous Melanoma AJCC v7; Stage IIIB Cutaneous Melanoma AJCC v7; Stage IIIC Cutaneous Melanoma AJCC v7; Stage IV Cutaneous Melanoma AJCC v6 and v7

Palm Mutants in DNA Polymerases α and η Alter DNA Replication Fidelity and Translesion Activity

PubMed Central

Niimi, Atsuko; Limsirichaikul, Siripan; Yoshida, Shonen; Iwai, Shigenori; Masutani, Chikahide; Hanaoka, Fumio; Kool, Eric T.; Nishiyama, Yukihiro; Suzuki, Motoshi

2004-01-01

We isolated active mutants in Saccharomyces cerevisiae DNA polymerase α that were associated with a defect in error discrimination. Among them, L868F DNA polymerase α has a spontaneous error frequency of 3 in 100 nucleotides and 570-fold lower replication fidelity than wild-type (WT) polymerase α. In vivo, mutant DNA polymerases confer a mutator phenotype and are synergistic with msh2 or msh6, suggesting that DNA polymerase α-dependent replication errors are recognized and repaired by mismatch repair. In vitro, L868F DNA polymerase α catalyzes efficient bypass of a cis-syn cyclobutane pyrimidine dimer, extending the 3′ T 26,000-fold more efficiently than the WT. Phe34 is equivalent to residue Leu868 in translesion DNA polymerase η, and the F34L mutant of S. cerevisiae DNA polymerase η has reduced translesion DNA synthesis activity in vitro. These data suggest that high-fidelity DNA synthesis by DNA polymerase α is required for genomic stability in yeast. The data also suggest that the phenylalanine and leucine residues in translesion and replicative DNA polymerases, respectively, might have played a role in the functional evolution of these enzyme classes. PMID:15024063

Iron dysregulation combined with aging prevents sepsis-induced apoptosis

PubMed Central

Javadi, Pardis; Buchman, Timothy G.; Stromberg, Paul E.; Turnbull, Isaiah R.; Vyas, Dinesh; Hotchkiss, Richard S.; Karl, Irene E.; Coopersmith, Craig M.

2005-01-01

Background Sepsis, iron loading and aging cause independent increases in gut epithelial and splenic apoptosis. It is unknown how their combination will affect apoptosis and systemic cytokine levels. Methods Hfe−/− mice (a murine homolog of hemochromatosis) abnormally accumulate iron in their tissues. Aged (24–26 months) or mature (16–18 months) Hfe−/− mice and wild type (WT) littermates were subjected to cecal ligation and puncture (CLP) or sham laparotomy. Intestine, spleen, and blood were harvested 24 hours later and assessed for apoptosis and cytokine levels. Results Gut epithelial and splenic apoptosis were low in both aged septic and sham Hfe−/− mice, regardless of the amount of iron in their diet. Mature septic WT mice had increased apoptosis compared to age-matched sham WT mice. Mature septic Hfe−/− mice had similar levels of intestinal cell death to age-matched septic WT mice but higher levels of splenic apoptosis. Apoptosis was significantly lower in septic aged Hfe−/− mice than septic mature Hfe−/− animals. Interleukin-6 was elevated in septic aged Hfe−/− mice compared to sham mice. Conclusions Although sepsis, chronic iron dysregulation, and aging each increase gut and splenic apoptosis, their combination yields cell death levels similar to sham animals despite the fact that aged Hfe−/− mice are able to mount an inflammatory response following CLP and mature Hfe−/− mice have elevated sepsis-induced apoptosis. Combining sepsis with two risk factors that ordinarily increase cell death and increase mortality in CLP yields an apoptotic response that could not have been predicted based upon each element in isolation. PMID:15921699

Preconditioning by isoflurane elicits mitochondrial protective mechanisms independent of sarcolemmal KATP channel in mouse cardiomyocytes

PubMed Central

Muravyeva, Maria; Sedlic, Filip; Dolan, Nicholas; Bosnjak, Zeljko J; Stadnicka, Anna

2013-01-01

Cardiac mitochondria and the sarcolemmal (sarc)KATP channels contribute to cardioprotective signaling of anesthetic-induced preconditioning (APC). Changes in mitochondrial bioenergetics influence the sarcKATP channel function, but whether this channel has impacts on mitochondria is uncertain. We used the mouse model with deleted pore-forming Kir6.2 subunit of sarcKATP channel (Kir6.2 KO) to investigate whether the functional sarcKATP channels are necessary for isoflurane activation of mitochondrial protective mechanisms. Ventricular cardiomyocytes were isolated from C57Bl6 wild type (WT) and Kir6.2 KO mouse hearts. Flavoprotein autofluorescence, mitochondrial ROS production and mitochondrial membrane potential were monitored by laser-scanning confocal microscopy in intact cardiomyocytes. Cell survival was assessed using H2O2-induced stress. Isoflurane (0.5 mM) increased flavoprotein fluorescence to 180±14% and 190±15% and ROS production to 118±2% and 124±6% of baseline in WT and Kir6.2 KO myocytes, respectively. TMRE fluorescence decreased to 84±6% in WT and to 86±4% in Kir6.2 KO myocytes. This effect was abolished by 5HD. Pretreatment with isoflurane decreased the stress-induced cell death from 31±1% to 21±1% in WT and from 44±2% to 35±2% in Kir6.2 KO myocytes. In conclusion, Kir6.2 deletion increases sensitivity of intact cardiomyocytes t o oxidative stress, but does not alter the isoflurane-elicited protective mitochondrial mechanisms, suggesting independent roles for cardiac mitochondria and sarcKATP channels in APC by isoflurane. PMID:23318991

[Molecular pathogenesis of Waardenburg syndrome type II resulting from SOX10 gene mutation].

PubMed

Zhang, Hua; Chen, Hongsheng; Feng, Yong; Qian, Minfei; Li, Jiping; Liu, Jun; Zhang, Chun

2016-08-01

To explore the molecular mechanism of Waardenburg syndrome type II (WS2) resulting from SOX10 gene mutation E248fs through in vitro experiment. 293T cells were transiently transfected with wild type (WT) SOX10 and mutant type (MT) E248fs plasmids. The regulatory effect of WT/MT SOX10 on the transcriptional activity of MITF gene and influence of E248fs on WT SOX10 function were determined with a luciferase activity assay. The DNA binding capacity of the WT/MT SOX10 with the promoter of the MITF gene was determined with a biotinylated double-stranded oligonucleotide probe containing the SOX10 binding sequence cattgtc to precipitate MITF and E248fs, respectively. The stability of SOX10 and E248fs were also analyzed. As a loss-of-function mutation, the E248fs mutant failed to transactivate the MITF promoter as compared with the WT SOX10 (P<0.01), which also showed a dominant-negative effect on WT SOX10. The WT SOX10 and E248fs mutant were also able to bind specifically to the cattgtc motif in the MITF promoter, whereas E248fs had degraded faster than WT SOX10. Despite the fact that the E248fs has a dominant-negative effect on SOX10, its reduced stability may down-regulate the transcription of MITF and decrease the synthesis of melanin, which may result in haploinsufficiency of SOX10 protein and cause the milder WS2 phenotype.

Toll-like receptor 2-independent host innate immune response against an epidemic strain of Streptococcus suis that causes a toxic shock-like syndrome in humans.

PubMed

Lachance, Claude; Segura, Mariela; Gerber, Pehuén Pereyra; Xu, Jianguo; Gottschalk, Marcelo

2013-01-01

Streptococcus suis is an emerging zoonotic agent causing meningitis and septicemia. Outbreaks in humans in China with atypical cases of streptococcal toxic shock-like syndrome have been described to be caused by a clonal epidemic S. suis strain characterized as sequence type (ST) 7 by multilocus sequence typing, different from the classical ST1 usually isolated in Europe. Previous in vitro studies showed that Toll-like receptor (TLR) 2 plays a major role in S. suis ST1 interactions with host cells. In the present study, the in vivo role of TLR2 in systemic infections caused by S. suis ST1 or ST7 strains using TLR2 deficient (TLR2(-/-)) mice was evaluated. TLR2-mediated recognition significantly contributes to the acute disease caused by the highly virulent S. suis ST1 strain, since the TLR2(-/-) mice remained unaffected when compared to wild type (WT) mice. The lack of mortality could not be associated with a lower bacterial burden; however, a significant decrease in the induction of pro-inflammatory mediators, as evaluated by microarray, real-time PCR and protein assays, was observed. On the other hand, TLR2(-/-) mice infected with the epidemic ST7 strain presented no significant differences regarding survival and expression of pro-inflammatory mediators when compared to the WT mice. Together, these results show a TLR2-independent host innate immune response to S. suis that depends on the strain.

Differential composition of culture supernatants from wild-type Brucella abortus and its isogenic virB mutants.

PubMed

Delpino, M Victoria; Comerci, Diego J; Wagner, Mary Ann; Eschenbrenner, Michel; Mujer, Cesar V; Ugalde, Rodolfo A; Fossati, Carlos A; Baldi, Pablo C; Delvecchio, Vito G

2009-07-01

The virB genes coding type IV secretion system are necessary for the intracellular survival and replication of Brucella spp. In this study, extracellular proteins from B. abortus 2308 (wild type, WT) and its isogenic virB10 polar mutant were compared. Culture supernatants harvested in the early stationary phase were concentrated and subjected to 2D electrophoresis. Spots present in the WT strain but absent in the virB10 mutant (differential spots) were considered extracellular proteins released in a virB-related manner, and were identified by MALDI-TOF analysis and matching with Brucella genomes. Among the 11 differential proteins identified, DnaK chaperone (Hsp70), choloylglycine hydrolase (CGH) and a peptidyl-prolyl cis-trans isomerase (PPIase) were chosen for further investigation because of their homology with extracellular and/or virulence factors from other bacteria. The three proteins were obtained in recombinant form and specific monoclonal antibodies (mAbs) were prepared. By Western blot with these mAbs, the three proteins were detected in supernatants from the WT but not in those from the virB10 polar mutant or from strains carrying non-polar mutations in virB10 or virB11 genes. These results suggest that the expression of virB genes affects the extracellular release of DnaK, PPIase and CGH, and possibly other proteins from B. abortus.

Changes in oil content of transgenic soybeans expressing the yeast SLC1 gene.

PubMed

Rao, Suryadevara S; Hildebrand, David

2009-10-01

The wild type (Wt) and mutant form of yeast (sphingolipid compensation) genes, SLC1 and SLC1-1, have been shown to have lysophosphatidic acid acyltransferase (LPAT) activities (Nageic et al. in J Biol Chem 269:22156-22163, 1993). Expression of these LPAT genes was reported to increase oil content in transgenic Arabidopsis and Brassica napus. It is of interest to determine if the TAG content increase would also be seen in soybeans. Therefore, the wild type SLC1 was expressed in soybean somatic embryos under the control of seed specific phaseolin promoter. Some transgenic somatic embryos and in both T2 and T3 transgenic seeds showed higher oil contents. Compared to controls, the average increase in triglyceride values went up by 1.5% in transgenic somatic embryos. A maximum of 3.2% increase in seed oil content was observed in a T3 line. Expression of the yeast Wt LPAT gene did not alter the fatty acid composition of the seed oil.

Mutants of the pentose-fermenting yeast Pachysolen tannophilus tolerant to hardwood spent sulfite liquor and acetic acid.

PubMed

Harner, Nicole K; Bajwa, Paramjit K; Habash, Marc B; Trevors, Jack T; Austin, Glen D; Lee, Hung

2014-01-01

A strain development program was initiated to improve the tolerance of the pentose-fermenting yeast Pachysolen tannophilus to inhibitors in lignocellulosic hydrolysates. Several rounds of UV mutagenesis followed by screening were used to select for mutants of P. tannophilus NRRL Y2460 with improved tolerance to hardwood spent sulfite liquor (HW SSL) and acetic acid in separate selection lines. The wild type (WT) strain grew in 50 % (v/v) HW SSL while third round HW SSL mutants (designated UHW301, UHW302 and UHW303) grew in 60 % (v/v) HW SSL, with two of these isolates (UHW302 and UHW303) being viable and growing, respectively, in 70 % (v/v) HW SSL. In defined liquid media containing acetic acid, the WT strain grew in 0.70 % (w/v) acetic acid, while third round acetic acid mutants (designated UAA301, UAA302 and UAA303) grew in 0.80 % (w/v) acetic acid, with one isolate (UAA302) growing in 0.90 % (w/v) acetic acid. Cross-tolerance of HW SSL-tolerant mutants to acetic acid and vice versa was observed with UHW303 able to grow in 0.90 % (w/v) acetic acid and UAA302 growing in 60 % (v/v) HW SSL. The UV-induced mutants retained the ability to ferment glucose and xylose to ethanol in defined media. These mutants of P. tannophilus are of considerable interest for bioconversion of the sugars in lignocellulosic hydrolysates to ethanol.

Embryonic catalase protects against ethanol embryopathies in acatalasemic mice and transgenic human catalase-expressing mice in embryo culture.

PubMed

Miller-Pinsler, Lutfiya; Wells, Peter G

2015-09-15

Reactive oxygen species (ROS) have been implicated in the mechanism of ethanol (EtOH) teratogenicity, but the protective role of the embryonic antioxidative enzyme catalase is unclear, as embryonic activity is only about 5% of maternal levels. We addressed this question in a whole embryo culture model. C57BL/6 mouse embryos expressing human catalase (hCat) or their wild-type (C57BL/6 WT) controls, and C3Ga.Cg-Cat(b)/J catalase-deficient, acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug=GD 1), exposed for 24h to 2 or 4mg/mL EtOH or vehicle, and evaluated for functional and morphological changes. hCat and C57BL/6 WT vehicle-exposed embryos developed normally, while EtOH was embryopathic in C57BL/6 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed, turning and head length, whereas hCat embryos were protected (p<0.001). Maternal pretreatment of C57BL/6 WT dams with 50kU/kg PEG-catalase (PEG-cat) 8h prior to embryo culture, which increases embryonic catalase activity, blocked all EtOH embryopathies (p<0.001). Vehicle-exposed aCat mouse embryos had lower yolk sac diameters compared to WT controls, suggesting that endogenous ROS are embryopathic. EtOH was more embryopathic in aCat embryos than WT controls, evidenced by reduced head length and somite development (p<0.01), and trends for reduced anterior neuropore closure, turning and crown-rump length. Maternal pretreatment of aCat dams with PEG-Cat blocked all EtOH embryopathies (p<0.05). These data suggest that embryonic catalase is a determinant of risk for EtOH embryopathies. Copyright © 2015 Elsevier Inc. All rights reserved.

Genotype differences in anxiety and fear learning and memory of WT and ApoE4 mice associated with enhanced generation of hippocampal reactive oxygen species.

PubMed

Villasana, Laura E; Weber, Sydney; Akinyeke, Tunde; Raber, Jacob

2016-09-01

Apolipoprotein E (apoE), involved in cholesterol and lipid metabolism, also influences cognitive function and injury repair. In humans, apoE is expressed in three isoforms. E4 is a risk factor for age-related cognitive decline and Alzheimer's disease, particularly in women. E4 might also be a risk factor for developing behavioral and cognitive changes following (56) Fe irradiation, a component of the space environment astronauts are exposed to during missions. These changes might be related to enhanced generation of reactive oxygen species (ROS). In this study, we compared the behavioral and cognitive performance of sham-irradiated and irradiated wild-type (WT) mice and mice expressing the human E3 or E4 isoforms, and assessed the generation of ROS in hippocampal slices from these mice. E4 mice had greater anxiety-like and conditioned fear behaviors than WT mice, and these genotype differences were associated with greater levels of ROS in E4 than WT mice. The greater generation of ROS in the hippocampus of E4 than WT mice might contribute to their higher anxiety levels and enhanced fear conditioning. In E4, but not WT, mice, phorbol-12-myristate-13-acetate-treated hippocampal slices showed more dihydroxy ethidium oxidation in sham-irradiated than irradiated mice and hippocampal heme oxygenase-1 levels were higher in irradiated than sham-irradiated E4 mice. Mice with apolipoprotein E4 (E4), a risk factor for Alzheimer's disease, have greater anxiety-like and conditioned fear behaviors than wild-type (WT) mice. Generation of reactive oxygen species (ROS, in red) 3 months following (56) Fe irradiation, a component of the space environment astronauts are exposed to, is more pronounced in the hippocampus of E4 than WT mice. In E4, but not WT, mice, hippocampal levels of the oxidative stress-relevant marker heme oxygenase-1 are higher in irradiated than sham-irradiated E4 mice. © 2016 International Society for Neurochemistry.

Glutaminase and MMP-9 Downregulation in Cortex and Hippocampus of LPA1 Receptor Null Mice Correlate with Altered Dendritic Spine Plasticity

PubMed Central

Peñalver, Ana; Campos-Sandoval, José A.; Blanco, Eduardo; Cardona, Carolina; Castilla, Laura; Martín-Rufián, Mercedes; Estivill-Torrús, Guillermo; Sánchez-Varo, Raquel; Alonso, Francisco J.; Pérez-Hernández, Mercedes; Colado, María I.; Gutiérrez, Antonia; de Fonseca, Fernando Rodríguez; Márquez, Javier

2017-01-01

Lysophosphatidic acid (LPA) is an extracellular lipid mediator that regulates nervous system development and functions acting through G protein-coupled receptors (GPCRs). Here we explore the crosstalk between LPA1 receptor and glutamatergic transmission by examining expression of glutaminase (GA) isoforms in different brain areas isolated from wild-type (WT) and KOLPA1 mice. Silencing of LPA1 receptor induced a severe down-regulation of Gls-encoded long glutaminase protein variant (KGA) (glutaminase gene encoding the kidney-type isoforms, GLS) protein expression in several brain regions, particularly in brain cortex and hippocampus. Immunohistochemical assessment of protein levels for the second type of glutaminase (GA) isoform, glutaminase gene encoding the liver-type isoforms (GLS2), did not detect substantial differences with regard to WT animals. The regional mRNA levels of GLS were determined by real time RT-PCR and did not show significant variations, except for prefrontal and motor cortex values which clearly diminished in KO mice. Total GA activity was also significantly reduced in prefrontal and motor cortex, but remained essentially unchanged in the hippocampus and rest of brain regions examined, suggesting activation of genetic compensatory mechanisms and/or post-translational modifications to compensate for KGA protein deficit. Remarkably, Golgi staining of hippocampal regions showed an altered morphology of glutamatergic pyramidal cells dendritic spines towards a less mature filopodia-like phenotype, as compared with WT littermates. This structural change correlated with a strong decrease of active matrix-metalloproteinase (MMP) 9 in cerebral cortex and hippocampus of KOLPA1 mice. Taken together, these results demonstrate that LPA signaling through LPA1 influence expression of the main isoenzyme of glutamate biosynthesis with strong repercussions on dendritic spines maturation, which may partially explain the cognitive and learning defects previously reported for this colony of KOLPA1 mice. PMID:28928633

Comparison of Ehrlichia muris strains isolated from wild mice and ticks and serologic survey of humans and animals with E. muris as antigen.

PubMed

Kawahara, M; Ito, T; Suto, C; Shibata, S; Rikihisa, Y; Hata, K; Hirai, K

1999-04-01

In metropolitan Tokyo, the Ehrlichia muris seropositivity rate of 24 wild mice was 63% in Hinohara Village, but in the surrounding areas, it was 0 to 5%. This finding suggests that the reservoir of E. muris is focal. Among the 15 seropositive mice, ehrlichiae were isolated from 9 Apodemus speciosus mice and 1 A. argenteus mouse, respectively. Five ehrlichial isolates were obtained from 10 ticks (Haemaphysalis flava) collected in Asuke Town, Aichi Prefecture, where the E. muris type strain had been isolated. These new isolates were compared with the E. muris type strain. The mouse virulence and ultrastructure of the new isolates were similar to those of the type strain, and all of them were cross-reactive with each other, as well as with the type strain, by indirect immunofluorescent-antibody test. The levels of similarity of the base sequences of the 16S rRNA gene of one of the A. speciosus isolates and one of the tick isolates to that of the E. muris type strain were 99.79 and 99.93%, respectively. We suggest that all of these isolates are E. muris; that E. muris is not limited to Eothenomys kageus but infects other species of mice; and that E. muris is present at locations other than Aichi Prefecture. It appears that H. flava is a potential vector of E. muris. Twenty (1%) of 1803 humans from metropolitan Tokyo were found to be seropositive for E. muris antibodies. A serological survey revealed that exposure to E. muris or organisms antigenically cross-reactive to E. muris occurred among dogs, wild mice, monkeys, bears, deer, and wild boars in Gifu Prefecture, nearby prefectures, and Nagoya City, central Japan. However, human beings and Rattus norvegicus rats in this area were seronegative. These results indicate broader geographic distribution of and human and animal species exposure to E. muris or related Ehrlichia spp. in Japan.

Replication of type 5 adenovirus promotes middle ear infection by Streptococcus pneumoniae in the chinchilla model of otitis media

PubMed Central

Murrah, Kyle A.; Turner, Roberta L.; Pang, Bing; Perez, Antonia C.; Reimche, Jennifer L.; King, Lauren B.; Wren, John; Gandhi, Uma; Swords, W. Edward; Ornelles, David A.

2015-01-01

Adenoviral infection is a major risk factor for otitis media. We hypothesized that adenovirus promotes bacterial ascension into the middle ear through the disruption of normal function in the Eustachian tubes due to inflammation-induced changes. An intranasal infection model of the chinchilla was used to test the ability of type 5 adenovirus to promote middle ear infection by Streptococcus pneumoniae. The hyperinflammatory adenovirus mutant dl327 and the nonreplicating adenovirus mutant H5wt300ΔpTP were used to test the role of inflammation and viral replication, respectively, in promotion of pneumococcal middle ear infection. Precedent infection with adenovirus resulted in a significantly greater incidence of middle ear disease by S. pneumoniae as compared to nonadenovirus infected animals. Infection with the adenovirus mutant dl327 induced a comparable degree of bacterial ascension into the middle ear as did infection with the wild-type virus. By contrast, infection with the nonreplicating adenovirus mutant H5wt300ΔpTP resulted in less extensive middle ear infection compared to the wild-type adenovirus. We conclude that viral replication is necessary for adenoviral-induced pneumococcal middle ear disease. PMID:25251686

Grain Filling Characteristics and Their Relations with Endogenous Hormones in Large- and Small-Grain Mutants of Rice.

PubMed

Zhang, Weiyang; Cao, Zhuanqin; Zhou, Qun; Chen, Jing; Xu, Gengwen; Gu, Junfei; Liu, Lijun; Wang, Zhiqin; Yang, Jianchang; Zhang, Hao

2016-01-01

This study determined if the variation in grain filling parameters between two different spikelet types of rice (Oryza sativa L.) is regulated by the hormonal levels in the grains. Two rice mutants, namely, a large-grain mutant (AZU-M) and a small-grain mutant (ZF802-M), and their respective wild types (AZU-WT and ZF802-WT) were grown in the field. The endosperm cell division rate, filling rate, and hormonal levels: zeatin + zeatin riboside (Z+ZR), indo-3-acetic acid (IAA), polyamines (PAs), and abscisic acid (ABA) were determined. The results showed that there was no significant difference between the filling and endosperm cell division rates. These rates were synchronous between the superior and inferior spikelets for both mutants. However, the abovementioned parameters were significantly different between the two spikelet types for the two wild types. The superior spikelets filled faster and their filling rate was higher compared to the inferior ones. Changes in the concentrations of plant hormones were consistent with the observed endosperm cell division rate and the filling rate for both types of spikelets of mutant and wild type plants. Regression analysis showed a significant positive correlation between cell division and filling rates with the concentrations of the investigated hormones. Exogenous chemical application verified the role of ABA, IAA, and PAs in grain filling. The results indicate that poor filling of inferior spikelets in rice occurs primarily due to the reduced hormone concentrations therein, leading to lower division rate of endosperm cells, fewer endosperm cells, slower filling rate, and smaller grain weight.

Grain Filling Characteristics and Their Relations with Endogenous Hormones in Large- and Small-Grain Mutants of Rice

PubMed Central

Zhang, Weiyang; Cao, Zhuanqin; Zhou, Qun; Chen, Jing; Xu, Gengwen; Gu, Junfei; Liu, Lijun; Wang, Zhiqin; Yang, Jianchang; Zhang, Hao

2016-01-01

This study determined if the variation in grain filling parameters between two different spikelet types of rice (Oryza sativa L.) is regulated by the hormonal levels in the grains. Two rice mutants, namely, a large-grain mutant (AZU-M) and a small-grain mutant (ZF802-M), and their respective wild types (AZU-WT and ZF802-WT) were grown in the field. The endosperm cell division rate, filling rate, and hormonal levels: zeatin + zeatin riboside (Z+ZR), indo-3-acetic acid (IAA), polyamines (PAs), and abscisic acid (ABA) were determined. The results showed that there was no significant difference between the filling and endosperm cell division rates. These rates were synchronous between the superior and inferior spikelets for both mutants. However, the abovementioned parameters were significantly different between the two spikelet types for the two wild types. The superior spikelets filled faster and their filling rate was higher compared to the inferior ones. Changes in the concentrations of plant hormones were consistent with the observed endosperm cell division rate and the filling rate for both types of spikelets of mutant and wild type plants. Regression analysis showed a significant positive correlation between cell division and filling rates with the concentrations of the investigated hormones. Exogenous chemical application verified the role of ABA, IAA, and PAs in grain filling. The results indicate that poor filling of inferior spikelets in rice occurs primarily due to the reduced hormone concentrations therein, leading to lower division rate of endosperm cells, fewer endosperm cells, slower filling rate, and smaller grain weight. PMID:27780273

In vitro and in vivo host range of Anticarsia gemmatalis multiple nuclear polyhedrosis virus.

PubMed

Grasela, J J; McIntosh, A H

1998-01-01

A clone of the wild type (wt) Anticarsia gemmatalis multiple nuclear polyhedrosis virus AgMNPV, derived from a geographical isolate (Hondrina, Brazil) and designated AgMNPV-CL4-3A1, was used to determine the host range of this virus in six established lepidopteran cell lines: Anticarsia gemmatalis (BCIRL-AG-AM1), Helicoverpa zea (BCIRL-HZ-AM1), Heliothis virescens (BCIRL-HV-AM1), Helicoverpa armigera (BCIRL-HA-AM1), Trichoplusia ni (TN-CL1), Bombyx mori (BMN), and a coleopteran cell line Anthonomus grandis (BRL-AG-1). In addition, the in vivo host range of this clone was also assayed in larvae of Helicoverpa zea, Heliothis virescens, Trichoplusia ni, and the homologous species Anticarsia gemmatalis by probit analysis. On the basis of temporal studies of TCID50 values, BCIRL-HV-AM1 cells gave the highest extracellular virus (ECV) titer (9.7 x 10(6) TCID50/ml) followed by BCIRL-HA-AM1 cells (8.3 x 10(5) TCID50/ml) and BCIRL-AG-AM1 cells (3.2 x 10(5) TCID50/ml). In addition, a low ECV titer of 1.37 x 10(3) TCID50/ml was detected from TN-CL1 cells 96 h postinoculation, while BRL-AG-1, BMN, and BCIRL-HZ-AM1 cells were nonpermissive to AgMNPV-CL4-3A1 on the basis of TCID50 results. AgMNPV-CL4-3A1 and the wild type AgMNPV had similar restriction profiles that were different from wild type AcMNPV. The LC50 values were 96.9, 564.6, 733.3, and 1.1 x 10(4) occlusion bodies/cm2 of diet for A. gemmatalis, Helicoverpa zea, Heliothis virescens, and T. ni, respectively.

Action potentials and ion conductances in wild-type and CALHM1-knockout type II taste cells

PubMed Central

Saung, Wint Thu; Foskett, J. Kevin

2017-01-01

Taste bud type II cells fire action potentials in response to tastants, triggering nonvesicular ATP release to gustatory neurons via voltage-gated CALHM1-associated ion channels. Whereas CALHM1 regulates mouse cortical neuron excitability, its roles in regulating type II cell excitability are unknown. In this study, we compared membrane conductances and action potentials in single identified TRPM5-GFP-expressing circumvallate papillae type II cells acutely isolated from wild-type (WT) and Calhm1 knockout (KO) mice. The activation kinetics of large voltage-gated outward currents were accelerated in cells from Calhm1 KO mice, and their associated nonselective tail currents, previously shown to be highly correlated with ATP release, were completely absent in Calhm1 KO cells, suggesting that CALHM1 contributes to all of these currents. Calhm1 deletion did not significantly alter resting membrane potential or input resistance, the amplitudes and kinetics of Na+ currents either estimated from action potentials or recorded from steady-state voltage pulses, or action potential threshold, overshoot peak, afterhyperpolarization, and firing frequency. However, Calhm1 deletion reduced the half-widths of action potentials and accelerated the deactivation kinetics of transient outward currents, suggesting that the CALHM1-associated conductance becomes activated during the repolarization phase of action potentials. NEW & NOTEWORTHY CALHM1 is an essential ion channel component of the ATP neurotransmitter release mechanism in type II taste bud cells. Its contribution to type II cell resting membrane properties and excitability is unknown. Nonselective voltage-gated currents, previously associated with ATP release, were absent in cells lacking CALHM1. Calhm1 deletion was without effects on resting membrane properties or voltage-gated Na+ and K+ channels but contributed modestly to the kinetics of action potentials. PMID:28202574

Action potentials and ion conductances in wild-type and CALHM1-knockout type II taste cells.

PubMed

Ma, Zhongming; Saung, Wint Thu; Foskett, J Kevin

2017-05-01

Taste bud type II cells fire action potentials in response to tastants, triggering nonvesicular ATP release to gustatory neurons via voltage-gated CALHM1-associated ion channels. Whereas CALHM1 regulates mouse cortical neuron excitability, its roles in regulating type II cell excitability are unknown. In this study, we compared membrane conductances and action potentials in single identified TRPM5-GFP-expressing circumvallate papillae type II cells acutely isolated from wild-type (WT) and Calhm1 knockout (KO) mice. The activation kinetics of large voltage-gated outward currents were accelerated in cells from Calhm1 KO mice, and their associated nonselective tail currents, previously shown to be highly correlated with ATP release, were completely absent in Calhm1 KO cells, suggesting that CALHM1 contributes to all of these currents. Calhm1 deletion did not significantly alter resting membrane potential or input resistance, the amplitudes and kinetics of Na + currents either estimated from action potentials or recorded from steady-state voltage pulses, or action potential threshold, overshoot peak, afterhyperpolarization, and firing frequency. However, Calhm1 deletion reduced the half-widths of action potentials and accelerated the deactivation kinetics of transient outward currents, suggesting that the CALHM1-associated conductance becomes activated during the repolarization phase of action potentials. NEW & NOTEWORTHY CALHM1 is an essential ion channel component of the ATP neurotransmitter release mechanism in type II taste bud cells. Its contribution to type II cell resting membrane properties and excitability is unknown. Nonselective voltage-gated currents, previously associated with ATP release, were absent in cells lacking CALHM1. Calhm1 deletion was without effects on resting membrane properties or voltage-gated Na + and K + channels but contributed modestly to the kinetics of action potentials. Copyright © 2017 the American Physiological Society.

Differential Responses of Polyamines and Antioxidants to Drought in a Centipedegrass Mutant in Comparison to Its Wild Type Plants

PubMed Central

Liu, Mingxi; Chen, Jingjing; Guo, Zhenfei; Lu, Shaoyun

2017-01-01

Centipedegrass (Eremochloa ophiuroides [Munro] Hack.) is an important warm-season turfgrass species with low turf maintenance requirements. However, our knowledge on physiological adaptation of centipedegrass to drought stress is limited. Physiological responses to drought in a gamma-ray-induced mutant 22-1 as compared with two wild type (WT) lines were analyzed for understanding of drought tolerance mechanism of centipedegrass. The mutant showed an elevated drought tolerance with higher levels of relative water content, net photosynthetic rate (A) and stomatal conductance (gs) and lower levels of ion leakage and malondialdehyde (MDA) under drought stress as compared with WT plants. A showed significant correlation with gs and MDA. Higher levels of antioxidant enzymes activities, non-enzyme antioxidants, and polyamines including putrescine (Put), spermidine (Spd), and spermine (Spm) were maintained in 22-1 than in WT plants. Superoxide dismutase (SOD), catalase (CAT), ascorbate-peroxidase (APX), and glutathione reductase (GR) activities and ascorbic acid (AsA) content were significantly correlated with both Put and Spd levels, and reduced glutathione level was correlated with Put during drought stress. Exogenous application of Put, Spd, and Spm increased drought tolerance and activities of SOD, CAT, APX, and GR in WT plants. The results suggest that higher levels of polyamines and antioxidant defense system are associated with the elevated drought tolerance in 22-1, which may improve protection on photosynthesis against drought induced oxidative damage. PMID:28559909

CAR-Engineered NK Cells Targeting Wild-Type EGFR and EGFRvIII Enhance Killing of Glioblastoma and Patient-Derived Glioblastoma Stem Cells.

PubMed

Han, Jianfeng; Chu, Jianhong; Keung Chan, Wing; Zhang, Jianying; Wang, Youwei; Cohen, Justus B; Victor, Aaron; Meisen, Walter H; Kim, Sung-hak; Grandi, Paola; Wang, Qi-En; He, Xiaoming; Nakano, Ichiro; Chiocca, E Antonio; Glorioso, Joseph C; Kaur, Balveen; Caligiuri, Michael A; Yu, Jianhua

2015-07-09

Glioblastoma (GB) remains the most aggressive primary brain malignancy. Adoptive transfer of chimeric antigen receptor (CAR)-modified immune cells has emerged as a promising anti-cancer approach, yet the potential utility of CAR-engineered natural killer (NK) cells to treat GB has not been explored. Tumors from approximately 50% of GB patients express wild-type EGFR (wtEGFR) and in fewer cases express both wtEGFR and the mutant form EGFRvIII; however, previously reported CAR T cell studies only focus on targeting EGFRvIII. Here we explore whether both wtEGFR and EGFRvIII can be effectively targeted by CAR-redirected NK cells to treat GB. We transduced human NK cell lines NK-92 and NKL, and primary NK cells with a lentiviral construct harboring a second generation CAR targeting both wtEGFR and EGFRvIII and evaluated the anti-GB efficacy of EGFR-CAR-modified NK cells. EGFR-CAR-engineered NK cells displayed enhanced cytolytic capability and IFN-γ production when co-cultured with GB cells or patient-derived GB stem cells in an EGFR-dependent manner. In two orthotopic GB xenograft mouse models, intracranial administration of NK-92-EGFR-CAR cells resulted in efficient suppression of tumor growth and significantly prolonged the tumor-bearing mice survival. These findings support intracranial administration of NK-92-EGFR-CAR cells represents a promising clinical strategy to treat GB.

Improvement of enzyme activity of β-1,3-1,4-glucanase from Paenibacillus sp. X4 by error-prone PCR and structural insights of mutated residues.

PubMed

Baek, Seung Cheol; Ho, Thien-Hoang; Lee, Hyun Woo; Jung, Won Kyeong; Gang, Hyo-Seung; Kang, Lin-Woo; Kim, Hoon

2017-05-01

β-1,3-1,4-Glucanase (BGlc8H) from Paenibacillus sp. X4 was mutated by error-prone PCR or truncated using termination primers to improve its enzyme properties. The crystal structure of BGlc8H was determined at a resolution of 1.8 Å to study the possible roles of mutated residues and truncated regions of the enzyme. In mutation experiments, three clones of EP 2-6, 2-10, and 5-28 were finally selected that exhibited higher specific activities than the wild type when measured using their crude extracts. Enzyme variants of BG 2-6 , BG 2-10 , and BG 5-28 were mutated at two, two, and six amino acid residues, respectively. These enzymes were purified homogeneously by Hi-Trap Q and CHT-II chromatography. Specific activity of BG 5-28 was 2.11-fold higher than that of wild-type BG wt , whereas those of BG 2-6 and BG 2-10 were 0.93- and 1.19-fold that of the wild type, respectively. The optimum pH values and temperatures of the variants were nearly the same as those of BG wt (pH 5.0 and 40 °C, respectively). However, the half-life of the enzyme activity and catalytic efficiency (k cat /K m ) of BG 5-28 were 1.92- and 2.12-fold greater than those of BG wt at 40 °C, respectively. The catalytic efficiency of BG 5-28 increased to 3.09-fold that of BG wt at 60 °C. These increases in the thermostability and catalytic efficiency of BG 5-28 might be useful for the hydrolysis of β-glucans to produce fermentable sugars. Of the six mutated residues of BG 5-28 , five residues were present in mature BGlc8H protein, and two of them were located in the core scaffold of BGlc8H and the remaining three residues were in the substrate-binding pocket forming loop regions. In truncation experiments, three forms of C-terminal truncated BGlc8H were made, which comprised 360, 286, and 215 amino acid residues instead of the 409 residues of the wild type. No enzyme activity was observed for these truncated enzymes, suggesting the complete scaffold of the α 6 /α 6 -double-barrel structure is essential for enzyme activity.

Interleukin-12- and interferon-gamma-mediated natural killer cell activation by Agaricus blazei Murill.

PubMed

Yuminamochi, Eri; Koike, Taisuke; Takeda, Kazuyoshi; Horiuchi, Isao; Okumura, Ko

2007-06-01

Dried fruiting bodies of Agaricus blazei Murill (A. blazei) and its extracts have generally used as complementary and alternative medicines (CAMs). Here, we report that the oral administration of A. blazei augmented cytotoxicity of natural killer (NK) cells in wild-type (WT) C57BL/6, C3H/HeJ, and BALB/c mice. Augmented cytotoxicity was demonstrated by purified NK cells from treated wild-type (WT) and RAG-2-deficient mice, but not from interferon-gamma (IFN-gamma) deficient mice. NK cell activation and IFN-gamma production was also observed in vitro when dendritic cell (DC)-rich splenocytes of WT mice were coincubation with an extract of A. blazei. Both parameters were largely inhibited by neutralizing anti-interleukin-12 (IL-12) monoclonal antibody (mAb) and completely inhibited when anti-IL-12 mAb and anti-IL-18 mAb were used in combination. An aqueous extract of the hemicellulase-digested compound of A. blazei particle; (ABPC) induced IFN-gamma production more effectively, and this was completely inhibited by anti-IL-12 mAb alone. NK cell cytotoxicty was augmented with the same extracts, again in an IL-12 and IFN-gamma-dependent manner. These results clearly demonstrated that A. blazei and ABPC augmented NK cell activation through IL-12-mediated IFN-gamma production.

Leaf hydraulic conductance varies with vein anatomy across Arabidopsis thaliana wild-type and leaf vein mutants.

PubMed

Caringella, Marissa A; Bongers, Franca J; Sack, Lawren

2015-12-01

Leaf venation is diverse across plant species and has practical applications from paleobotany to modern agriculture. However, the impact of vein traits on plant performance has not yet been tested in a model system such as Arabidopsis thaliana. Previous studies analysed cotyledons of A. thaliana vein mutants and identified visible differences in their vein systems from the wild type (WT). We measured leaf hydraulic conductance (Kleaf ), vein traits, and xylem and mesophyll anatomy for A. thaliana WT (Col-0) and four vein mutants (dot3-111 and dot3-134, and cvp1-3 and cvp2-1). Mutant true leaves did not possess the qualitative venation anomalies previously shown in the cotyledons, but varied quantitatively in vein traits and leaf anatomy across genotypes. The WT had significantly higher mean Kleaf . Across all genotypes, there was a strong correlation of Kleaf with traits related to hydraulic conductance across the bundle sheath, as influenced by the number and radial diameter of bundle sheath cells and vein length per area. These findings support the hypothesis that vein traits influence Kleaf , indicating the usefulness of this mutant system for testing theory that was primarily established comparatively across species, and supports a strong role for the bundle sheath in influencing Kleaf . © 2015 John Wiley & Sons Ltd.

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

Agostoni, Marco; Lucker, Ben F.; Smith, Matthew A. Y.

Phycobilisomes (PBSs) are pigment-rich super-complexes required for efficient harvest and transfer of light energy to photosynthetic reaction centers of cyanobacteria. The model cyanobacterium Fremyella diplosiphon is able to adjust PBS pigmentation and size in response to the prevailing light spectrum through a process called complementary chromatic acclimation to optimize spectral light absorption, concomitantly optimizing photosynthesis and growth. We explored the fitness costs versus advantages of modulating antennae size and composition under sinusoidal continuous and fluctuating light conditions in F. diplosiphon by comparing growth of wild-type (WT) cells with a mutant strain deficient in PBSs in both monoculture and polyculture conditions.more » Comparative analyses of WT and the PBS-deficient FdCh1 strain under continuous vs. fluctuating sinusoidal light suggest a potential fitness advantage for maintaining PBSs in WT cells during continuous light and a fitness cost during transitions to and acclimation under fluctuating light. Here, we explored the physiological changes correlated with the observed differential growth to understand the dynamics and biochemical bases of comparative fitness of distinct strains under defined growth conditions. Wild-type F. diplosiphon appears to accumulate longer PBS rods and exhibits higher oxidative stress under fluctuating light conditions than continuous sinusoidal light, which may impact responses and the fitness of cells that do not adapt to rapid changes in external light.« less

Efficient replication, and evolution of Sindbis virus genomes with non-canonical 3'A/U-rich elements (NC3ARE) in neonatal mice.

PubMed

James, Frederick D; Hietala, Katie A; Eldar, Dganit; Guess, Tiffany E; Cone, Cecil; Mundell, Nathan A; Mundall, Nathan; Barnett, Joey V; Raju, Ramaswamy

2007-12-01

Sindbis virus (SIN) is a mosquito-transmitted animal RNA virus. We previously reported that SIN genomes lacking a canonical 19 nt 3'CSE undergo novel repair processes in BHK cells to generate a library of stable atypical SIN genomes with non-canonical 3'A/U-rich elements (NC3AREs) adjacent to the 3' poly(A) tail [1]. To determine the stability and evolutionary pressures on the SIN genomes with NC3AREs to regain a 3'CSE, five representative SIN isolates and a wild type SIN were tested in newborn mice. The key findings of this study are: (a) all six SIN isolates, including those that have extensive NC3AREs in the 3'NTRs, replicate well and produce high titer viremia in newborn mice; (b) 7-9 successive passages of these isolates in newborn mice produced comparable levels of viremia; (c) while all isolates produced only small-sized plaques during primary infection in animals, both small- and large-sized plaques were generated in all other passages; (d) polymerase stuttering occurs on select 3' oligo(U) motifs to add more U residues within the NC3AREs; (e) the S3-8 isolate with an internal UAUUU motif in the 3'poly(A) tail maintains this element even after 9 passages in animals; (f) despite differences in 3'NTRs and variable tissue distribution, all SIN isolates appear to produce similar tissue pathology in infected animals. Competition experiments with wt SIN and atypical SIN isolates in BHK cells show dominance of wt SIN. As shown for BHK cells in culture, the 3'CSE of the SIN genome is not required for virus replication and genome stability in live animals. Since the NC3AREs of atypical SIN genomes are not specific to SIN replicases, alternate RNA motifs of alphavirus genome must confer specificity in template selection. These studies fulfill the need to confirm the long-term viability of atypical SIN genomes in newborn mice and offer a basis for exploring the use of atypical SIN genomes in biotechnology.

PDZ domain-binding motif of Tax sustains T-cell proliferation in HTLV-1-infected humanized mice.

PubMed

Pérès, Eléonore; Blin, Juliana; Ricci, Emiliano P; Artesi, Maria; Hahaut, Vincent; Van den Broeke, Anne; Corbin, Antoine; Gazzolo, Louis; Ratner, Lee; Jalinot, Pierre; Duc Dodon, Madeleine

2018-03-01

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), an aggressive malignant proliferation of activated CD4+ T lymphocytes. The viral Tax oncoprotein is critically involved in both HTLV-1-replication and T-cell proliferation, a prerequisite to the development of ATLL. In this study, we investigated the in vivo contribution of the Tax PDZ domain-binding motif (PBM) to the lymphoproliferative process. To that aim, we examined T-cell proliferation in humanized mice (hu-mice) carrying a human hemato-lymphoid system infected with either a wild type (WT) or a Tax PBM-deleted (ΔPBM) provirus. We observed that the frequency of CD4+ activated T-cells in the peripheral blood and in the spleen was significantly higher in WT than in ΔPBM hu-mice. Likewise, human T-cells collected from WT hu-mice and cultivated in vitro in presence of interleukin-2 were proliferating at a higher level than those from ΔPBM animals. We next examined the association of Tax with the Scribble PDZ protein, a prominent regulator of T-cell polarity, in human T-cells analyzed either after ex vivo isolation or after in vitro culture. We confirmed the interaction of Tax with Scribble only in T-cells from the WT hu-mice. This association correlated with the presence of both proteins in aggregates at the leading edge of the cells and with the formation of long actin filopods. Finally, data from a comparative genome-wide transcriptomic analysis suggested that the PBM-PDZ association is implicated in the expression of genes regulating proliferation, apoptosis and cytoskeletal organization. Collectively, our findings suggest that the Tax PBM is an auxiliary motif that contributes to the sustained growth of HTLV-1 infected T-cells in vivo and in vitro and is essential to T-cell immortalization.

PDZ domain-binding motif of Tax sustains T-cell proliferation in HTLV-1-infected humanized mice

PubMed Central

Artesi, Maria; Jalinot, Pierre

2018-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), an aggressive malignant proliferation of activated CD4+ T lymphocytes. The viral Tax oncoprotein is critically involved in both HTLV-1-replication and T-cell proliferation, a prerequisite to the development of ATLL. In this study, we investigated the in vivo contribution of the Tax PDZ domain-binding motif (PBM) to the lymphoproliferative process. To that aim, we examined T-cell proliferation in humanized mice (hu-mice) carrying a human hemato-lymphoid system infected with either a wild type (WT) or a Tax PBM-deleted (ΔPBM) provirus. We observed that the frequency of CD4+ activated T-cells in the peripheral blood and in the spleen was significantly higher in WT than in ΔPBM hu-mice. Likewise, human T-cells collected from WT hu-mice and cultivated in vitro in presence of interleukin-2 were proliferating at a higher level than those from ΔPBM animals. We next examined the association of Tax with the Scribble PDZ protein, a prominent regulator of T-cell polarity, in human T-cells analyzed either after ex vivo isolation or after in vitro culture. We confirmed the interaction of Tax with Scribble only in T-cells from the WT hu-mice. This association correlated with the presence of both proteins in aggregates at the leading edge of the cells and with the formation of long actin filopods. Finally, data from a comparative genome-wide transcriptomic analysis suggested that the PBM-PDZ association is implicated in the expression of genes regulating proliferation, apoptosis and cytoskeletal organization. Collectively, our findings suggest that the Tax PBM is an auxiliary motif that contributes to the sustained growth of HTLV-1 infected T-cells in vivo and in vitro and is essential to T-cell immortalization. PMID:29566098

Population genetics of the wild yeast Saccharomyces paradoxus.

PubMed Central

Johnson, Louise J; Koufopanou, Vassiliki; Goddard, Matthew R; Hetherington, Richard; Schäfer, Stefanie M; Burt, Austin

2004-01-01

Saccharomyces paradoxus is the closest known relative of the well-known S. cerevisiae and an attractive model organism for population genetic and genomic studies. Here we characterize a set of 28 wild isolates from a 10-km(2) sampling area in southern England. All 28 isolates are homothallic (capable of mating-type switching) and wild type with respect to nutrient requirements. Nine wild isolates and two lab strains of S. paradoxus were surveyed for sequence variation at six loci totaling 7 kb, and all 28 wild isolates were then genotyped at seven polymorphic loci. These data were used to calculate nucleotide diversity and number of segregating sites in S. paradoxus and to investigate geographic differentiation, population structure, and linkage disequilibrium. Synonymous site diversity is approximately 0.3%. Extensive incompatibilities between gene genealogies indicate frequent recombination between unlinked loci, but there is no evidence of recombination within genes. Some localized clonal growth is apparent. The frequency of outcrossing relative to inbreeding is estimated at 1.1% on the basis of heterozygosity. Thus, all three modes of reproduction known in the lab (clonal replication, inbreeding, and outcrossing) have been important in molding genetic variation in this species. PMID:15020405

De novo establishment of wild-type song culture in the zebra finch

PubMed Central

Feher, Olga; Wang, Haibin; Saar, Sigal; Mitra, Partha P.; Tchernichovski, Ofer

2009-01-01

What sort of culture would evolve in an island colony of naive founders? This question cannot be studied experimentally in humans. We performed the analogous experiment using socially learned birdsong. Culture is typically viewed as consisting of traits inherited epigenetically, via social learning. However, cultural diversity has species-typical constraints1, presumably of genetic origin. A celebrated, if contentious, example is whether a universal grammar constrains syntactic diversity in human languages2. Oscine songbirds exhibit song learning and provide biologically tractable models of culture: members of a species show individual variation in song3 and geographically separated groups have local song dialects 4,5. Different species exhibit distinct song cultures6,7, suggestive of genetic constraints8,9. Absent such constraints, innovations and copying errors should cause unbounded variation over multiple generations or geographical distance, contrary to observations9. We asked if wild-type song culture might emerge over multiple generations in an isolated colony founded by isolates, and if so, how this might happen and what type of social environment is required10. Zebra finch isolates, unexposed to singing males during development, produce song with characteristics that differ from the wild-type song found in laboratory11 or natural colonies. In tutoring lineages starting from isolate founders, we quantified alterations in song across tutoring generations in two social environments: tutor-pupil pairs in sound-isolated chambers and an isolated semi-natural colony. In both settings, juveniles imitated the isolate tutors, but changed certain characteristics of the songs. These alterations accumulated over learning generations. Consequently, songs evolved toward the wild-type in 3–4 generations. Thus, species-typical song culture can appear de novo. Our study has parallels with language change and evolution12,13. In analogy to models in quantitative genetics14,15, we model song culture as a multi-generational phenotype, partly encoded genetically in an isolate founding population, influenced by environmental variables, and taking multiple generations to emerge. PMID:19412161

Accuracy of Sensititre YeastOne Echinocandins Epidemiological Cut-off Values for Identification of FKS mutant Candida albicans and Candida glabrata: A Ten Year National Survey of the Fungal Infection Network of Switzerland (FUNGINOS).

PubMed

Kritikos, A; Neofytos, D; Khanna, N; Schreiber, P W; Boggian, K; Bille, J; Schrenzel, J; Mühlethaler, K; Zbinden, R; Bruderer, T; Goldenberger, D; Pfyffer, G; Conen, A; Van Delden, C; Zimmerli, S; Sanglard, D; Bachmann, D; Marchetti, O; Lamoth, F

2018-06-14

Echinocandins represent the first-line treatment of candidemia. Acquired echinocandin resistance is mainly observed among Candida albicans and glabrata and is associated with FKS hotspot mutations. The commercial Sensititre YeastOne TM (SYO) kit is widely used for antifungal susceptibility testing, but interpretive clinical breakpoints are not well defined. We determined echinocandins epidemiological cut-off values (ECV) for C. albicans/glabrata tested by SYO and assessed their ability to identify FKS mutants in a national survey of candidemia. Bloodstream isolates of C. albicans and C. glabrata were collected in 25 Swiss hospitals from 2004 to 2013 and tested by SYO. FKS hotspot sequencing was performed for isolates with a minimal inhibitory concentration (MIC) ≥ECV for any echinocandin. 1277 C. albicans and 347 C. glabrata were included. ECV 97.5% [μg/ml] of caspofungin, anidulafungin and micafungin were 0.12, 0.06, 0.03 for C. albicans, and 0.25, 0.12, 0.03 for C. glabrata. FKS hotspot sequencing was performed for 70 isolates. No mutation was found in the 52 "limit wild-type" isolates (MIC=ECV for ≥1 echinocandin). Among the 18 "non wild-type" isolates (MIC>ECV for ≥1 echinocandin), FKS mutations were recovered in the only two isolates with MIC>ECV for all 3 echinocandins, but not in those exhibiting a "non wild-type" phenotype for only one or two echinocandins. This 10-year nationwide survey showed that the rate of echinocandin resistance among C. albicans and C. glabrata remains low in Switzerland despite increased echinocandin use. SYO-ECV could discriminate FKS mutants from wild-type isolates tested by SYO in this population. Copyright © 2018. Published by Elsevier Ltd.

Mesenchymal stem cells induce epithelial proliferation within the inflamed stomach.

PubMed

Donnelly, Jessica M; Engevik, Amy; Feng, Rui; Xiao, Chang; Boivin, Gregory P; Li, Jing; Houghton, JeanMarie; Zavros, Yana

2014-06-15

Bone marrow-derived mesenchymal stem cells (MSCs) sustain cancer cells by creating a microenvironment favorable for tumor growth. In particular, MSCs have been implicated in gastric cancer development. There is extensive evidence suggesting that Hedgehog signaling regulates tumor growth. However, very little is known regarding the precise roles of Hedgehog signaling and MSCs in tumor development within the stomach. The current study tests that hypothesis that Sonic Hedgehog (Shh), secreted from MSCs, provides a proliferative stimulus for the gastric epithelium in the presence of inflammation. Red fluorescent protein-expressing MSCs transformed in vitro (stMSCs) were transduced with lentiviral constructs containing a vector control (stMSC(vect)) or short hairpin RNA (shRNA) targeting the Shh gene (stMSC(ShhKO)). Gastric submucosal transplantation of wild-type MSCs (wtMSCs), wild-type MSCs overexpressing Shh (wtMSC(Shh)), stMSC(vect), or stMSC(ShhKO) cells in C57BL/6 control (BL/6) or gastrin-deficient (GKO) mice was performed and mice analyzed 30 and 60 days posttransplantation. Compared with BL/6 mice transplanted with wtMSC(Shh) and stMSC(vect) cells, inflamed GKO mice developed aggressive gastric tumors. Tumor development was not observed in mouse stomachs transplanted with wtMSC or stMSC(ShhKO) cells. Compared with stMSC(ShhKO)-transplanted mice, within the inflamed GKO mouse stomach, Shh-expressing stMSC(vect)- and wtMSC(Shh)-induced proliferation of CD44-positive cells. CD44-positive cells clustered in gland-like structures within the tumor stroma and were positive for Patched (Ptch) expression. We conclude that Shh, secreted from MSCs, provides a proliferative stimulus for the gastric epithelium that is associated with tumor development, a response that is sustained by chronic inflammation. Copyright © 2014 the American Physiological Society.

Mesenchymal stem cells induce epithelial proliferation within the inflamed stomach

PubMed Central

Donnelly, Jessica M.; Engevik, Amy; Feng, Rui; Xiao, Chang; Boivin, Gregory P.; Li, Jing; Houghton, JeanMarie

2014-01-01

Bone marrow-derived mesenchymal stem cells (MSCs) sustain cancer cells by creating a microenvironment favorable for tumor growth. In particular, MSCs have been implicated in gastric cancer development. There is extensive evidence suggesting that Hedgehog signaling regulates tumor growth. However, very little is known regarding the precise roles of Hedgehog signaling and MSCs in tumor development within the stomach. The current study tests that hypothesis that Sonic Hedgehog (Shh), secreted from MSCs, provides a proliferative stimulus for the gastric epithelium in the presence of inflammation. Red fluorescent protein-expressing MSCs transformed in vitro (stMSCs) were transduced with lentiviral constructs containing a vector control (stMSCvect) or short hairpin RNA (shRNA) targeting the Shh gene (stMSCShhKO). Gastric submucosal transplantation of wild-type MSCs (wtMSCs), wild-type MSCs overexpressing Shh (wtMSCShh), stMSCvect, or stMSCShhKO cells in C57BL/6 control (BL/6) or gastrin-deficient (GKO) mice was performed and mice analyzed 30 and 60 days posttransplantation. Compared with BL/6 mice transplanted with wtMSCShh and stMSCvect cells, inflamed GKO mice developed aggressive gastric tumors. Tumor development was not observed in mouse stomachs transplanted with wtMSC or stMSCShhKO cells. Compared with stMSCShhKO-transplanted mice, within the inflamed GKO mouse stomach, Shh-expressing stMSCvect- and wtMSCShh-induced proliferation of CD44-positive cells. CD44-positive cells clustered in gland-like structures within the tumor stroma and were positive for Patched (Ptch) expression. We conclude that Shh, secreted from MSCs, provides a proliferative stimulus for the gastric epithelium that is associated with tumor development, a response that is sustained by chronic inflammation. PMID:24789207

Bone turnover in wild type and pleiotrophin-transgenic mice housed for three months in the International Space Station (ISS).

PubMed

Tavella, Sara; Ruggiu, Alessandra; Giuliani, Alessandra; Brun, Francesco; Canciani, Barbara; Manescu, Adrian; Marozzi, Katia; Cilli, Michele; Costa, Delfina; Liu, Yi; Piccardi, Federica; Tasso, Roberta; Tromba, Giuliana; Rustichelli, Franco; Cancedda, Ranieri

2012-01-01

Bone is a complex dynamic tissue undergoing a continuous remodeling process. Gravity is a physical force playing a role in the remodeling and contributing to the maintenance of bone integrity. This article reports an investigation on the alterations of the bone microarchitecture that occurred in wild type (Wt) and pleiotrophin-transgenic (PTN-Tg) mice exposed to a near-zero gravity on the International Space Station (ISS) during the Mice Drawer System (MDS) mission, to date, the longest mice permanence (91 days) in space. The transgenic mouse strain over-expressing pleiotrophin (PTN) in bone was selected because of the PTN positive effects on bone turnover. Wt and PTN-Tg control animals were maintained on Earth either in a MDS payload or in a standard vivarium cage. This study revealed a bone loss during spaceflight in the weight-bearing bones of both strains. For both Tg and Wt a decrease of the trabecular number as well as an increase of the mean trabecular separation was observed after flight, whereas trabecular thickness did not show any significant change. Non weight-bearing bones were not affected. The PTN-Tg mice exposed to normal gravity presented a poorer trabecular organization than Wt mice, but interestingly, the expression of the PTN transgene during the flight resulted in some protection against microgravity's negative effects. Moreover, osteocytes of the Wt mice, but not of Tg mice, acquired a round shape, thus showing for the first time osteocyte space-related morphological alterations in vivo. The analysis of specific bone formation and resorption marker expression suggested that the microgravity-induced bone loss was due to both an increased bone resorption and a decreased bone deposition. Apparently, the PTN transgene protection was the result of a higher osteoblast activity in the flight mice.

Endothelin-1 mediated induction of extracellular matrix genes in strial marginal cells underlies strial pathology in Alport mice.

PubMed

Meehan, Daniel T; Delimont, Duane; Dufek, Brianna; Zallocchi, Marisa; Phillips, Grady; Gratton, Michael Anne; Cosgrove, Dominic

2016-11-01

Alport syndrome, a type IV collagen disorder, manifests as glomerular disease associated with hearing loss with thickening of the glomerular and strial capillary basement membranes (SCBMs). We have identified a role for endothelin-1 (ET-1) activation of endothelin A receptors (ET A Rs) in glomerular pathogenesis. Here we explore whether ET-1 plays a role in strial pathology. Wild type (WT) and Alport mice were treated with the ET A R antagonist, sitaxentan. The stria vascularis was analyzed for SCBM thickness and for extracellular matrix (ECM) proteins. Additional WT and Alport mice were exposed to noise or hypoxia and the stria analyzed for hypoxia-related and ECM genes. A strial marginal cell line cultured under hypoxic conditions, or stimulated with ET-1 was analyzed for expression of hypoxia-related and ECM transcripts. Noise exposure resulted in significantly elevated ABR thresholds in Alport mice relative to wild type littermates. Alport stria showed elevated expression of collagen α1(IV), laminin α2, and laminin α5 proteins relative to WT. SCBM thickening and elevated ECM protein expression was ameliorated by ET A R blockade. Stria from normoxic Alport mice and hypoxic WT mice showed upregulation of hypoxia-related, ECM, and ET-1 transcripts. Both ET-1 stimulation and hypoxia up-regulated ECM transcripts in cultured marginal cells. We conclude that ET-1 mediated activation of ET A Rs on strial marginal cells results in elevated expression of ECM genes and thickening of the SCBMs in Alport mice. SCBM thickening results in hypoxic stress further elevating ECM and ET-1 gene expression, exacerbating strial pathology. Copyright © 2016 Elsevier B.V. All rights reserved.

Nuclease-mediated double-strand break (DSB) enhancement of small fragment homologous recombination (SFHR) gene modification in human-induced pluripotent stem cells (hiPSCs).

PubMed

Sargent, R Geoffrey; Suzuki, Shingo; Gruenert, Dieter C

2014-01-01

Recent developments in methods to specifically modify genomic DNA using sequence-specific endonucleases and donor DNA have opened the door to a new therapeutic paradigm for cell and gene therapy of inherited diseases. Sequence-specific endonucleases, in particular transcription activator-like (TAL) effector nucleases (TALENs), have been coupled with polynucleotide small/short DNA fragments (SDFs) to correct the most common mutation in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene, a 3-base-pair deletion at codon 508 (delF508), in induced pluripotent stem (iPS) cells. The studies presented here describe the generation of candidate TALENs and their co-transfection with wild-type (wt) CFTR-SDFs into CF-iPS cells homozygous for the delF508 mutation. Using an allele-specific PCR (AS-PCR)-based cyclic enrichment protocol, clonal populations of corrected CF-iPS cells were isolated and expanded.

Molecular characterization and genetic diversity of avian paramyxovirus type 4 isolated in South Korea from 2013 to 2017.

PubMed

Tseren-Ochir, Erdene-Ochir; Kwon, Jung-Hoon; Noh, Jin-Yong; Jeong, Jei-Hyun; Jeong, Sol; Kim, Kyu-Jik; Lee, Ji-Ho; Kim, Jun-Beom; Kim, Yu-Jin; Lee, Sun-Hak; Kim, Jun-Young; Song, Chang-Seon

2018-07-01

In recent years, avian paramyxovirus type 4 (APMV-4) frequently isolated from wild and domestic bird populations particularly waterfowls worldwide. However, molecular characteristics and genetic diversity of APMV-4 are uncertain, owing to the limited availability of sequence information. A total of 11 APMV-4 strains from 9850 fecal, swab, and environmental samples were isolated during the surveillance program in wintering seasons of 2013-2017 in South Korea. We performed genetic characterization and phylogenetic analysis to investigate the genetic diversity and relatedness between isolates from the region. We report high APMV-4 genetic diversity (multiple genotypes and sub-genotypes) among wild bird and poultry populations in Korea and that the potential virus exchange occurs between neighboring countries via wild bird migration. Furthermore, our study results suggest the possibility of transcontinental transmission of APMV-4 between Asia and Europe. Copyright © 2018 Elsevier B.V. All rights reserved.

Structural dynamics of native and V260E mutant C-terminal domain of HIV-1 integrase

NASA Astrophysics Data System (ADS)

Sangeetha, Balasubramanian; Muthukumaran, Rajagopalan; Amutha, Ramaswamy

2015-04-01

The C-terminal domain (CTD) of HIV-1 integrase is a five stranded β-barrel resembling an SH3 fold. Mutational studies on isolated CTD and full-length IN have reported V260E mutant as either homo-dimerization defective or affecting the stability and folding of CTD. In this study, molecular dynamics simulation techniques were used to unveil the effect of V260E mutation on isolated CTD monomer and dimer. Both monomeric and dimeric forms of wild type and V260E mutant are highly stable during the simulated period. However, the stabilizing π-stacking interaction between Trp243 and Trp243' at the dimer interface is highly disturbed in CTD-V260E (>6 Å apart). The loss in entropy for dimerization is -30 and -25 kcal/mol for CTD-wt and CTD-V260E respectively signifying a weak hydrophobic interaction and its perturbation in CTD-V260E. The mutant Glu260 exhibits strong attraction/repulsion with all the basic/acidic residues of CTD. In addition to this, the dynamics of CTD-wild type and V260E monomers at 498 K was analyzed to elucidate the effect of V260E mutation on CTD folding. Increase in SASA and reduction in the number of contacts in CTD-V260E during simulation highlights the instability caused by the mutation. In general, V260E mutation affects both multimerization and protein folding with a pronounced effect on protein folding rather than multimerization. This study emphasizes the importance of the hydrophobic nature and SH3 fold of CTD in proper functioning of HIV integrase and perturbing this nature would be a rational approach toward designing more selective and potent allosteric anti-HIV inhibitors.

The effects of Capn1 gene inactivation on the differential expression of genes in skeletal muscle

USDA-ARS?s Scientific Manuscript database

Protein turnover is required for muscle growth and regeneration and several proteolytic enzymes, including the calpains, degrade myofibrillar proteins during this process. In a previous experiment, phenotypic differences were observed between µ-calpain knockout (KO) and wild type (WT) mice, includin...

B cells have distinct roles in host protection against different nematode parasites

USDA-ARS?s Scientific Manuscript database

B cells may mediate protective responses against nematode parasites by supporting Th2 cell development and/or by producing antibodies. To examine this, B cell-deficient mice were inoculated with Nippostrongylus brasiliensis (Nb) or Heligmosomoides polygyrus (Hp). B cell-deficient and wild type (WT...

Revealing the drug-resistant mechanism for diarylpyrimidine analogue inhibitors of HIV-1 reverse transcriptase.

PubMed

Zhang, Hao; Qin, Fang; Ye, Wei; Li, Zeng; Ma, Songyao; Xia, Yan; Jiang, Yi; Zhu, Jiayi; Li, Yixue; Zhang, Jian; Chen, Hai-Feng

2011-09-01

Diaryltriazine (DATA) and diarylpyrimidine (DAPY) were two category inhibitors with highly potent activity for wild type (wt) and four principal mutant types (L100I, K103N, Y181C and Y188L) of HIV-1 reverse transcriptase (RT). We had revealed the drug-resistant mechanism of DATA analogue inhibitors with molecular dynamics simulation and three-dimensional quantitative structure-activity relationship (3D-QSAR) methods. In this work, we investigated the drug-resistant mechanism of DAPY analogue inhibitors. It was found that DAPY analogue inhibitors form more hydrogen bonds and hydrophobic contacts with wild type and mutants of HIV-1 RT than DATA inhibitors. This could explain that DAPY analogue inhibitors are more potent than DATA for the wild type and mutants of HIV-1 RT. Then, 3D-QSAR models were constructed for these inhibitors of wild type and four principal mutant types HIV-1 RT and evaluated by test set compounds. These combined models can be used to design new chemical entities and make quantitative prediction of the bioactivities for HIV-1 RT inhibitors before resorting to in vitro and in vivo experiment. © 2011 John Wiley & Sons A/S.

[Isolation and characterization of a Streptococcus suis serotype 9 from a wild cat].

PubMed

Tang, Fang; Pan, Zihao; Li, Dezhi; Ma, Lin; Xiong, Yi; Lu, Chengping

2016-02-04

Streptococcus suis (S. suis) is an emerging zoonotic pathogenic bacterium capable of infecting piglets and human and with sporadic infections in a variety of mammalian species. The aim of this study is to investigate the prevalence of S. suis in wild cats. We isolated an S. suis strain from a wild cat. We tested the serotype of the isolated strain by anti-serum agglutination and PCR. We determined the sequence type (ST) of the isolated strain by multilocus sequence typing tests (MLST). We constructed the 16S rRNA phylogenetic tree of the isolation and S. suis strains in NCBI database to demonstrated genetic relationship of different strains. We measured the antibiotic resistance of the isolated strain by triple disk diffusion method. We detected the virulence of the isolated strain by mice infection experiments. We isolated an S. suis strain m70 from a wild cat, which belongs to serotype 9. MLST showed that m70 fell into a new ST. The 16S rRNA phylogenetic tree of m70 and S. suis strains in NCBI database demonstrated that m70 was in a separate cluster. m70 was resistant to tetracycline, intermediate to erythromycin, and sensitive to ampicillin, corresponding to clinical S. suis isolates in China. The mortality of mice infected with 10(8) CFU of m70 was achieved 60%-80% (3/5-4/5). The mean LD50 of mice infected with m70 was 5.1 x 10(7) CFU, while the mean LD50 of virulent S. suis strain HA9801 was 3.9 x 10(7) CFU. There is no significant difference between the LD50 of the two strains (P < 0.05). We isolated an S. suis strain from a wild cat, which belongs to the prevalent serotype and was a virulent strain, indicating the potential of transmission of S. suis from wild cats to humans, especially some prevalent serotype strains.

Oxidative Capacity and Fatigability in Run Trained Malignant Hyperthermia Susceptible Mice

PubMed Central

Rouviere, Clement; Corona, Benjamin T.; Ingalls, Christopher P.

2011-01-01

Introduction The purpose of this study was to test the hypothesis that Malignant Hyperthermia model mice (RyR1Y522S/wt) are more vulnerable to exercise-induced muscle injury and fatigability and adapt less to run training. Methods Following 6 weeks of voluntary wheel running, we measured anterior crural muscle fatigability, muscle injury, and cytochrome oxidase (COX) and citrate synthase (CS). Results Although RyR1Y522S/wt mice ran without experiencing MH episodes, they ran 42% less distance than wild type (WT) mice. Muscles from WT mice exhibited increased fatigue resistance and COX content after training. Muscles from RyR1Y522S/wt mice demonstrated no significant change in fatigability or COX and CS after training. However, muscles from RyR1Y522S/wt mice displayed less intrinsic fatigability and greater COX/CS content and muscle damage than WT mice. Discussion RyR1Y522S/wt mice can run without experiencing rhabdomyolysis, and their inability to adapt to training appears to stem from intrinsic enhancement of mitochondrial enzymes and fatigue resistance. PMID:22431093

Oxidative capacity and fatigability in run-trained malignant hyperthermia-susceptible mice.

PubMed

Rouviere, Clement; Corona, Benjamin T; Ingalls, Christopher P

2012-04-01

The purpose of this study was to test the hypothesis that malignant hyperthermia model mice (RyR1Y522S/wt) are more vulnerable to exercise-induced muscle injury and fatigability and adapt less to run training. After 6 weeks of voluntary wheel running, we measured anterior crural muscle fatigability, muscle injury, and cytochrome oxidase (COX) and citrate synthase (CS). Although RyR1Y522S/wt mice ran without undergoing MH episodes, they ran 42% less distance than wild-type (WT) mice. Muscles from WT mice exhibited increased fatigue resistance and COX content after training. Muscles from RyR1Y522S/wt mice demonstrated no significant change in fatigability or COX and CS after training. However, muscles from RyR1Y522S/wt mice displayed less intrinsic fatigability and greater COX/CS content and muscle damage than WT mice. RyR1Y522S/wt mice can run without having rhabdomyolysis, and their inability to adapt to training appears to stem from intrinsic enhancement of mitochondrial enzymes and fatigue resistance. Copyright © 2012 Wiley Periodicals, Inc.

Pharmacokinetic properties of radiolabeled mutant Interleukin-2v: a PET imaging study

PubMed Central

Hartimath, Siddesh V.; Manuelli, Valeria; Zijlma, Rolf; Signore, Alberto; Nayak, Tapan K.; Freimoser-Grundschober, Anne; Klein, Christian; Dierckx, Rudi A.J.O.; de Vries, Erik F.J.

2018-01-01

Interleukin-2 (IL2) is a cytokine that can stimulate cytotoxic immune cells to attack infected and malignant cells. Unfortunately, IL2 can also cause serious immune-related toxicity. Recently, a mutant of IL2 (IL2v) with abolished CD25 binding, increased plasma half-life and less toxicity was engineered. Unlike wild-type IL2 (wt-IL2), mutant IL2v does not bind to the α-subunit (CD25) of the high affinity IL2αβγ receptor, but only to its β and γ subunit. Here, we investigated the biological properties of IL2v and compared with the wt-IL2 using fluorine-18 and PET. [18F]FB-IL2v binds specifically to IL2 receptors (IL2R) on activated human peripheral blood monocytes (hPBMCs) and is cleared mainly by the kidneys (Balb/c mice). [18F]FB-IL2v PET studies in SCID mice injected with hPBMCs revealed high uptake in the implant (0.85 ± 0.15 SUV), which was significantly reduced after pretreatment with wt-IL2 or mutant IL2v (SUV 0.26 ± 0.1 and 0.46 ± 0.1, p < 0.01). Compartment modeling and Logan graphical analysis in wistar rats inoculated with hPBMCs indicated that the binding of [18F]FB-IL2v to IL2R was reversible. The volume of distribution (VT) and the non-displaceable binding potential (BPnd) of mutant [18F]FB-IL2v in the implant were approximately 3 times lower than those of wild-type [18F]FB-IL2 (p < 0.01). Pretreatment with wt-IL2 significantly reduced the VT and BPnd of mutant [18F]FB-IL2v in the implant (p < 0.001). This demonstrates that wild-type [18F]FB-IL2 binds stronger to IL2R and has faster kinetics than [18F]FB-IL2v, which makes it less suitable as a therapeutic drug. [18F]FB-IL2v, on the other hand, seems to have better properties for use as a therapeutic drug. PMID:29467958

Pharmacokinetic properties of radiolabeled mutant Interleukin-2v: a PET imaging study.

PubMed

Hartimath, Siddesh V; Manuelli, Valeria; Zijlma, Rolf; Signore, Alberto; Nayak, Tapan K; Freimoser-Grundschober, Anne; Klein, Christian; Dierckx, Rudi A J O; de Vries, Erik F J

2018-01-23

Interleukin-2 (IL2) is a cytokine that can stimulate cytotoxic immune cells to attack infected and malignant cells. Unfortunately, IL2 can also cause serious immune-related toxicity. Recently, a mutant of IL2 (IL2v) with abolished CD25 binding, increased plasma half-life and less toxicity was engineered. Unlike wild-type IL2 (wt-IL2), mutant IL2v does not bind to the α-subunit (CD25) of the high affinity IL2αβγ receptor, but only to its β and γ subunit. Here, we investigated the biological properties of IL2v and compared with the wt-IL2 using fluorine-18 and PET. [ 18 F]FB-IL2v binds specifically to IL2 receptors (IL2R) on activated human peripheral blood monocytes (hPBMCs) and is cleared mainly by the kidneys (Balb/c mice). [ 18 F]FB-IL2v PET studies in SCID mice injected with hPBMCs revealed high uptake in the implant (0.85 ± 0.15 SUV), which was significantly reduced after pretreatment with wt-IL2 or mutant IL2v (SUV 0.26 ± 0.1 and 0.46 ± 0.1, p < 0.01). Compartment modeling and Logan graphical analysis in wistar rats inoculated with hPBMCs indicated that the binding of [ 18 F]FB-IL2v to IL2R was reversible. The volume of distribution (V T ) and the non-displaceable binding potential (BP nd ) of mutant [ 18 F]FB-IL2v in the implant were approximately 3 times lower than those of wild-type [ 18 F]FB-IL2 ( p < 0.01). Pretreatment with wt-IL2 significantly reduced the V T and BPnd of mutant [ 18 F]FB-IL2v in the implant ( p < 0.001). This demonstrates that wild-type [ 18 F]FB-IL2 binds stronger to IL2R and has faster kinetics than [18F]FB-IL2v, which makes it less suitable as a therapeutic drug. [ 18 F]FB-IL2v, on the other hand, seems to have better properties for use as a therapeutic drug.

Glutathione-S-transferase-omega [MMA(V) reductase] knockout mice: Enzyme and arsenic species concentrations in tissues after arsenate administration

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

Chowdhury, Uttam K.; Zakharyan, Robert A.; Hernandez, Alba

Inorganic arsenic is a human carcinogen to which millions of people are exposed via their naturally contaminated drinking water. Its molecular mechanisms of carcinogenicity have remained an enigma, perhaps because arsenate is biochemically transformed to at least five other arsenic-containing metabolites. In the biotransformation of inorganic arsenic, GSTO1 catalyzes the reduction of arsenate, MMA(V), and DMA(V) to the more toxic + 3 arsenic species. MMA(V) reductase and human (hGSTO1-1) are identical proteins. The hypothesis that GST-Omega knockout mice biotransformed inorganic arsenic differently than wild-type mice has been tested. The livers of male knockout (KO) mice, in which 222 bp ofmore » Exon 3 of the GSTO1 gene were eliminated, were analyzed by PCR for mRNA. The level of transcripts of the GSTO1 gene in KO mice was 3.3-fold less than in DBA/1lacJ wild-type (WT) mice. The GSTO2 transcripts were about two-fold less in the KO mouse. When KO and WT mice were injected intramuscularly with Na arsenate (4.16 mg As/kg body weight); tissues removed at 0.5, 1, 2, 4, 8, and 12 h after arsenate injection; and the arsenic species measured by HPLC-ICP-MS, the results indicated that the highest concentration of the recently discovered and very toxic MMA(III), a key biotransformant, was in the kidneys of both KO and WT mice. The highest concentration of DMA(III) was in the urinary bladder tissue for both the KO and WT mice. The MMA(V) reducing activity of the liver cytosol of KO mice was only 20% of that found in wild-type mice. There appears to be another enzyme(s) other than GST-O able to reduce arsenic(V) species but to a lesser extent. This and other studies suggest that each step of the biotransformation of inorganic arsenic has an alternative enzyme to biotransform the arsenic substrate.« less

Use of SLAM and PVRL4 and identification of pro-HB-EGF as cell entry receptors for wild type phocine distemper virus.

PubMed

Melia, Mary M; Earle, John Philip; Abdullah, Haniah; Reaney, Katherine; Tangy, Frederic; Cosby, Sara Louise

2014-01-01

Signalling lymphocyte activation molecule (SLAM) has been identified as an immune cell receptor for the morbilliviruses, measles (MV), canine distemper (CDV), rinderpest and peste des petits ruminants (PPRV) viruses, while CD46 is a receptor for vaccine strains of MV. More recently poliovirus like receptor 4 (PVRL4), also known as nectin 4, has been identified as a receptor for MV, CDV and PPRV on the basolateral surface of polarised epithelial cells. PVRL4 is also up-regulated by MV in human brain endothelial cells. Utilisation of PVRL4 as a receptor by phocine distemper virus (PDV) remains to be demonstrated as well as confirmation of use of SLAM. We have observed that unlike wild type (wt) MV or wtCDV, wtPDV strains replicate in African green monkey kidney Vero cells without prior adaptation, suggesting the use of a further receptor. We therefore examined candidate molecules, glycosaminoglycans (GAG) and the tetraspan proteins, integrin β and the membrane bound form of heparin binding epithelial growth factor (proHB-EGF),for receptor usage by wtPDV in Vero cells. We show that wtPDV replicates in Chinese hamster ovary (CHO) cells expressing SLAM and PVRL4. Similar wtPDV titres are produced in Vero and VeroSLAM cells but more limited fusion occurs in the latter. Infection of Vero cells was not inhibited by anti-CD46 antibody. Removal/disruption of GAG decreased fusion but not the titre of virus. Treatment with anti-integrin β antibody increased rather than decreased infection of Vero cells by wtPDV. However, infection was inhibited by antibody to HB-EGF and the virus replicated in CHO-proHB-EGF cells, indicating use of this molecule as a receptor. Common use of SLAM and PVRL4 by morbilliviruses increases the possibility of cross-species infection. Lack of a requirement for wtPDV adaptation to Vero cells raises the possibility of usage of proHB-EGF as a receptor in vivo but requires further investigation.

Selective Gene Regulation by Androgen Receptor in Prostate Cancer

DTIC Science & Technology

2012-10-01

empty vector, wt AR, AR-E255K and AR- R753Q cells were transfected with an ARE- responsive reporter and renilla as control. Cells were treated with...empty vector (empty), wild-type AR (WT), AR-E255K or AR-R753Q were transfected with ARE-luciferase and renilla . Cells were treated with 0 or 1 nm...R1881, harvested after 24 hrs to read luciferase and renilla actiivity. % G ro w th (D ay 5 / D ay 1 ) Vector WTAR E255KR753Q 600 700 800 900

Predominance of Cryptococcus neoformans var. grubii multilocus sequence type 5 and emergence of isolates with non-wild-type minimum inhibitory concentrations to fluconazole: a multi-centre study in China.

PubMed

Fan, X; Xiao, M; Chen, S; Kong, F; Dou, H-T; Wang, H; Xiao, Y-L; Kang, M; Sun, Z-Y; Hu, Z-D; Wan, Z; Chen, S-L; Liao, K; Chu, Y-Z; Hu, T-S; Zou, G-L; Hou, X; Zhang, L; Zhao, Y-P; Xu, Y-C; Liu, Z-Y

2016-10-01

There are few data on the molecular epidemiology of cryptococcosis in China. Here we investigated the species distribution, molecular types and antifungal susceptibilities of 312 Cryptococcus neoformans species complex isolates from ten hospitals over 5 years. Isolates were identified by internal transcribed spacer (ITS) sequencing and by two matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) systems. Multilocus sequence typing (MLST) was used to verify species/variety and to designate molecular types. Susceptibility to six antifungal drugs was determined by the Sensititre YeastOne™ method. Cryptococcus neoformans was the predominant species (305/312 isolates (97.8%), all were ITS type 1, serotype A), of which 89.2% (272/305) were C. neoformans var. grubii MLST sequence type (ST) 5 and 6.2% (19/305) were ST31. Other C. neoformans var. grubii STs were rare but included six novel STs. Only two strains were C. neoformans var. neoformans (both serotype AD). Cryptococcus gattii was uncommon (n = 7, four ITS types) and comprised five MLST STs including one novel ST. For C. neoformans var. grubii, the proportion of isolates with non-wild-type MICs to fluconazole significantly rose in the fourth study year (from 0% (0/56 isolates) in the first year to 23.9% (17/71) in the fourth year), including five isolates with fluconazole MICs of ≥32 mg/L. The study has provided useful data on the species epidemiology and their genetic diversity and antifungal susceptibility. The proportional increase in isolates with non-wild-type MICs to fluconazole is noted. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Development of an FHbp-CTB holotoxin-like chimera and the elicitation of bactericidal antibodies against serogroup B Neisseria meningitidis.

PubMed

Price, Gregory A; Bash, Margaret C

2018-01-29

The Neisseria meningitidis factor H binding protein (FHbp) is an important virulence factor and vaccine antigen contained in both USA licensed serogroup B meningococcal vaccines. Recent studies in human factor H (hFH) transgenic mice suggest that hFH-FHbp interactions lower FHbp-elicited immunogenicity. To provide tools with which to characterize and potentially improve FHbp immunogenicity, we developed an FHbp-cholera holotoxin-like chimera vaccine expression system in Escherichia coli that utilizes cholera toxin B (CTB) as both a scaffold and adjuvant for FHbp. We developed FHbp-CTB chimeras using a wild-type (WT) FHbp and a low hFH-binding FHbp mutant R41S. Both chimeras bound to G M1 ganglioside and were recognized by the FHbp-specific monoclonal antibody JAR4. The R41S mutant had greatly reduced hFH binding compared to the WT FHbp-CTB chimera. WT and R41S FHbp-CTB chimeric antigens were compared to equimolar amounts of FHbp admixed with CTB or FHbp alone in mouse immunogenicity studies. The chimeras were significantly more immunogenic than FHbp alone or mixed with CTB, and elicited bactericidal antibodies against a panel of MenB isolates. This study demonstrates a unique and simple method for studying FHbp immunogenicity. The chimeric approach may facilitate studies of other protein-based antigens targeting pathogenic Neisseria and lay groundwork for the development of new protein based vaccines against meningococcal and gonococcal disease. Published by Elsevier Ltd.

Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS

PubMed Central

Bosco, Daryl A.; Morfini, Gerardo; Karabacak, N. Murat; Song, Yuyu; Gros-Louis, Francois; Pasinelli, Piera; Goolsby, Holly; Fontaine, Benjamin A.; Lemay, Nathan; McKenna-Yasek, Diane; Frosch, Matthew P.; Agar, Jeffery N.; Julien, Jean-Pierre; Brady, Scott T.; Brown, Robert H.

2010-01-01

Many mutations confer upon copper/zinc superoxide dismutase-1 (SOD1) one or more toxic function(s) that impair motor neuron viability and cause familial amyotrophic lateral sclerosis (FALS). Using a conformation-specific antibody that detects misfolded SOD1 (C4F6), we demonstrate that oxidized WT-SOD1 and mutant-SOD1 share a conformational epitope that is not present in normal WT-SOD1. In a subset of human sporadic ALS (SALS) cases, motor neurons in the lumbosacral spinal cord displayed striking C4F6 immunoreactivity, denoting the presence of aberrant WT-SOD1 species. Recombinant, oxidized WT-SOD1 and WT-SOD1 immunopurified from SALS tissues inhibited kinesin-based fast axonal transport in a manner similar to FALS-linked mutant SOD1. Studies here suggest that WT-SOD1 can be pathogenic in SALS and identifies an SOD1-dependent pathogenic mechanism common to FALS and SALS. PMID:20953194

Variation in biological properties of cauliflower mosaic virus clones.

PubMed

al-Kaff, N; Covey, S N

1994-11-01

Infectious clones were prepared from virion DNA of three cauliflower mosaic virus (CaMV) isolates, 11/3, Xinjiang (XJ), and Aust, to investigate pathogenic variation in virus populations. Of 10 infectious clones obtained for isolate 11/3, four pathotypes were identified, each producing symptoms in turnip that differed from those of the 11/3 wild-type. Virus from two clonal groups of 11/3 was transmissible by aphids whereas that from two others was not. Of the five infectious clones obtained from isolate XJ, two groups were identified, one of which differed symptomatically from the wild-type. Only one infectious clone was obtained from isolate Aust and this had properties similar to the wild-type. Restriction enzyme polymorphisms were found in some clonal groups and these correlated with symptoms. Other groups with different pathogenic properties could not be distinguished apart by restriction site polymorphisms. Further variation was observed in the nucleotide sequences of gene II (coding for aphid transmission factor) from these viruses as compared with other CaMV isolates. In the aphid non-transmissible clones of isolate 11/3, one had a Gly to Arg mutation in gene II similar to that of other non-deleted non-transmissible CaMV isolates. The second had a 322 bp deletion at the site of a small direct repeat similar to that of isolate CM4-184 although occurring in a different position. The gene II deletion of isolate 11/3 produced a frame-shift that separated genes II and III by 60 bp. Most CaMV clones studied remained biologically stable producing similar symptoms during subsequent passages. However, one clone (11/3-7) produced two new biotypes during its first passage suggesting that it was relatively unstable. Our results show that wild-type populations of CaMV contain a range of infectious genome variants with contrasting biological properties and differing stability. We suggest that a variety of significant viral phenotypic changes can occur during each infection cycle resulting from relatively small genome changes.

The characterization of low pathogenic avian influenza viruses isolated from wild birds in northern Vietnam from 2006 to 2009.

PubMed

Takakuwa, Hiroki; Yamashiro, Tetsu; Le, Mai Q; Phuong, Lien S; Ozaki, Hiroichi; Tsunekuni, Ryota; Usui, Tatsufumi; Ito, Hiroshi; Yamaguchi, Tsuyoshi; Ito, Toshihiro; Murase, Toshiyuki; Ono, Etsuro; Otsuki, Koichi

2013-12-01

Due to concerns that wild birds could possibly spread H5N1 viruses, surveillance was conducted to monitor the types of avian influenza viruses circulating among the wild birds migrating to or inhabiting in northern Vietnam from 2006 to 2009. An H5N2 virus isolated from a Eurasian woodcock had a close phylogenetic relationship to H5 viruses recently isolated in South Korea and Japan, suggesting that H5N2 has been shared between Vietnam, South Korea, and Japan. An H9N2 virus isolated from a Chinese Hwamei was closely related to two H9N2 viruses that were isolated from humans in Hong Kong in 2009, suggesting that an H9N2 strain relevant to the human isolates had been transmitted to and maintained among the wild bird population in Vietnam and South China. The results support the idea that wild bird species play a significant role in the spread and maintenance of avian influenza and that this also occurs in Vietnam. Copyright © 2013 Elsevier Ltd. All rights reserved.

Microevolution of Group A Streptococci In Vivo: Capturing Regulatory Networks Engaged in Sociomicrobiology, Niche Adaptation, and Hypervirulence

PubMed Central

Aziz, Ramy K.; Kansal, Rita; Aronow, Bruce J.; Taylor, William L.; Rowe, Sarah L.; Kubal, Michael; Chhatwal, Gursharan S.; Walker, Mark J.; Kotb, Malak

2010-01-01

The onset of infection and the switch from primary to secondary niches are dramatic environmental changes that not only alter bacterial transcriptional programs, but also perturb their sociomicrobiology, often driving minor subpopulations with mutant phenotypes to prevail in specific niches. Having previously reported that M1T1 Streptococcus pyogenes become hypervirulent in mice due to selection of mutants in the covRS regulatory genes, we set out to dissect the impact of these mutations in vitro and in vivo from the impact of other adaptive events. Using a murine subcutaneous chamber model to sample the bacteria prior to selection or expansion of mutants, we compared gene expression dynamics of wild type (WT) and previously isolated animal-passaged (AP) covS mutant bacteria both in vitro and in vivo, and we found extensive transcriptional alterations of pathoadaptive and metabolic gene sets associated with invasion, immune evasion, tissue-dissemination, and metabolic reprogramming. In contrast to the virulence-associated differences between WT and AP bacteria, Phenotype Microarray analysis showed minor in vitro phenotypic differences between the two isogenic variants. Additionally, our results reflect that WT bacteria's rapid host-adaptive transcriptional reprogramming was not sufficient for their survival, and they were outnumbered by hypervirulent covS mutants with SpeB−/Sdahigh phenotype, which survived up to 14 days in mice chambers. Our findings demonstrate the engagement of unique regulatory modules in niche adaptation, implicate a critical role for bacterial genetic heterogeneity that surpasses transcriptional in vivo adaptation, and portray the dynamics underlying the selection of hypervirulent covS mutants over their parental WT cells. PMID:20418946

Desalted deep-sea water improves cognitive function in mice by increasing the production of insulin-like growth factor-I in the hippocampus.

PubMed

Harada, Naoaki; Zhao, Juan; Kurihara, Hiroki; Nakagata, Naomi; Okajima, Kenji

2011-08-01

The stimulation of sensory neurons in the gastrointestinal (GI) tract improves cognitive function by increasing the hippocampal production of insulin-like growth factor-I (IGF-I) in mice. In the current study, we examined whether oral administration of desalted deep-sea water (DSW) increases the hippocampal production of IGF-I by stimulating sensory neurons in the GI tract, thereby improving cognitive function in mice. Desalted DSW increased calcitonin gene-related peptide (CGRP) release from dorsal root ganglion (DRG) neurons isolated from wild-type (WT) mice by activating transient receptor potential vanilloid 1. The plasma levels of IGF-I and tissue levels of CGRP, IGF-I, and IGF-I mRNA in the hippocampus were increased by oral administration of desalted DSW in WT mice. In these animals, nociceptive information originating from the GI tract was transmitted to the hippocampus via the spinothalamic pathway. Improvement of spatial learning was observed in WT mice after administration of desalted DSW. Distilled DSW showed results similar to those of desalted DSW in vitro and in vivo. None of the effects of desalted DSW in WT mice were observed after the administration of desalted DSW in CGRP-knockout (CGRP-/-) mice. No volatile compounds were detected in distilled DSW on GC-MS analysis. These observations suggest that desalted DSW may increase the hippocampal IGF-I production via sensory neuron stimulation in the Gl tract, thereby improving cognitive function in mice. Such effects of desalted DSW might not be dependent on the minerals but are dependent on the function of the water molecule itself. Copyright © 2011 Mosby, Inc. All rights reserved.

Phospholemman is not required for the acute stimulation of Na⁺-K⁺-ATPase α₂-activity during skeletal muscle fatigue.

PubMed

Manoharan, Palanikumar; Radzyukevich, Tatiana L; Hakim Javadi, Hesamedin; Stiner, Cory A; Landero Figueroa, Julio A; Lingrel, Jerry B; Heiny, Judith A

2015-12-15

The Na(+)-K(+)-ATPase α2-isoform in skeletal muscle is rapidly stimulated during muscle use and plays a critical role in fatigue resistance. The acute mechanisms that stimulate α2-activity are not completely known. This study examines whether phosphorylation of phospholemman (PLM/FXYD1), a regulatory subunit of Na(+)-K(+)-ATPase, plays a role in the acute stimulation of α2 in working muscles. Mice lacking PLM (PLM KO) have a normal content of the α2-subunit and show normal exercise capacity, in contrast to the greatly reduced exercise capacity of mice that lack α2 in the skeletal muscles. Nerve-evoked contractions in vivo did not induce a change in total PLM or PLM phosphorylated at Ser63 or Ser68, in either WT or PLM KO. Isolated muscles of PLM KO mice maintain contraction and resist fatigue as well as wild type (WT). Rb(+) transport by the α2-Na(+)-K(+)-ATPase is stimulated to the same extent in contracting WT and contracting PLM KO muscles. Phosphorylation of sarcolemmal membranes prepared from WT but not PLM KO skeletal muscles stimulates the activity of both α1 and α2 in a PLM-dependent manner. The stimulation occurs by an increase in Na(+) affinity without significant change in Vmax and is more effective for α1 than α2. These results demonstrate that phosphorylation of PLM is capable of stimulating the activity of both isozymes in skeletal muscle; however, contractile activity alone is not sufficient to induce PLM phosphorylation. Importantly, acute stimulation of α2, sufficient to support exercise and oppose fatigue, does not require PLM or its phosphorylation. Copyright © 2015 the American Physiological Society.

Glycogen synthase kinase 3α regulates urine concentrating mechanism in mice

PubMed Central

Nørregaard, Rikke; Tao, Shixin; Nilsson, Line; Woodgett, James R.; Kakade, Vijayakumar; Yu, Alan S. L.; Howard, Christiana

2015-01-01

In mammals, glycogen synthase kinase (GSK)3 comprises GSK3α and GSK3β isoforms. GSK3β has been shown to play a role in the ability of kidneys to concentrate urine by regulating vasopressin-mediated water permeability of collecting ducts, whereas the role of GSK3α has yet to be discerned. To investigate the role of GSK3α in urine concentration, we compared GSK3α knockout (GSK3αKO) mice with wild-type (WT) littermates. Under normal conditions, GSK3αKO mice had higher water intake and urine output. GSK3αKO mice also showed reduced urine osmolality and aquaporin-2 levels but higher urinary vasopressin. When water deprived, they failed to concentrate their urine to the same level as WT littermates. The addition of 1-desamino-8-d-arginine vasopressin to isolated inner medullary collecting ducts increased the cAMP response in WT mice, but this response was reduced in GSK3αKO mice, suggesting reduced responsiveness to vasopressin. Gene silencing of GSK3α in mpkCCD cells also reduced forskolin-induced aquaporin-2 expression. When treated with LiCl, an isoform nonselective inhibitor of GSK3 and known inducer of polyuria, WT mice developed significant polyuria within 6 days. However, in GSK3αKO mice, the polyuric response was markedly reduced. This study demonstrates, for the first time, that GSK3α could play a crucial role in renal urine concentration and suggest that GSK3α might be one of the initial targets of Li+ in LiCl-induced nephrogenic diabetes insipidus. PMID:25608967

Relationships of bone characteristics in MYO9B deficient femurs.

PubMed

Kim, Do-Gyoon; Jeong, Yong-Hoon; McMichael, Brooke K; Bähler, Martin; Bodnyk, Kyle; Sedlar, Ryan; Lee, Beth S

2018-08-01

The objective of this study was to examine relationships among a variety of bone characteristics, including volumetric, mineral density, geometric, dynamic mechanical analysis, and static fracture mechanical properties. As MYO9B is an unconventional myosin in bone cells responsible for normal skeletal growth, bone characteristics of wild-type (WT), heterozygous (HET), and MYO9B knockout (KO) mice groups were compared as an animal model to express different bone quantity and quality. Forty-five sex-matched 12-week-old mice were used in this study. After euthanization, femurs were isolated and scanned using microcomputed tomography (micro-CT) to assess bone volumetric, tissue mineral density (TMD), and geometric parameters. Then, a non-destructive dynamic mechanical analysis (DMA) was performed by applying oscillatory bending displacement on the femur. Finally, the same femur was subject to static fracture testing. KO group had significantly lower length, bone mineral density (BMD), bone mass and volume, dynamic and static stiffness, and strength than WT and HET groups (p < 0.019). On the other hand, TMD parameters of KO group were comparable with those of WT group. HET group showed volumetric, geometric, and mechanical properties similar to WT group, but had lower TMD (p < 0.014). Non-destructive micro-CT and DMA parameters had significant positive correlations with strength (p < 0.015) without combined effect of groups and sex on the correlations (p > 0.077). This comprehensive characterization provides a better understanding of interactive behavior between the tissue- and organ-level of the same femur. The current findings elucidate that MYO9B is responsible for controlling bone volume to determine the growth rate and fracture risk of bone. Copyright © 2018 Elsevier Ltd. All rights reserved.

Neuronal expression of pathological tau accelerates oligodendrocyte progenitor cell differentiation

PubMed Central

Ossola, Bernardino; Zhao, Chao; Compston, Alastair; Pluchino, Stefano; Franklin, Robin J. M.

2015-01-01

Oligodendrocyte progenitor cell (OPC) differentiation is an important therapeutic target to promote remyelination in multiple sclerosis (MS). We previously reported hyperphosphorylated and aggregated microtubule‐associated protein tau in MS lesions, suggesting its involvement in axonal degeneration. However, the influence of pathological tau‐induced axonal damage on the potential for remyelination is unknown. Therefore, we investigated OPC differentiation in human P301S tau (P301S‐htau) transgenic mice, both in vitro and in vivo following focal demyelination. In 2‐month‐old P301S‐htau mice, which show hyperphosphorylated tau in neurons, we found atrophic axons in the spinal cord in the absence of prominent axonal degeneration. These signs of early axonal damage were associated with microgliosis and an upregulation of IL‐1β and TNFα. Following in vivo focal white matter demyelination we found that OPCs differentiated more efficiently in P301S‐htau mice than wild type (Wt) mice. We also found an increased level of myelin basic protein within the lesions, which however did not translate into increased remyelination due to higher susceptibility of P301S‐htau axons to demyelination‐induced degeneration compared to Wt axons. In vitro experiments confirmed higher differentiation capacity of OPCs from P301S‐htau mice compared with Wt mice‐derived OPCs. Because the OPCs from P301S‐htau mice do not ectopically express the transgene, and when isolated from newborn mice behave like Wt mice‐derived OPCs, we infer that their enhanced differentiation capacity must have been acquired through microenvironmental priming. Our data suggest the intriguing concept that damaged axons may signal to OPCs and promote their differentiation in the attempt at rescue by remyelination. GLIA 2016;64:457–471 PMID:26576485

Physiological investigation of C4-phosphoenolpyruvate-carboxylase-introduced rice line shows that sucrose metabolism is involved in the improved drought tolerance.

PubMed

Zhang, Chen; Li, Xia; He, Yafei; Zhang, Jinfei; Yan, Ting; Liu, Xiaolong

2017-06-01

We compared the drought tolerance of wild-type (WT) and transgenic rice plants (PC) over-expressing the maize C 4 PEPC gene, which encodes phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) gene, and evaluated the roles of saccharide and sugar-related enzymes in the drought response. Pot-grown seedlings were subjected to real drought conditions outdoors, and the yield components were compared between PC and untransformed wild-type (WT) plants. The stable yield from PC plants was associated with higher net photosynthetic rate under the real drought treatment. The physiological characters of WT and PC seedlings under a simulated drought treatment (25% (w/v) polyethylene glycol-6000 for 3 h; PEG 6000 treatment) were analyzed in detail for the early response of drought. The relative water content was higher in PC than in WT, and PEPC activity and the C 4 -PEPC transcript level in PC were elevated under the simulated drought conditions. The endogenous saccharide responses also differed between PC and WT under simulated drought stress. The higher sugar decomposition rate in PC than in WT under drought analog stress was related to the increased activities of sucrose phosphate synthase, sucrose synthase, acid invertase, and neutral invertase, increased transcript levels of VIN1, CIN1, NIN1, SUT2, SUT4, and SUT5, and increased activities of superoxide dismutase and peroxidase in the leaves. The greater antioxidant defense capacity of PC and its relationship with saccharide metabolism was one of the reasons for the improved drought tolerance. In conclusion, PEPC effectively alleviated oxidative damage and enhanced the drought tolerance in rice plants, which were more related to the increase of the endogenous saccharide decomposition. These findings show that components of C 4 photosynthesis can be used to increase the yield of rice under drought conditions. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Detection of wild-type EGFR amplification and EGFRvIII mutation in CSF-derived extracellular vesicles of glioblastoma patients.

PubMed

Figueroa, Javier M; Skog, Johan; Akers, Johnny; Li, Hongying; Komotar, Ricardo; Jensen, Randy; Ringel, Florian; Yang, Isaac; Kalkanis, Steven; Thompson, Reid; LoGuidice, Lori; Berghoff, Emily; Parsa, Andrew; Liau, Linda; Curry, William; Cahill, Daniel; Bettegowda, Chetan; Lang, Frederick F; Chiocca, E Antonio; Henson, John; Kim, Ryan; Breakefield, Xandra; Chen, Clark; Messer, Karen; Hochberg, Fred; Carter, Bob S

2017-10-19

RNAs within extracellular vesicles (EVs) have potential as diagnostic biomarkers for patients with cancer and are identified in a variety of biofluids. Glioblastomas (GBMs) release EVs containing RNA into cerebrospinal fluid (CSF). Here we describe a multi-institutional study of RNA extracted from CSF-derived EVs of GBM patients to detect the presence of tumor-associated amplifications and mutations in epidermal growth factor receptor (EGFR). CSF and matching tumor tissue were obtained from patients undergoing resection of GBMs. We determined wild-type (wt)EGFR DNA copy number amplification, as well as wtEGFR and EGFR variant (v)III RNA expression in tumor samples. We also characterized wtEGFR and EGFRvIII RNA expression in CSF-derived EVs. EGFRvIII-positive tumors had significantly greater wtEGFR DNA amplification (P = 0.02) and RNA expression (P = 0.03), and EGFRvIII-positive CSF-derived EVs had significantly more wtEGFR RNA expression (P = 0.004). EGFRvIII was detected in CSF-derived EVs for 14 of the 23 EGFRvIII tissue-positive GBM patients. Conversely, only one of the 48 EGFRvIII tissue-negative patients had the EGFRvIII mutation detected in their CSF-derived EVs. These results yield a sensitivity of 61% and a specificity of 98% for the utility of CSF-derived EVs to detect an EGFRvIII-positive GBM. Our results demonstrate CSF-derived EVs contain RNA signatures reflective of the underlying molecular genetic status of GBMs in terms of wtEGFR expression and EGFRvIII status. The high specificity of the CSF-derived EV diagnostic test gives us an accurate determination of positive EGFRvIII tumor status and is essentially a less invasive "liquid biopsy" that might direct mutation-specific therapies for GBMs. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Transcervical Inoculation with Chlamydia trachomatis Induces Infertility in HLA-DR4 Transgenic and Wild-Type Mice.

PubMed

Pal, Sukumar; Tifrea, Delia F; Zhong, Guangming; de la Maza, Luis M

2018-01-01

Chlamydia trachomatis is the leading cause of infection-induced infertility in women. Attempts to control this epidemic with screening programs and antibiotic therapy have failed. Currently, a vaccine to prevent C. trachomatis infections is not available. In order to develop an animal model for evaluating vaccine antigens that can be applied to humans, we used C. trachomatis serovar D (strain UW-3/Cx) to induce infertility in mice whose major histocompatibility complex class II antigen was replaced with the human leukocyte antigen DR4 (HLA-DR4). Transcervical inoculation of medroxyprogesterone-treated HLA-DR4 transgenic mice with 5 × 10 5 C. trachomatis D inclusion forming units (IFU) induced a significant reduction in fertility, with a mean number of embryos/mouse of 4.4 ± 1.3 compared to 7.8 ± 0.5 for the uninfected control mice ( P < 0.05). A similar fertility reduction was elicited in the wild-type (WT) C57BL/6 mice (4.3 ± 1.4 embryos/mouse) compared to the levels of the WT controls (9.1 ± 0.4 embryos/mouse) ( P < 0.05). Following infection, WT mice mounted more robust humoral and cellular immune responses than HLA-DR4 mice. As determined by vaginal shedding, HLA-DR4 mice were more susceptible to a transcervical C. trachomatis D infection than WT mice. To assess if HLA-DR4 transgenic and WT mice could be protected by vaccination, 10 4 IFU of C. trachomatis D was delivered intranasally, and mice were challenged transcervically 6 weeks later with 5 × 10 5 IFU of C. trachomatis D. As determined by severity and length of vaginal shedding, WT C57BL/6 and HLA-DR4 mice were significantly protected by vaccination. The advantages and limitations of the HLA-DR4 transgenic mouse model for evaluating human C. trachomatis vaccine antigens are discussed. Copyright © 2017 American Society for Microbiology.

Rapid Phosphoproteomic Effects of Abscisic Acid (ABA) on Wild-Type and ABA Receptor-Deficient A. thaliana Mutants*

PubMed Central

Minkoff, Benjamin B.; Stecker, Kelly E.; Sussman, Michael R.

2015-01-01

Abscisic acid (ABA)1 is a plant hormone that controls many aspects of plant growth, including seed germination, stomatal aperture size, and cellular drought response. ABA interacts with a unique family of 14 receptor proteins. This interaction leads to the activation of a family of protein kinases, SnRK2s, which in turn phosphorylate substrates involved in many cellular processes. The family of receptors appears functionally redundant. To observe a measurable phenotype, four of the fourteen receptors have to be mutated to create a multilocus loss-of-function quadruple receptor (QR) mutant, which is much less sensitive to ABA than wild-type (WT) plants. Given these phenotypes, we asked whether or not a difference in ABA response between the WT and QR backgrounds would manifest on a phosphorylation level as well. We tested WT and QR mutant ABA response using isotope-assisted quantitative phosphoproteomics to determine what ABA-induced phosphorylation changes occur in WT plants within 5 min of ABA treatment and how that phosphorylation pattern is altered in the QR mutant. We found multiple ABA-induced phosphorylation changes that occur within 5 min of treatment, including three SnRK2 autophosphorylation events and phosphorylation on SnRK2 substrates. The majority of robust ABA-dependent phosphorylation changes observed were partially diminished in the QR mutant, whereas many smaller ABA-dependent phosphorylation changes observed in the WT were not responsive to ABA in the mutant. A single phosphorylation event was increased in response to ABA treatment in both the WT and QR mutant. A portion of the discovery data was validated using selected reaction monitoring-based targeted measurements on a triple quadrupole mass spectrometer. These data suggest that different subsets of phosphorylation events depend upon different subsets of the ABA receptor family to occur. Altogether, these data expand our understanding of the model by which the family of ABA receptors directs rapid phosphoproteomic changes. PMID:25693798

Antigenic and molecular characterization of wild type 1 poliovirus causing outbreaks of poliomyelitis in Albania and neighboring countries in 1996.

PubMed

Fiore, L; Genovese, D; Diamanti, E; Catone, S; Ridolfi, B; Ibrahimi, B; Konomi, R; van der Avoort, H G; Hovi, T; Crainic, R; Simeoni, P; Amato, C

1998-07-01

Mass vaccination has led poliomyelitis to become a rare disease in a large part of the world, including Western Europe. However, in the past 20 years wild polioviruses imported from countries where polio is endemic have been responsible for outbreaks in otherwise polio-free European countries. We report on the characterization of poliovirus isolates from a large outbreak of poliomyelitis that occurred in Albania in 1996 and that also spread to the neighboring countries of Yugoslavia and Greece. The epidemics involved 145 subjects, mostly young adults, and caused persisting paralysis in 87 individuals and 16 deaths. The agent responsible for the outbreak was isolated from 74 patients and was identified as wild type 1 poliovirus by both immunological and molecular methods. Sequence analysis of the genome demonstrated the involvement of a single virus strain throughout the epidemics, and genotyping analysis showed 95% homology of the strain with a wild type 1 poliovirus strain isolated in Pakistan in 1995. Neutralization assays with both human sera and monoclonal antibodies were performed to analyze the antigenic structure of the epidemic strain, suggesting its peculiar antigenic characteristics. The presented data underline the current risks of outbreaks due to imported wild poliovirus and emphasize the need to improve vaccination efforts and also the need to implement surveillance in countries free of indigenous wild poliovirus.

Antigenic and Molecular Characterization of Wild Type 1 Poliovirus Causing Outbreaks of Poliomyelitis in Albania and Neighboring Countries in 1996

PubMed Central

Fiore, L.; Genovese, D.; Diamanti, E.; Catone, S.; Ridolfi, B.; Ibrahimi, B.; konomi, R.; van der Avoort, H. G. A. M.; Hovi, T.; Crainic, R.; Simeoni, P.; Amato, C.

1998-01-01

Mass vaccination has led poliomyelitis to become a rare disease in a large part of the world, including Western Europe. However, in the past 20 years wild polioviruses imported from countries where polio is endemic have been responsible for outbreaks in otherwise polio-free European countries. We report on the characterization of poliovirus isolates from a large outbreak of poliomyelitis that occurred in Albania in 1996 and that also spread to the neighboring countries of Yugoslavia and Greece. The epidemics involved 145 subjects, mostly young adults, and caused persisting paralysis in 87 individuals and 16 deaths. The agent responsible for the outbreak was isolated from 74 patients and was identified as wild type 1 poliovirus by both immunological and molecular methods. Sequence analysis of the genome demonstrated the involvement of a single virus strain throughout the epidemics, and genotyping analysis showed 95% homology of the strain with a wild type 1 poliovirus strain isolated in Pakistan in 1995. Neutralization assays with both human sera and monoclonal antibodies were performed to analyze the antigenic structure of the epidemic strain, suggesting its peculiar antigenic characteristics. The presented data underline the current risks of outbreaks due to imported wild poliovirus and emphasize the need to improve vaccination efforts and also the need to implement surveillance in countries free of indigenous wild poliovirus. PMID:9650935

COMPARISON OF IN VITRO-CULTURED AND WILD-TYPE PERKINSUS MARINUS I: PATHOGEN VIRULENCE

EPA Science Inventory

Perkinsus marinus is a highly contagious pathogen of the eastern oyster Crassostrea virginica. Until recently, transmission studies have employed wild-type parasites isolated directly from infected oysters. Newly developed methods to propagate P. marinus in vitro have led to usin...

Phosphorylation at tyrosine 114 of Proliferating Cell Nuclear Antigen (PCNA) is required for adipogenesis in response to high fat diet

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

Lo, Yuan-Hung; Ho, Po-Chun; Chen, Min-Shan

Highlights: Black-Right-Pointing-Pointer Proliferating Cell Nuclear Antigen (PCNA) is phosphorylated at Y114. Black-Right-Pointing-Pointer Phospho-Y114 of PCNA is not required for cell proliferation for normal growth. Black-Right-Pointing-Pointer MCE during adipogenesis is abolished in the lack of the phosphorylation. Black-Right-Pointing-Pointer Homozygous Y114F mice are resistant to high fat diet induced obesity. Black-Right-Pointing-Pointer Our results shed light on the interface between proliferation and differentiation. -- Abstract: Clonal proliferation is an obligatory component of adipogenesis. Although several cell cycle regulators are known to participate in the transition between pre-adipocyte proliferation and terminal adipocyte differentiation, how the core DNA synthesis machinery is coordinately regulated in adipogenesismore » remains elusive. PCNA (Proliferating Cell Nuclear Antigen) is an indispensable component for DNA synthesis during proliferation. Here we show that PCNA is subject to phosphorylation at the highly conserved tyrosine residue 114 (Y114). Replacing the Y114 residue with phenylalanine (Y114F), which is structurally similar to tyrosine but cannot be phosphorylated, does not affect normal animal development. However, when challenged with high fat diet, mice carrying homozygous Y114F alleles (PCNA{sup F/F}) are resistant to adipose tissue enlargement in comparison to wild-type (WT) mice. Mouse embryonic fibroblasts (MEFs) harboring WT or Y114F mutant PCNA proliferate at similar rates. However, when subjected to adipogenesis induction in culture, PCNA{sup F/F} MEFs are not able to re-enter the cell cycle and fail to form mature adipocytes, while WT MEFs undergo mitotic clonal expansion in response to the adipogenic stimulation, accompanied by enhanced Y114 phosphorylation of PCNA, and differentiate to mature adipocytes. Consistent with the function of Y114 phosphorylation in clonal proliferation in adipogenesis, fat tissues isolated from WT mice contain significantly more adipocytes than those isolated from PCNA{sup F/F} mice. This study identifies a critical role for PCNA in adipose tissue development, and for the first time identifies a role of the core DNA replication machinery at the interface between proliferation and differentiation.« less

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

Svensson, Emelie; Eriksson, Helena; Gekas, Christos

The Wilms tumor gene 1 (WT1) encodes a zinc-finger-containing transcription factor highly expressed in immature hematopoietic progenitor cells. Overexpression and presence of somatic mutations in acute leukemia indicate a role for WT1 in the pathogenesis of leukemia. CD34{sup +} progenitor cells were transduced with one splice variant of human WT1 without the KTS insert in the zinc-finger domain, WT1(+/-), and with a deleted mutant of WT1 lacking the entire zinc-finger region, WT1(delZ), thus incapable of binding DNA. We show that inhibition of erythroid colony formation and differentiation is absolutely dependent on the DNA-binding zinc-finger domain of WT1. Unexpectedly, however, WT1(delZ)more » was equally effective as wild type protein in the reduction of myeloid clonogenic growth as well as in stimulation of myeloid differentiation, as judged by the expression of cell surface CD11b. Expression of neither WT1(+/-) nor WT1(delZ) upregulated mRNA for the cdk inhibitor p21{sup Waf1/Cip1} or p27{sup Kip1}. Our results demonstrate that WT1 affects proliferation and differentiation in erythroid and myeloid cells by different molecular mechanisms, and suggest that mutations affecting the zinc-finger domain of WT1 could interfere with normal differentiation in the pathogenesis of leukemia.« less

In Vitro and in Vivo Analysis of the Binding of the C Terminus of the HDL Receptor Scavenger Receptor Class B, Type I (SR-BI), to the PDZ1 Domain of Its Adaptor Protein PDZK1*

PubMed Central

Kocher, Olivier; Birrane, Gabriel; Tsukamoto, Kosuke; Fenske, Sara; Yesilaltay, Ayce; Pal, Rinku; Daniels, Kathleen; Ladias, John A. A.; Krieger, Monty

2010-01-01

The PDZ1 domain of the four PDZ domain-containing protein PDZK1 has been reported to bind the C terminus of the HDL receptor scavenger receptor class B, type I (SR-BI), and to control hepatic SR-BI expression and function. We generated wild-type (WT) and mutant murine PDZ1 domains, the mutants bearing single amino acid substitutions in their carboxylate binding loop (Lys14-Xaa4-Asn19-Tyr-Gly-Phe-Phe-Leu24), and measured their binding affinity for a 7-residue peptide corresponding to the C terminus of SR-BI (503VLQEAKL509). The Y20A and G21Y substitutions abrogated all binding activity. Surprisingly, binding affinities (Kd) of the K14A and F22A mutants were 3.2 and 4.0 μm, respectively, similar to 2.6 μm measured for the WT PDZ1. To understand these findings, we determined the high resolution structure of WT PDZ1 bound to a 5-residue sequence from the C-terminal SR-BI (505QEAKL509) using x-ray crystallography. In addition, we incorporated the K14A and Y20A substitutions into full-length PDZK1 liver-specific transgenes and expressed them in WT and PDZK1 knock-out mice. In WT mice, the transgenes did not alter endogenous hepatic SR-BI protein expression (intracellular distribution or amount) or lipoprotein metabolism (total plasma cholesterol, lipoprotein size distribution). In PDZK1 knock-out mice, as expected, the K14A mutant behaved like wild-type PDZK1 and completely corrected their hepatic SR-BI and plasma lipoprotein abnormalities. Unexpectedly, the 10–20-fold overexpressed Y20A mutant also substantially, but not completely, corrected these abnormalities. The results suggest that there may be an additional site(s) within PDZK1 that bind(s) SR-BI and mediate(s) productive SR-BI-PDZK1 interaction previously attributed exclusively to the canonical binding of the C-terminal SR-BI to PDZ1. PMID:20739281

In vitro and in vivo Analysis of the Binding of the C Terminus of the HDL Receptor Scavenger Receptor Class B type I (SR-BI) to the PDZ1 Domain of its Cytoplasmic Adaptor Protein PDZK1

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

O Kocher; G Birrane; K Tsukamoto

2011-12-31

The PDZ1 domain of the four PDZ domain-containing protein PDZK1 has been reported to bind the C terminus of the HDL receptor scavenger receptor class B, type I (SR-BI), and to control hepatic SR-BI expression and function. We generated wild-type (WT) and mutant murine PDZ1 domains, the mutants bearing single amino acid substitutions in their carboxylate binding loop (Lys(14)-Xaa(4)-Asn(19)-Tyr-Gly-Phe-Phe-Leu(24)), and measured their binding affinity for a 7-residue peptide corresponding to the C terminus of SR-BI ((503)VLQEAKL(509)). The Y20A and G21Y substitutions abrogated all binding activity. Surprisingly, binding affinities (K(d)) of the K14A and F22A mutants were 3.2 and 4.0 ?M,more » respectively, similar to 2.6 ?M measured for the WT PDZ1. To understand these findings, we determined the high resolution structure of WT PDZ1 bound to a 5-residue sequence from the C-terminal SR-BI ((505)QEAKL(509)) using x-ray crystallography. In addition, we incorporated the K14A and Y20A substitutions into full-length PDZK1 liver-specific transgenes and expressed them in WT and PDZK1 knock-out mice. In WT mice, the transgenes did not alter endogenous hepatic SR-BI protein expression (intracellular distribution or amount) or lipoprotein metabolism (total plasma cholesterol, lipoprotein size distribution). In PDZK1 knock-out mice, as expected, the K14A mutant behaved like wild-type PDZK1 and completely corrected their hepatic SR-BI and plasma lipoprotein abnormalities. Unexpectedly, the 10-20-fold overexpressed Y20A mutant also substantially, but not completely, corrected these abnormalities. The results suggest that there may be an additional site(s) within PDZK1 that bind(s) SR-BI and mediate(s) productive SR-BI-PDZK1 interaction previously attributed exclusively to the canonical binding of the C-terminal SR-BI to PDZ1.« less

Ovariectomy modify local renin-angiotensin-aldosterone system gene expressions in the heart of ApoE (-/-) mice.

PubMed

Borges, Celina Carvalho; Penna-de-Carvalho, Aline; Medeiros Junior, Jorge L; Aguila, Marcia Barbosa; Mandarim-de-Lacerda, Carlos A

2017-12-15

The evaluation of the local Renin-Angiotensin-Aldosterone system (RAAS) gene expressions in the heart of ovariectomized (OVX) apolipoprotein E deficient mice (ApoE). Four-months old C57BL/6 female mice (wild-type, wt, n=20), and ApoE female mice (n=20), were submitted to OVX or a surgical procedure without ovary removal (SHAM) and formed four groups (n=10/group): SHAM/wt, SHAM/ApoE, OVX/wt, and OVX/ApoE. OVX led to greater body mass, plasma triglycerides (TG) and total cholesterol, and resulted in insulin resistance and altered RAAS gene expressions in the heart tissue. The gene expression of angiotensin-converting enzyme (ACE)-2 was lower in OVX/wt than in SHAM/wt (P=0.0004), Mas receptor (MASr) was lower in OVX/wt compared to SHAM/wt (P<0.0001). Also, angiotensin II receptor type 1 (AT1r) was higher in OVX/wt than in SHAM/wt (P=0.0229), and AT2r was lower in OVX/wt than in SHAM/wt (P=0.0121). OVX and ApoE deficiency showed interaction potentializing the insulin resistance, increasing TG levels and altering ACE and MASr gene expressions. ACE gene expression was higher in OVX/ApoE than in OVX/wt (P<0.0001), and MASr gene expression was lower in OVX/ApoE than in OVX/wt (P<0.0001). The impact of OVX on local RAAS cascade in the heart of ApoE deficient animals, besides the metabolic changes culminating with insulin resistance, involves an upregulation of renin, ACE, and AT1r gene expressions. The findings may contribute to clarify the mechanisms of development of postmenopausal hypertension and the link between RAAS and apolipoprotein E. Copyright © 2017 Elsevier Inc. All rights reserved.

Molecular Characterization of Avian Paramyxovirus Types 4 and 8 Isolated from Wild Migratory Waterfowl in Mongolia.

PubMed

Tseren-Ochir, Erdene-Ochir; Yuk, Seong-Su; Khishgee, Bodisaikhan; Kwon, Jung-Hoon; Noh, Jin-Yong; Hong, Woo-Tack; Jeong, Jei-Hyun; Gwon, Gyeong-Bin; Jeong, Sol; Kim, Yu-Jin; Kim, Jun-Beom; Lee, Ji-Ho; Kim, Kyu-Jik; Damdinjav, Batchuluun; Song, Chang-Seon

2018-04-01

  Avian paramyxoviruses (APMVs) constitute some of the most globally prevalent avian viruses and are frequently isolated from wild migratory bird species. Using 1,907 fresh fecal samples collected during the 2012 avian influenza surveillance program, we identified two serotypes of APMV: APMV-4 ( n=10) and APMV-8 ( n=1). Sequences for these isolates phylogenetically clustered with Asian APMV-4 and APMV-8 recently isolated from wild birds in Korea, Japan, China, and Kazakhstan. Analysis by DNA barcoding indicated that the Mongolian APMV-4 and APMV-8 strains were isolated from Anseriformes species including Mallards ( Anas platyrhynchos) and Whooper Swans ( Cygnus cygnus). The close genetic relatedness to Asian isolates, and to similar host species, suggested that wild bird species in the Anatidae family might play an important role as a natural reservoir in the spread of APMV-4 and APMV-8. However, we did not find conclusive evidence to support this hypothesis owing to the limited number of strains that could be isolated. Enhanced surveillance of poultry and wild bird populations in Asia is therefore crucial for the understanding of global AMPV transmission, ecology, evolution, and epidemiology.

Molecular Epidemiology of Leptospira Serogroup Pomona Infections Among Wild and Domestic Animals in Spain.

PubMed

Arent, Z J; Gilmore, C; San-Miguel Ayanz, J M; Neyra, L Quevedo; García-Peña, F J

2017-03-01

Strains of Leptospira serogroup Pomona are known to cause widespread animal infections in many parts of the world. Forty-three isolates retrieved from domestic animals and wild small mammals suggest that serogroup Pomona is epidemiologically relevant in Spain. This is supported by the high prevalence of serovar Pomona antibodies in livestock and wild animals. In this study, the strains were serologically and genetically characterized in an attempt to elucidate their epidemiology. Serological typing was based on the microscopic agglutination test but molecular typing involved species-specific polymerase chain reaction, restriction endonuclease analysis, and multiple-locus variable-number tandem repeat analysis. The study revealed that the infections are caused by two serovars, namely Pomona and Mozdok. Serovar Pomona was derived only from farm animals and may be adapted to pigs, which are recognized as the maintenance host. The results demonstrated that serovar Pomona is genetically heterogeneous and three different types were recognized. This heterogeneity was correlated with different geographical distributions of the isolates. All strains derived from small wild mammals were identified as serovar Mozdok. Some isolates of this serovar retrieved from cattle confirm that this serovar may also be the cause of infections in food-producing animals for which these wild species may be source of infection.

p38 mitogen-activated protein kinase is involved in arginase-II-mediated eNOS-Uncoupling in Obesity

PubMed Central

2014-01-01

Background Endothelial nitric oxide synthase (eNOS)-uncoupling links obesity-associated insulin resistance and type-II diabetes to the increased incidence of cardiovascular disease. Studies have indicated that increased arginase is involved in eNOS-uncoupling through competing with the substrate L-arginine. Given that arginase-II (Arg-II) exerts some of its biological functions through crosstalk with signal transduction pathways, and that p38 mitogen-activated protein kinase (p38mapk) is involved in eNOS-uncoupling, we investigated here whether p38mapk is involved in Arg-II-mediated eNOS-uncoupling in a high fat diet (HFD)-induced obesity mouse model. Methods Obesity was induced in wild type (WT) and Arg-II-deficient (Arg-II-/-) mice on C57BL/6 J background by high-fat diet (HFD, 55% fat) for 14 weeks starting from age of 7 weeks. The entire aortas were isolated and subjected to 1) immunoblotting analysis of the protein level of eNOS, Arg-II and p38mapk activation; 2) arginase activity assay; 3) endothelium-dependent and independent vasomotor responses; 4) en face staining of superoxide anion and NO production with Dihydroethidium and 4,5-Diaminofluorescein Diacetate, respectively, to assess eNOS-uncoupling. To evaluate the role of p38mapk, isolated aortas were treated with p38mapk inhibitor SB203580 (10 μmol/L, 1 h) prior to the analysis. In addition, the role of p38mapk in Arg-II-induced eNOS-uncoupling was investigated in cultured human endothelial cells overexpressing Arg-II in the absence or presence of shRNA against p38mapk. Results HFD enhanced Arg-II expression/activity and p38mapk activity, which was associated with eNOS-uncoupling as revealed by decreased NO and enhanced L-NAME-inhibitable superoxide in aortas of WT obese mice. In accordance, WT obese mice revealed decreased endothelium-dependent relaxations to acetylcholine despite of higher eNOS protein level, whereas Arg-II-/- obese mice were protected from HFD-induced eNOS-uncoupling and endothelial dysfunction, which was associated with reduced p38mapk activation in aortas of the Arg-II-/- obese mice. Moreover, overexpression of Arg-II in human endothelial cells caused eNOS-uncoupling and augmented p38mapk activation. The Arg-II-induced eNOS-uncoupling was prevented by silencing p38mapk. Furthermore, pharmacological inhibition of p38mapk recouples eNOS in isolated aortas from WT obese mice. Conclusions Taking together, we demonstrate here for the first time that Arg-II causes eNOS-uncoupling through activation of p38 mapk in HFD-induced obesity. PMID:25034973

Contribution of Neuraminidase of Influenza Viruses to the Sensitivity to Sera Inhibitors and Reassortment Efficiency

PubMed Central

Kiseleva, Irina; Larionova, Natalie; Fedorova, Ekaterina; Bazhenova, Ekaterina; Dubrovina, Irina; Isakova-Sivak, Irina; Rudenko, Larisa

2014-01-01

Live attenuated influenza vaccine (LAIV) represent reassortant viruses with hemagglutinin (HA) and neuraminidase (NA) gene segments inherited from circulating wild-type (WT) parental influenza viruses recommended for inclusion into seasonal vaccine formulation, and the 6 internal protein-encoding gene segments from cold-adapted attenuated master donor viruses (genome composition 6:2). In this study, we describe the obstacles in developing LAIV strains while taking into account the phenotypic peculiarities of WT viruses used for reassortment. Genomic composition analysis of 849 seasonal LAIV reassortants revealed that over 80% of reassortants based on inhibitor-resistant WT viruses inherited WT NA, compared to 26% of LAIV reassortants based on inhibitor-sensitive WT viruses. In addition, the highest percentage of LAIV genotype reassortants was achieved when WT parental viruses were resistant to non-specific serum inhibitors. We demonstrate that NA may play a role in influenza virus sensitivity to non-specific serum inhibitors. Replacing NA of inhibitor-sensitive WT virus with the NA of inhibitor-resistant master donor virus significantly decreased the sensitivity of the resulting reassortant virus to serum heat-stable inhibitors. PMID:25132869

Impact of the A2V Mutation on the Heterozygous and Homozygous Aβ1-40 Dimer Structures from Atomistic Simulations.

PubMed

Nguyen, Phuong H; Sterpone, Fabio; Campanera, Josep M; Nasica-Labouze, Jessica; Derreumaux, Philippe

2016-06-15

The A2V mutation was reported to protect from Alzheimer's disease in its heterozygous form and cause an early Alzheimer's disease type dementia in its homozygous form. Experiments showed that the aggregation rate follows the order A2V > WT (wild-type) > A2V-WT. To understand the impact of this mutation, we carried out replica exchange molecular dynamics simulations of Aβ1-40 WT-A2V and A2V-A2V dimers and compared to the WT dimer. Our atomistic simulations reveal that the mean secondary structure remains constant, but there are substantial differences in the intramolecular and intermolecular conformations upon single and double A2V mutation. Upon single mutation, the intrinsic disorder is reduced, the intermolecular potential energies are reduced, the population of intramolecular three-stranded β-sheets is increased, and the number of all α dimer topologies is decreased. Taken together, these results offer an explanation for the reduced aggregation rate of the Aβ1-40 A2V-WT peptides and the protective effect of A2V in heterozygotes.

Lipid biosensor interactions with wild type and matrix deletion HIV-1 Gag proteins.

PubMed

Barklis, Eric; Staubus, August O; Mack, Andrew; Harper, Logan; Barklis, Robin Lid; Alfadhli, Ayna

2018-05-01

The matrix (MA) domain of the HIV-1 precursor Gag protein (PrGag) has been shown interact with the HIV-1 envelope (Env) protein, and to direct PrGag proteins to plasma membrane (PM) assembly sites by virtue of its affinity to phosphatidylinositol-4,5-bisphosphate (PI[4,5]P2). Unexpectedly, HIV-1 viruses with large MA deletions (ΔMA) have been shown to be conditionally infectious as long as they are matched with Env truncation mutant proteins or alternative viral glycoproteins. To characterize the interactions of wild type (WT) and ΔMA Gag proteins with PI(4,5)P2 and other acidic phospholipids, we have employed a set of lipid biosensors as probes. Our investigations showed marked differences in WT and ΔMA Gag colocalization with biosensors, effects on biosensor release, and association of biosensors with virus-like particles. These results demonstrate an alternative approach to the analysis of viral protein-lipid associations, and provide new data as to the lipid compositions of HIV-1 assembly sites. Copyright © 2018 Elsevier Inc. All rights reserved.

Synthesis, Biological Activity, and Crystal Structure of Potent Nonnucleoside Inhibitors of HIV-1 Reverse Transcriptase That Retain Activity against Mutant Forms of the Enzyme†

PubMed Central

Morningstar, Marshall L.; Roth, Thomas; Farnsworth, David W.; Smith, Marilyn Kroeger; Watson, Karen; Buckheit, Robert W.; Das, Kalyan; Zhang, Wanyi; Arnold, Eddy; Julias, John G.; Hughes, Stephen H.; Michejda, Christopher J.

2010-01-01

In an ongoing effort to develop novel and potent nonnucleoside HIV-1 reverse transcriptase (RT) inhibitors that are effective against the wild type (WT) virus and clinically observed mutants, 1,2-bis-substituted benzimidazoles were synthesized and tested. Optimization of the N1 and C2 positions of benzimidazole led to the development of 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)-4-methylbenzimidazole (1) (IC50 = 0.2 μM, EC50 = 0.44 μM, and TC50 ≥ 100 against WT). This paper describes how substitution on the benzimidazole ring profoundly affects activity. Substituents at the benzimidazole C4 dramatically enhanced potency, while at C5 or C6 substituents were generally detrimental or neutral to activity, respectively. A 7-methyl analogue did not inhibit HIV-1 RT. Determination of the crystal structure of 1 bound to RT provided the basis for accurate modeling of additional analogues, which were synthesized and tested. Several derivatives were nanomolar inhibitors of wild-type virus and were effective against clinically relevant HIV-1 mutants. PMID:17663538

Rootcap structure in wild type and in a starchless mutant of Arabidopsis

NASA Technical Reports Server (NTRS)

Sack, F. D.; Kiss, J. Z.

1989-01-01

Rootcaps of the wild type (WT) and of a starchless, gravitropic mutant (TC7) of Arabidopsis thaliana L. were examined by electron microscopy to identify cellular polarities with respect to gravity. In columella cells, nuclei are located proximally, and the nuclear envelope is continuous with endoplasmic reticulum (ER) that is in turn connected to nearby plasmodesmata. Impregnation of ER with osmium ferricyanide revealed numerous contacts between columella plastids and ER in both genotypes. ER is present mostly in the outer regions of the columella protoplast except in older columella cells that are developing into peripheral cells. In vertical roots, only columella cells that are intermediate in development (story 2 cells) have a higher surface density (S) of ER in the distal compared to proximal regions of the cell. The distal but not the proximal S of the ER is constant throughout columella development. Plastids are less sedimented in TC7 columella cells compared to those of the WT. It is hypothesized that plastid contact with the ER plays a role in gravity perception in both genotypes.

Reconstitution of wild type viral DNA in simian cells transfected with early and late SV40 defective genomes.

PubMed

O'Neill, F J; Gao, Y; Xu, X

1993-11-01

The DNAs of polyomaviruses ordinarily exist as a single circular molecule of approximately 5000 base pairs. Variants of SV40, BKV and JCV have been described which contain two complementing defective DNA molecules. These defectives, which form a bipartite genome structure, contain either the viral early region or the late region. The defectives have the unique property of being able to tolerate variable sized reiterations of regulatory and terminus region sequences, and portions of the coding region. They can also exchange coding region sequences with other polyomaviruses. It has been suggested that the bipartite genome structure might be a stage in the evolution of polyomaviruses which can uniquely sustain genome and sequence diversity. However, it is not known if the regulatory and terminus region sequences are highly mutable. Also, it is not known if the bipartite genome structure is reversible and what the conditions might be which would favor restoration of the monomolecular genome structure. We addressed the first question by sequencing the reiterated regulatory and terminus regions of E- and L-SV40 DNAs. This revealed a large number of mutations in the regulatory regions of the defective genomes, including deletions, insertions, rearrangements and base substitutions. We also detected insertions and base substitutions in the T-antigen gene. We addressed the second question by introducing into permissive simian cells, E- and L-SV40 genomes which had been engineered to contain only a single regulatory region. Analysis of viral DNA from transfected cells demonstrated recombined genomes containing a wild type monomolecular DNA structure. However, the complete defectives, containing reiterated regulatory regions, could often compete away the wild type genomes. The recombinant monomolecular genomes were isolated, cloned and found to be infectious. All of the DNA alterations identified in one of the regulatory regions of E-SV40 DNA were present in the recombinant monomolecular genomes. These and other findings indicate that the bipartite genome state can sustain many mutations which wtSV40 cannot directly sustain. However, the mutations can later be introduced into the wild type genomes when the E- and L-SV40 DNAs recombine to generate a new monomolecular genome structure.

Genetic relationships and epidemiological links between wild type 1 poliovirus isolates in Pakistan and Afghanistan

PubMed Central

2012-01-01

Background/Aim Efforts have been made to eliminate wild poliovirus transmission since 1988 when the World Health Organization began its global eradication campaign. Since then, the incidence of polio has decreased significantly. However, serotype 1 and serotype 3 still circulate endemically in Pakistan and Afghanistan. Both countries constitute a single epidemiologic block representing one of the three remaining major global reservoirs of poliovirus transmission. In this study we used genetic sequence data to investigate transmission links among viruses from diverse locations during 2005-2007. Methods In order to find the origins and routes of wild type 1 poliovirus circulation, polioviruses were isolated from faecal samples of Acute Flaccid Paralysis (AFP) patients. We used viral cultures, two intratypic differentiation methods PCR, ELISA to characterize as vaccine or wild type 1 and nucleic acid sequencing of entire VP1 region of poliovirus genome to determine the genetic relatedness. Results One hundred eleven wild type 1 poliovirus isolates were subjected to nucleotide sequencing for genetic variation study. Considering the 15% divergence of the sequences from Sabin 1, Phylogenetic analysis by MEGA software revealed that active inter and intra country transmission of many genetically distinct strains of wild poliovirus type 1 belonged to genotype SOAS which is indigenous in this region. By grouping wild type 1 polioviruses according to nucleotide sequence homology, three distinct clusters A, B and C were obtained with multiple chains of transmission together with some silent circulations represented by orphan lineages. Conclusion Our results emphasize that there was a persistent transmission of wild type1 polioviruses in Pakistan and Afghanistan during 2005-2007. The epidemiologic information provided by the sequence data can contribute to the formulation of better strategies for poliomyelitis control to those critical areas, associated with high risk population groups which include migrants, internally displaced people, and refugees. The implication of this study is to maintain high quality mass immunization with oral polio vaccine (OPV) in order to interrupt chains of virus transmission in both countries to endorse substantial progress in Eastern-Mediterranean region. PMID:22353446

Staurosporine scaffold-based rational discovery of the wild-type sparing reversible inhibitors of EGFR T790M gatekeeper mutant in lung cancer with analog-sensitive kinase technology.

PubMed

Song, Xiaoyun; Liu, Xingcai; Ding, Xi

2017-04-01

The human epidermal growth factor receptor (EGFR) has been established as an attractive target for lung cancer therapy. However, an acquired EGFR T790M gatekeeper mutation is frequently observed in patients treated with first-line anticancer agents such as gefitinib and erlotinib to cause drug resistance, largely limiting the application of small-molecule kinase inhibitors in EGFR-targeted chemotherapy. Previously, the reversible pan-kinase inhibitor staurosporine and its several analogs such as Gö6976 and K252a have been reported to selectively inhibit the EGFR T790M mutant (EGFR T790M ) over wild-type kinase (EGFR WT ), suggesting that the staurosporine scaffold is potentially to develop the wild-type sparing reversible inhibitors of EGFR T790M . Here, we systematically evaluated the inhibitor response of 28 staurosporine scaffold-based compounds to EGFR T790M mutation at structural, energetic, and molecular levels by using an integrated in silico-in vitro analog-sensitive (AS) kinase technology. With the strategy, we were able to identify 4 novel wild-type sparing inhibitors UCN-01, UCN-02, AFN941, and SB-218078 with high or moderate selectivity of 30-, 45-, 5-, and 8-fold for EGFR T790M over EGFR WT , respectively, which are comparable with or even better than that of the parent compound staurosporine (24-fold). Molecular modeling and structural analysis revealed that van der Waals contacts and hydrophobic forces can form between the side chain of mutated residue Met790 and the pyrrolidinone moiety of inhibitor ligand UCN-02, which may simultaneously improve the favorable interaction energy between the kinase and inhibitor, and reduce the unfavorable desolvation penalty upon the kinase-inhibitor binding. A hydroxyl group of UCN-02 additional to staurosporine locates at the pyrrolidinone moiety, which can largely alter the electronic distribution of pyrrolidinone moiety and thus promote the intermolecular interaction with Met790 residue. This can well explain the measured higher selectivity of UCN-02 than staurosporine for mutant over wild-type kinase. Copyright © 2016 John Wiley & Sons, Ltd.

The SBP-Box Gene VpSBP11 from Chinese Wild Vitis Is Involved in Floral Transition and Affects Leaf Development.

PubMed

Hou, Hongmin; Yan, Xiaoxiao; Sha, Ting; Yan, Qin; Wang, Xiping

2017-07-13

Flowering occurs in angiosperms during a major developmental transition from vegetative growth to the reproductive phase. Squamosa promoter binding protein (SBP)-box genes have been found to play critical roles in regulating flower and fruit development, but their roles in grapevine have remained unclear. To better understand the functions of the grape SBP-box genes in both vegetative and reproductive growth phases, a full-length complementary DNA (cDNA) sequence of the putative SBP-box transcription factor gene, VpSBP11 , was obtained from Chinese wild grapevine Vitis pseudoreticulata Wen Tsai Wang (W. T. Wang) clone 'Baihe-35-1'. VpSBP11 encoded a putative polypeptide of 170 amino acids with a highly conserved SBP-domain with two zinc-binding sites of the Cx2C-x3-H-x11-C-x6-H (C2HCH) type and a nuclear localization signal. We confirmed that the VpSBP11 protein was targeted to the nucleus and possessed transcriptional activation activity by subcellular localization and trans -activation assay. Over-expression of VpSBP11 in Arabidopsis thaliana was shown to activate the FUL gene, and subsequently the AP1 and LFY genes, all of which were floral meristem identity genes, and to cause earlier flowering than in wild type (WT) plants. The pattern of vegetative growth was also different between the transgenic and WT plants. For example, in the VpSBP11 over-expressing transgenic plants, the number of rosette leaves was less than that of WT; the petiole was significantly elongated; and the rosette and cauline leaves curled upwards or downwards. These results were consistent with VpSBP11 acting as a transcription factor during the transition from the vegetative stage to the reproductive stage.

The Epidemiologic and Pharmacodynamic Cutoff Values of Tilmicosin against Haemophilus parasuis.

PubMed

Zhang, Peng; Hao, Haihong; Li, Jun; Ahmad, Ijaz; Cheng, Guyue; Chen, Dongmei; Tao, Yanfei; Huang, Lingli; Wang, Yulian; Dai, Menghong; Liu, Zhenli; Yuan, Zonghui

2016-01-01

The aim of this study was to establish antimicrobial susceptibility breakpoints for tilmicosin against Haemophilus parasuis, which is an important pathogen of respiratory tract infections. The minimum inhibitory concentrations (MICs) of 103 H. parasuis isolates were determined by the agar dilution method. The wild type (WT) distribution and epidemiologic cutoff value (ECV) were evaluated by statistical analysis. The new bronchoaveolar lavage was used to establish intrapulmonary pharmacokinetic (PK) model in swine. The pharmacokinetic (PK) parameters of tilmicosin, both in pulmonary epithelial lining fluid (PELF) and in plasma, were determined using high performance liquid chromatography method and WinNonlin software. The pharmacodynamic cutoff (COPD) was calculated using Monte Carlo simulation. Our results showed that 100% of WT isolates were covered when the ECV was set at 16 μg/mL. The tilmicosin had concentration-dependent activity against H. parasuis. The PK data indicated that tilmicosin concentrations in PELF was rapidly increased to high levels at 4 h and kept stable until 48 h after drug administration, while the tilmicosin concentration in plasma reached maximum levels at 4 h and continued to decrease during 4-72 h. Using Monte Carlo simulation, COPD was defined as 1 μg/mL. Conclusively, the ECV and COPD of tilmicosin against H. parasuis were established for the first time based on the MIC distribution and PK-PD analysis in the target tissue, respectively. These values are of great importance for detection of tilmicosin-resistant H. parasuis and for effective treatment of clinical intrapulmonary infection caused by H. parasuis.

The Epidemiologic and Pharmacodynamic Cutoff Values of Tilmicosin against Haemophilus parasuis

PubMed Central

Zhang, Peng; Hao, Haihong; Li, Jun; Ahmad, Ijaz; Cheng, Guyue; Chen, Dongmei; Tao, Yanfei; Huang, Lingli; Wang, Yulian; Dai, Menghong; Liu, Zhenli; Yuan, Zonghui

2016-01-01

The aim of this study was to establish antimicrobial susceptibility breakpoints for tilmicosin against Haemophilus parasuis, which is an important pathogen of respiratory tract infections. The minimum inhibitory concentrations (MICs) of 103 H. parasuis isolates were determined by the agar dilution method. The wild type (WT) distribution and epidemiologic cutoff value (ECV) were evaluated by statistical analysis. The new bronchoaveolar lavage was used to establish intrapulmonary pharmacokinetic (PK) model in swine. The pharmacokinetic (PK) parameters of tilmicosin, both in pulmonary epithelial lining fluid (PELF) and in plasma, were determined using high performance liquid chromatography method and WinNonlin software. The pharmacodynamic cutoff (COPD) was calculated using Monte Carlo simulation. Our results showed that 100% of WT isolates were covered when the ECV was set at 16 μg/mL. The tilmicosin had concentration-dependent activity against H. parasuis. The PK data indicated that tilmicosin concentrations in PELF was rapidly increased to high levels at 4 h and kept stable until 48 h after drug administration, while the tilmicosin concentration in plasma reached maximum levels at 4 h and continued to decrease during 4–72 h. Using Monte Carlo simulation, COPD was defined as 1 μg/mL. Conclusively, the ECV and COPD of tilmicosin against H. parasuis were established for the first time based on the MIC distribution and PK-PD analysis in the target tissue, respectively. These values are of great importance for detection of tilmicosin-resistant H. parasuis and for effective treatment of clinical intrapulmonary infection caused by H. parasuis. PMID:27047487

Multicenter Evaluation of MIC Distributions for Epidemiologic Cutoff Value Definition To Detect Amphotericin B, Posaconazole, and Itraconazole Resistance among the Most Clinically Relevant Species of Mucorales

PubMed Central

Chakrabarti, A.; Chowdhary, A.; Cordoba, S.; Dannaoui, E.; Dufresne, P.; Fothergill, A.; Ghannoum, M.; Gonzalez, G. M.; Guarro, J.; Kidd, S.; Lass-Flörl, C.; Meis, J. F.; Pelaez, T.; Tortorano, A. M.; Turnidge, J.

2015-01-01

Clinical breakpoints (CBPs) have not been established for the Mucorales and any antifungal agent. In lieu of CBPs, epidemiologic cutoff values (ECVs) are proposed for amphotericin B, posaconazole, and itraconazole and four Mucorales species. Wild-type (WT) MIC distributions (organisms in a species-drug combination with no detectable acquired resistance mechanisms) were defined with available pooled CLSI MICs from 14 laboratories (Argentina, Australia, Canada, Europe, India, Mexico, and the United States) as follows: 10 Apophysomyces variabilis, 32 Cunninghamella bertholletiae, 136 Lichtheimia corymbifera, 10 Mucor indicus, 123 M. circinelloides, 19 M. ramosissimus, 349 Rhizopus arrhizus, 146 R. microsporus, 33 Rhizomucor pusillus, and 36 Syncephalastrum racemosum isolates. CLSI broth microdilution MICs were aggregated for the analyses. ECVs comprising ≥95% and ≥97.5% of the modeled populations were as follows: amphotericin B ECVs for L. corymbifera were 1 and 2 μg/ml, those for M. circinelloides were 1 and 2 μg/ml, those for R. arrhizus were 2 and 4 μg/ml, and those for R. microsporus were 2 and 2 μg/ml, respectively; posaconazole ECVs for L. corymbifera were 1 and 2, those for M. circinelloides were 4 and 4, those for R. arrhizus were 1 and 2, and those for R. microsporus were 1 and 2, respectively; both itraconazole ECVs for R. arrhizus were 2 μg/ml. ECVs may aid in detecting emerging resistance or isolates with reduced susceptibility (non-WT MICs) to the agents evaluated. PMID:25583714

Multicenter evaluation of MIC distributions for epidemiologic cutoff value definition to detect amphotericin B, posaconazole, and itraconazole resistance among the most clinically relevant species of Mucorales.

PubMed

Espinel-Ingroff, A; Chakrabarti, A; Chowdhary, A; Cordoba, S; Dannaoui, E; Dufresne, P; Fothergill, A; Ghannoum, M; Gonzalez, G M; Guarro, J; Kidd, S; Lass-Flörl, C; Meis, J F; Pelaez, T; Tortorano, A M; Turnidge, J

2015-03-01

Clinical breakpoints (CBPs) have not been established for the Mucorales and any antifungal agent. In lieu of CBPs, epidemiologic cutoff values (ECVs) are proposed for amphotericin B, posaconazole, and itraconazole and four Mucorales species. Wild-type (WT) MIC distributions (organisms in a species-drug combination with no detectable acquired resistance mechanisms) were defined with available pooled CLSI MICs from 14 laboratories (Argentina, Australia, Canada, Europe, India, Mexico, and the United States) as follows: 10 Apophysomyces variabilis, 32 Cunninghamella bertholletiae, 136 Lichtheimia corymbifera, 10 Mucor indicus, 123 M. circinelloides, 19 M. ramosissimus, 349 Rhizopus arrhizus, 146 R. microsporus, 33 Rhizomucor pusillus, and 36 Syncephalastrum racemosum isolates. CLSI broth microdilution MICs were aggregated for the analyses. ECVs comprising ≥95% and ≥97.5% of the modeled populations were as follows: amphotericin B ECVs for L. corymbifera were 1 and 2 μg/ml, those for M. circinelloides were 1 and 2 μg/ml, those for R. arrhizus were 2 and 4 μg/ml, and those for R. microsporus were 2 and 2 μg/ml, respectively; posaconazole ECVs for L. corymbifera were 1 and 2, those for M. circinelloides were 4 and 4, those for R. arrhizus were 1 and 2, and those for R. microsporus were 1 and 2, respectively; both itraconazole ECVs for R. arrhizus were 2 μg/ml. ECVs may aid in detecting emerging resistance or isolates with reduced susceptibility (non-WT MICs) to the agents evaluated. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Structural and functional cardiac cholinergic deficits in adult neurturin knockout mice.

PubMed

Mabe, Abigail M; Hoover, Donald B

2009-04-01

Previous work provided indirect evidence that the neurotrophic factor neurturin (NRTN) is required for normal cholinergic innervation of the heart. This study used nrtn knockout (KO) and wild-type (WT) mice to determine the effect of nrtn deletion on cardiac cholinergic innervation and function in the adult heart. Immunohistochemistry, confocal microscopy, and quantitative image analysis were used to directly evaluate intrinsic cardiac neuronal development. Atrial acetylcholine (ACh) levels were determined as an indirect index of cholinergic innervation. Cholinergic function was evaluated by measuring negative chronotropic responses to right vagal nerve stimulation in anaesthetized mice and responses of isolated atria to muscarinic agonists. KO hearts contained only 35% the normal number of cholinergic neurons, and the residual cholinergic neurons were 15% smaller than in WT. Cholinergic nerve density at the sinoatrial node was reduced by 87% in KOs, but noradrenergic nerve density was unaffected. Atrial ACh levels were substantially lower in KO mice (0.013 +/- 0.004 vs. 0.050 +/- 0.011 pmol/microg protein; P < 0.02) as expected from cholinergic neuron and nerve fibre deficits. Maximum bradycardia evoked by vagal stimulation was reduced in KO mice (38 +/- 6% vs. 69 +/- 3% decrease at 20 Hz; P < 0.001), and chronotropic responses took longer to develop and fade. In contrast to these deficits, isolated atria from KO mice had normal post-junctional sensitivity to carbachol and bethanechol. These findings demonstrate that NRTN is essential for normal cardiac cholinergic innervation and cholinergic control of heart rate. The presence of residual cardiac cholinergic neurons and vagal bradycardia in KO mice suggests that additional neurotrophic factors may influence this system.

Chlamydia muridarum Genital and Gastrointestinal Infection Tropism Is Mediated by Distinct Chromosomal Factors.

PubMed

Morrison, Sandra G; Giebel, Amanda M; Toh, Evelyn C; Spencer, Horace J; Nelson, David E; Morrison, Richard P

2018-07-01

Some members of the genus Chlamydia , including the human pathogen Chlamydia trachomatis , infect multiple tissues, including the genital and gastrointestinal (GI) tracts. However, it is unknown if bacterial targeting to these sites is mediated by multifunctional or distinct chlamydial factors. We previously showed that disruption of individual large clostridial toxin homologs encoded within the Chlamydia muridarum plasticity zone were not critical for murine genital tract infection. Here, we assessed whether cytotoxin genes contribute to C. muridarum GI tropism. Infectivity and shedding of wild-type (WT) C. muridarum and three mutants containing nonsense mutations in different cytotoxin genes, tc0437 , tc0438 , and tc0439 , were compared in mouse genital and GI infection models. One mutant, which had a nonsense mutation in tc0439 , was highly attenuated for GI infection and had a GI 50% infectious dose (ID 50 ) that was 1,000 times greater than that of the WT. GI inoculation with this mutant failed to elicit anti-chlamydial antibodies or to protect against subsequent genital tract infection. Genome sequencing of the tc0439 mutant revealed additional chromosomal mutations, and phenotyping of additional mutants suggested that the GI attenuation might be linked to a nonsense mutation in tc0600 The molecular mechanism underlying this dramatic difference in tissue-tropic virulence is not fully understood. However, isolation of these mutants demonstrates that distinct chlamydial chromosomal factors mediate chlamydial tissue tropism and provides a basis for vaccine initiatives to isolate chlamydia strains that are attenuated for genital infection but retain the ability to colonize the GI tract and elicit protective immune responses. Copyright © 2018 Morrison et al.

Interplay between VEGF and Nrf2 regulates angiogenesis due to intracranial venous hypertension.

PubMed

Li, Liwen; Pan, Hao; Wang, Handong; Li, Xiang; Bu, Xiaomin; Wang, Qiang; Gao, Yongyue; Wen, Guodao; Zhou, Yali; Cong, Zixiang; Yang, Youqing; Tang, Chao; Liu, Zhengwei

2016-11-21

Venous hypertension(VH) plays an important role in the pathogenesis of cerebral arteriovenous malformations (AVMs) and is closely associated with the HIF-1α/VEGF signaling pathway. Nuclear factor erythroid 2-related factor 2(Nrf2) significantly influences angiogenesis; however, the interplay between Nrf2 and VEGF under VH in brain AVMs remains unclear. Therefore, our study aimed to investigate the interplay between Nrf2 and VEGF due to VH in brain AVMs. Immunohistochemistry indicated that Nrf2 and VEGF were highly expressed in human brain AVM tissues. In vivo, we established a VH model in both wild-type (WT) and siRNA-mediated Nrf2 knockdown rats. VH significantly increased the expression of Nrf2 and VEGF. Loss of Nrf2 markedly inhibited the upregulation of VEGF, as determined by Western blot analysis and qRT-PCR. In vitro, primary brain microvascular endothelial cells (BMECs) were isolated from WT and Nrf2 -/- mice, and a VEGF-Nrf2 positive feed-back loop was observed in BMECs. By trans well assay and angiogenesis assay, Nrf2 knockout significantly inhibited the migration and vascular tube formation of BMECs. These findings suggest that the interplay between Nrf2 and VEGF can contribute to VH-induced angiogenesis in brain AVMs pathogenesis.

C/EBPβ promotes BCR–ABL-mediated myeloid expansion and leukemic stem cell exhaustion

PubMed Central

Hayashi, Y; Hirai, H; Kamio, N; Yao, H; Yoshioka, S; Miura, Y; Ashihara, E; Fujiyama, Y; Tenen, DG; Maekawa, T

2015-01-01

The BCR–ABL fusion oncoprotein accelerates differentiation and proliferation of myeloid cells during the chronic phase of chronic myeloid leukemia (CP-CML). Here, the role of CCAAT/enhancer binding protein β (C/EBPβ), a regulator for ‘emergency granulopoiesis,’ in the pathogenesis of CP-CML was examined. C/EBPβ expression was upregulated in Lineage− CD34+ CD38− hematopoietic stem cells (HSCs) and myeloid progenitors isolated from bone marrow of patients with CP-CML. In EML cells, a mouse HSC line, BCR–ABL upregulated C/EBPβ, at least in part, through the activation of STAT5. Myeloid differentiation and proliferation induced by BCR–ABL was significantly impaired in C/EBPβ-deficient bone marrow cells in vitro. Mice that were transplanted with BCR–ABL-transduced C/EBPβ knockout bone marrow cells survived longer than mice that received BCR–ABL-transduced wild-type (WT) bone marrow cells. Significantly higher levels of leukemic stem cells were maintained in BCR–ABL-transduced C/EBPβ-deficient cells than in BCR–ABL-transduced WT cells. These results suggest that C/EBPβ is involved in BCR–ABL-mediated myeloid expansion. Further elucidation of the molecular mechanisms underlying the C/EBPβ-mediated stem cell loss might reveal a novel therapeutic strategy for eradication of CML stem cells. PMID:22948537

Efficient selective breeding of live oil-rich Euglena gracilis with fluorescence-activated cell sorting

PubMed Central

Yamada, Koji; Suzuki, Hideyuki; Takeuchi, Takuto; Kazama, Yusuke; Mitra, Sharbanee; Abe, Tomoko; Goda, Keisuke; Suzuki, Kengo; Iwata, Osamu

2016-01-01

Euglena gracilis, a microalgal species of unicellular flagellate protists, has attracted much attention in both the industrial and academic sectors due to recent advances in the mass cultivation of E. gracilis that have enabled the cost-effective production of nutritional food and cosmetic commodities. In addition, it is known to produce paramylon (β-1,3-glucan in a crystalline form) as reserve polysaccharide and convert it to wax ester in hypoxic and anaerobic conditions–a promising feedstock for biodiesel and aviation biofuel. However, there remain a number of technical challenges to be solved before it can be deployed in the competitive fuel market. Here we present a method for efficient selective breeding of live oil-rich E. gracilis with fluorescence-activated cell sorting (FACS). Specifically, the selective breeding method is a repetitive procedure for one-week heterotrophic cultivation, staining intracellular lipids with BODIPY505/515, and FACS-based isolation of top 0.5% lipid-rich E. gracilis cells with high viability, after inducing mutation with Fe-ion irradiation to the wild type (WT). Consequently, we acquire a live, stable, lipid-rich E. gracilis mutant strain, named B1ZFeL, with 40% more lipid content on average than the WT. Our method paves the way for rapid, cost-effective, energy-efficient production of biofuel. PMID:27212384

ER/Golgi trafficking is facilitated by unbranched actin filaments containing Tpm4.2.

PubMed

Kee, Anthony J; Bryce, Nicole S; Yang, Lingyan; Polishchuk, Elena; Schevzov, Galina; Weigert, Roberto; Polishchuk, Roman; Gunning, Peter W; Hardeman, Edna C

2017-10-01

We have identified novel actin filaments defined by tropomyosin Tpm4.2 at the ER. EM analysis of mouse embryo fibroblasts (MEFs) isolated from mice expressing a mutant Tpm4.2 (Tpm4 Plt53/Plt53 ), incapable of incorporating into actin filaments, revealed swollen ER structures compared with wild-type (WT) MEFs (Tpm4 +/+ ). ER-to-Golgi, but not Golgi-to-ER trafficking was altered in the Tpm4 Plt53/Plt53 MEFs following the transfection of the temperature sensitive ER-associated ts045-VSVg construct. Exogenous Tpm4.2 was able to rescue the ER-to-Golgi trafficking defect in the Tpm4 Plt53/Plt53 cells. The treatment of WT MEFs with the myosin II inhibitor, blebbistatin, blocked the Tpm4.2-dependent ER-to-Golgi trafficking. The lack of an effect on ER-to-Golgi trafficking following treatment of MEFs with CK666 indicates that branched Arp2/3-containing actin filaments are not involved in anterograde vesicle trafficking. We propose that unbranched, Tpm4.2-containing filaments have an important role in maintaining ER/Golgi structure and that these structures, in conjunction with myosin II motors, mediate ER-to-Golgi trafficking. © 2017 Wiley Periodicals, Inc.

Mechanisms of HO-1 mediated attenuation of renal immune injury: a gene profiling study.

PubMed

Duann, Pu; Lianos, Elias A

2011-10-01

Using a mouse model of immune injury directed against the renal glomerular vasculature and resembling human forms of glomerulonephritis (GN), we assessed the effect of targeted expression of the cytoprotective enzyme heme oxygenase (HO)-1. A human (h) HO-1 complementary DNAN (cDNA) sequence was targeted to glomerular epithelial cells (GECs) using a GEC-specific murine nephrin promoter. Injury by administration of antibody against the glomerular basement membrane (anti-GBM) to transgenic (TG) mice with GEC-targeted hHO-1 was attenuated compared with wild-type (WT) controls. To explore changes in the expression of genes that could mediate this salutary effect, we performed gene expression profiling using a microarray analysis of RNA isolated from the renal cortex of WT or TG mice with or without anti-GBM antibody-induced injury. Significant increases in expression were detected in 9 major histocompatibility complex (MHC)-class II genes, 2 interferon-γ (IFN-γ)-inducible guanosine triphosphate (GTP)ases, and 3 genes of the ubiquitin-proteasome system. The increase in MHC-class II and proteasome gene expression in TG mice with injury was validated by real-time polymerase chain reaction (PCR) or Western blot analysis. The observations point to novel mechanisms underlying the cytoprotective effect of HO-1 in renal immune injury. Copyright © 2011. Published by Mosby, Inc.

Monitoring Photosynthesis in Individual Cells of Synechocystis sp. PCC 6803 on a Picosecond Timescale

PubMed Central

Krumova, S.B.; Laptenok, S.P.; Borst, J.W.; Ughy, B.; Gombos, Z.; Ajlani, G.; van Amerongen, H.

2010-01-01

Picosecond fluorescence kinetics of wild-type (WT) and mutant cells of Synechocystis sp. PCC 6803, were studied at the ensemble level with a streak-camera and at the cell level using fluorescence-lifetime-imaging microscopy (FLIM). The FLIM measurements are in good agreement with the ensemble measurements, but they (can) unveil variations between and within cells. The BE mutant cells, devoid of photosystem II (PSII) and of the light-harvesting phycobilisomes, allowed the study of photosystem I (PSI) in vivo for the first time, and the observed 6-ps equilibration process and 25-ps trapping process are the same as found previously for isolated PSI. No major differences are detected between different cells. The PAL mutant cells, devoid of phycobilisomes, show four lifetimes: ∼20 ps (PSI and PSII), ∼80 ps, ∼440 ps, and 2.8 ns (all due to PSII), but not all cells are identical and variations in the kinetics are traced back to differences in the PSI/PSII ratio. Finally, FLIM measurements on WT cells reveal that in some cells or parts of cells, phycobilisomes are disconnected from PSI/PSII. It is argued that the FLIM setup used can become instrumental in unraveling photosynthetic regulation mechanisms in the future. PMID:20858447

Generating Phenotypic Diversity in a Fungal Biocatalyst to Investigate Alcohol Stress Tolerance Encountered during Microbial Cellulosic Biofuel Production

PubMed Central

Hennessy, Rosanna C.; Doohan, Fiona; Mullins, Ewen

2013-01-01

Consolidated bioprocessing (CBP) of lignocellulosic biomass offers an alternative route to renewable energy. The crop pathogen Fusarium oxysporum is a promising fungal biocatalyst because of its broad host range and innate ability to co-saccharify and ferment lignocellulose to bioethanol. A major challenge for cellulolytic CBP-enabling microbes is alcohol inhibition. This research tested the hypothesis that Agrobacterium tumefaciens - mediated transformation (ATMT) could be exploited as a tool to generate phenotypic diversity in F. oxysporum to investigate alcohol stress tolerance encountered during CBP. A random mutagenesis library of gene disruption transformants (n=1,563) was constructed and screened for alcohol tolerance in order to isolate alcohol sensitive or tolerant phenotypes. Following three rounds of screening, exposure of select transformants to 6% ethanol and 0.75% n-butanol resulted respectively in increased (≥11.74%) and decreased (≤43.01%) growth compared to the wild –type (WT). Principal component analysis (PCA) quantified the level of phenotypic diversity across the population of genetically transformed individuals and isolated candidate strains for analysis. Characterisation of one strain, Tr. 259, ascertained a reduced growth phenotype under alcohol stress relative to WT and indicated the disruption of a coding region homologous to a putative sugar transporter (FOXG_09625). Quantitative PCR (RT-PCR) showed FOXG_09625 was differentially expressed in Tr. 259 compared to WT during alcohol-induced stress (P<0.05). Phylogenetic analysis of putative sugar transporters suggests diverse functional roles in F. oxysporum and other filamentous fungi compared to yeast for which sugar transporters form part of a relatively conserved family. This study has confirmed the potential of ATMT coupled with a phenotypic screening program to select for genetic variation induced in response to alcohol stress. This research represents a first step in the investigation of alcohol tolerance in F. oxysporum and has resulted in the identification of several novel strains, which will be of benefit to future biofuel research. PMID:24147009

Live, Attenuated Influenza A H5N1 Candidate Vaccines Provide Broad Cross-Protection in Mice and Ferrets

PubMed Central

Mills, Kimberly L; Jin, Hong; Duke, Greg; Lu, Bin; Luke, Catherine J; Murphy, Brian; Swayne, David E; Kemble, George; Subbarao, Kanta

2006-01-01

Background Recent outbreaks of highly pathogenic influenza A H5N1 viruses in humans and avian species that began in Asia and have spread to other continents underscore an urgent need to develop vaccines that would protect the human population in the event of a pandemic. Methods and Findings Live, attenuated candidate vaccines possessing genes encoding a modified H5 hemagglutinin (HA) and a wild-type (wt) N1 neuraminidase from influenza A H5N1 viruses isolated in Hong Kong and Vietnam in 1997, 2003, and 2004, and remaining gene segments derived from the cold-adapted (ca) influenza A vaccine donor strain, influenza A/Ann Arbor/6/60 ca (H2N2), were generated by reverse genetics. The H5N1 ca vaccine viruses required trypsin for efficient growth in vitro, as predicted by the modification engineered in the gene encoding the HA, and possessed the temperature-sensitive and attenuation phenotypes specified by the internal protein genes of the ca vaccine donor strain. More importantly, the candidate vaccines were immunogenic in mice. Four weeks after receiving a single dose of 106 50% tissue culture infectious doses of intranasally administered vaccines, mice were fully protected from lethality following challenge with homologous and antigenically distinct heterologous wt H5N1 viruses from different genetic sublineages (clades 1, 2, and 3) that were isolated in Asia between 1997 and 2005. Four weeks after receiving two doses of the vaccines, mice and ferrets were fully protected against pulmonary replication of homologous and heterologous wt H5N1 viruses. Conclusions The promising findings in these preclinical studies of safety, immunogenicity, and efficacy of the H5N1 ca vaccines against antigenically diverse H5N1 vaccines provide support for their careful evaluation in Phase 1 clinical trials in humans. PMID:16968127

Heat-killed BCG induces biphasic cyclooxygenase 2+ splenic macrophage formation--role of IL-10 and bone marrow precursors.

PubMed

Shibata, Yoshimi; Gabbard, Jon; Yamashita, Makiko; Tsuji, Shoutaro; Smith, Mike; Nishiyama, Akihito; Henriksen, Ruth Ann; Myrvik, Quentin N

2006-09-01

Previous studies have shown that prostaglandin E(2) (PGE(2)) release by splenic F4/80(+) cyclooxygenase (COX)-2(+) macrophages (MØ) isolated from mice, treated with mycobacterial components, plays a major role in the regulation of immune responses. However, splenic MØ, isolated from untreated mice and treated in vitro with lipopolysaccharide and interferon-gamma, express COX-1 and COX-2 within 1 day but release only minimal amounts of PGE(2) following elicitation with calcium ionophore A23187. For further characterization of in vivo requirements for development of PGE(2)-releasing MØ (PGE(2)-MØ), C57Bl/6 [wild-type (WT)], and interleukin (IL)-10-deficient (IL-10(-/-)) mice were treated intraperitoneally with heat-killed Mycobacterium bovis bacillus Calmette-Guerin (HK-BCG). One day following injection, COX-2 was induced in splenic MØ of both mouse strains. However, PGE(2) biosynthesis by these MØ was not increased. Thus, expression of COX-2 is not sufficient to induce PGE(2) production in vivo or in vitro. In sharp contrast, 14 days after HK-BCG treatment, PGE(2) release by COX-2(+) splenic MØ increased as much as sevenfold, and a greater increase was seen in IL-10(-/-) cells than in WT cells. To further determine whether the 14-day splenic PGE(2)-MØ could be derived from bone marrow precursors, we established a chimera in which bone marrow cells were transfused from green fluorescent protein (GFP)-transgenic donors to WT mice. Donors and recipients were treated with HK-BCG simultaneously, and marrow transfusion was performed on Days 1 and 2. On Day 14 after BCG treatment, a significant number of spleen cells coexpressed COX-2 and GFP, indicating that bone marrow-derived COX-2(+) MØ may be responsible for the increased PGE(2) production.

Role of B-type natriuretic peptide in epoxyeicosatrienoic acid-mediated improved post-ischaemic recovery of heart contractile function

PubMed Central

Chaudhary, Ketul R.; Batchu, Sri Nagarjun; Das, Dipankar; Suresh, Mavanur R.; Falck, John R.; Graves, Joan P.; Zeldin, Darryl C.; Seubert, John M.

2009-01-01

Aims This study examined the functional role of B-type natriuretic peptide (BNP) in epoxyeicosatrienoic acid (EET)-mediated cardioprotection in mice with targeted disruption of the sEH or Ephx2 gene (sEH null). Methods and results Isolated mouse hearts were perfused in the Langendorff mode and subjected to global no-flow ischaemia followed by reperfusion. Hearts were analysed for recovery of left ventricular developed pressure (LVDP), mRNA levels, and protein expression. Naïve hearts from sEH null mice had similar expression of preproBNP (Nppb) mRNA compared with wild-type (WT) hearts. However, significant increases in Nppb mRNA and BNP protein expression occurred during post-ischaemic reperfusion and correlated with improved post-ischaemic recovery of LVDP. Perfusion with the putative EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid prior to ischaemia reduced the preproBNP mRNA in sEH null hearts. Inhibitor studies demonstrated that perfusion with the natriuretic peptide receptor type-A (NPR-A) antagonist, A71915, limited the improved recovery in recombinant full-length mouse BNP (rBNP)- and 11,12-EET-perfused hearts as well as in sEH null mice. Increased expression of phosphorylated protein kinase C ε and Akt were found in WT hearts perfused with either 11,12-EET or rBNP, while mitochondrial glycogen synthase kinase-3β was significantly lower in the same samples. Furthermore, treatment with the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin abolished improved LVDP recovery in 11,12-EET-treated hearts but not did significantly inhibit recovery of rBNP-treated hearts. Conclusion Taken together, these data indicate that EET-mediated cardioprotection involves BNP and PI3K signalling events. PMID:19401302

Preparation of Graphene Oxide and Its Mechanism in Promoting Tomato Roots Growth.

PubMed

Jiao, Jingzhi; Cheng, Fan; Zhang, Xuekun; Xie, Lingli; Li, Zhiyang; Yuan, Chengfei; Xu, Benbo; Zhang, Liming

2016-04-01

Graphene oxide is a new kind of nanomaterial. The graphene oxide was prepared and its quality detected by atomic force microscopy (AFM) and transmission electron microscopy (TEM), for better understanding of effects of the nanomaterial on plants. Wild type. (WT) tomato (Solanum lycopersicum) germplasm 'New Yorker' and corresponding transgenic plants (Prd29A::LeNCED1) were treated with prepared graphene oxide. 9-cis-epoxycarotenoid dioxygenase (NCED) is a key gene for ABA biosynthesis and overexpression of the NCED resulted in ABA accumulation and higher drought tolerance. Seminal root length in the WT tomato was longer than that in the control samples when the seedlings were treated with 20 mg/L graphene oxide for 15 days. In contrast, the same treatment resulted in shorter seminal root length in the transgenic plants compared with control samples. The graphene oxide treatments led to lower Superoxide Dismutase (SOD), Peroxidase (POD), Catalase (CAT) activity and Malondialdehyde (MDA) content in the WT and transgenic plants. 20 mg/L graphene oxide treatment also affected the transcript levels of IAA7, IAA4 and IAA10 but the effect on the wild type and corresponding transgenic plants was different. IAA4 transcription level decreased both in the WT and Prd29A::LeNCED1 transgenic plants while the IAA7 transcription level decreased in the transgenic plants and increased in the WT tomato. The IAA10 transcription level decreased in the WT tomato and increased in the Prd29A::LeNCED1 transgenic plants. Graphene oxide treatments resulted in higher transcription level of ABCG25 and ABCG40 in the WT plants but had no significant effect on transgenic plants. The transcription level of NCED in the WT and Prd29A::LeNCED1 transgenic plants treated with graphene oxide increased significantly, however, it was higher in the transgenic plants than in the WT tomato after 15 d treatment, indicating that the graphene oxide activated the rd29A promoter as does drought and salt. The HD-ZIP transcription level only decreased significantly in the treated Prd29A::LeNCED1 transgenic plants. All these results suggested that there was a crosstalk between ABA and graphene oxide and the graphene oxide affected plant growth through the ABA and IAA pathway.

Interleukin-12- and interferon-γ-mediated natural killer cell activation by Agaricus blazei Murill

PubMed Central

Yuminamochi, Eri; Koike, Taisuke; Takeda, Kazuyoshi; Horiuchi, Isao; Okumura, Ko

2007-01-01

Dried fruiting bodies of Agaricus blazei Murill (A. blazei) and its extracts have generally used as complementary and alternative medicines (CAMs). Here, we report that the oral administration of A. blazei augmented cytotoxicity of natural killer (NK) cells in wild-type (WT) C57BL/6, C3H/HeJ, and BALB/c mice. Augmented cytotoxicity was demonstrated by purified NK cells from treated wild-type (WT) and RAG-2-deficient mice, but not from interferon-γ (IFN-γ) deficient mice. NK cell activation and IFN-γ production was also observed in vitro when dendritic cell (DC)-rich splenocytes of WT mice were coincubation with an extract of A. blazei. Both parameters were largely inhibited by neutralizing anti-interleukin-12 (IL-12) monoclonal antibody (mAb) and completely inhibited when anti-IL-12 mAb and anti-IL-18 mAb were used in combination. An aqueous extract of the hemicellulase-digested compound of A. blazei particle; (ABPC) induced IFN-γ production more effectively, and this was completely inhibited by anti-IL-12 mAb alone. NK cell cytotoxicty was augmented with the same extracts, again in an IL-12 and IFN-γ-dependent manner. These results clearly demonstrated that A. blazei and ABPC augmented NK cell activation through IL-12-mediated IFN-γ production. PMID:17346284

Site-directed mutagenesis of GH10 xylanase A from Penicillium canescens for determining factors affecting the enzyme thermostability.

PubMed

Denisenko, Yury A; Gusakov, Alexander V; Rozhkova, Aleksandra M; Osipov, Dmitry O; Zorov, Ivan N; Matys, Veronika Yu; Uporov, Igor V; Sinitsyn, Arkady P

2017-11-01

In order to investigate factors affecting the thermostability of GH10 xylanase A from Penicillium canescens (PcXylA) and to obtain its more stable variant, the wild-type (wt) enzyme and its mutant forms, carrying single amino acid substitutions, were cloned and expressed in Penicillium verruculosum B1-537 (niaD-) auxotrophic strain under the control of the cbh1 gene promoter. The recombinant PcXylA-wt and I6V, I6L, L18F, N77D, Y125R, H191R, S246P, A293P mutants were successfully expressed and purified for characterization. The mutations did not affect the enzyme specific activity against xylan from wheat as well as its pH-optimum of activity. One mutant (L18F) displayed a higher thermostability relative to the wild-type enzyme; its half-life time at 50-60°C was 2-2.5-fold longer than that for the PcXylA-wt, and the melting temperature was 60.0 and 56.1°C, respectively. Most of other mutations led to decrease in the enzyme thermostability. This study, together with data of other researchers, suggests that multiple mutations should be introduced into GH10 xylanases in order to dramatically improve their stability. Copyright © 2017 Elsevier B.V. All rights reserved.

Competition-based phenotyping reveals a fitness cost for maintaining phycobilisomes under fluctuating light in the cyanobacterium Fremyella diplosiphon

DOE PAGES

Agostoni, Marco; Lucker, Ben F.; Smith, Matthew A. Y.; ...

2016-02-21

Phycobilisomes (PBSs) are pigment-rich super-complexes required for efficient harvest and transfer of light energy to photosynthetic reaction centers of cyanobacteria. The model cyanobacterium Fremyella diplosiphon is able to adjust PBS pigmentation and size in response to the prevailing light spectrum through a process called complementary chromatic acclimation to optimize spectral light absorption, concomitantly optimizing photosynthesis and growth. We explored the fitness costs versus advantages of modulating antennae size and composition under sinusoidal continuous and fluctuating light conditions in F. diplosiphon by comparing growth of wild-type (WT) cells with a mutant strain deficient in PBSs in both monoculture and polyculture conditions.more » Comparative analyses of WT and the PBS-deficient FdCh1 strain under continuous vs. fluctuating sinusoidal light suggest a potential fitness advantage for maintaining PBSs in WT cells during continuous light and a fitness cost during transitions to and acclimation under fluctuating light. Here, we explored the physiological changes correlated with the observed differential growth to understand the dynamics and biochemical bases of comparative fitness of distinct strains under defined growth conditions. Wild-type F. diplosiphon appears to accumulate longer PBS rods and exhibits higher oxidative stress under fluctuating light conditions than continuous sinusoidal light, which may impact responses and the fitness of cells that do not adapt to rapid changes in external light.« less

Molecular dynamics simulations of wild type and mutants of botulinum neurotoxin A complexed with synaptic vesicle protein 2C.

PubMed

Wang, Feng; Wan, Hua; Hu, Jian-Ping; Chang, Shan

2015-01-01

Botulinum neurotoxins (BoNTs) are known as the most poisonous biological substances, and they are also used to treat a wide range of medical conditions as well as in the cosmetic applications. Recently, the complex structures of the BoNT/A receptor-binding domain (BoNT/A-RBD) and the synaptic vesicle protein 2C luminal domain (SV2C-LD) were determined by X-ray crystallography. In this article, the wild type (WT) and four mutants of the new structure are studied by molecular dynamics (MD) simulations. The differently decreased structural stabilities of the mutants relative to WT are shown to be consistent with the experimental data of binding affinities. The conformational changes of the five systems are explored by using principal component analysis (PCA) and free energy landscape (FEL) methods. Based on the calculation of interactions at the binding interface, we divide the interface between BoNT/A-RBD and SV2C-LD into two crucial binding regions. Through the comparison of WT and four mutants, we further propose the relationship between the conformational changes of BoNT/A-RBD:SV2C-LD and the interfacial interactions. This study would provide some new insights into the understanding of the dynamics and the interaction mechanism of BoNT/A-RBD:SV2C-LD.

18β-Glycyrrhetinic acid preferentially blocks late Na current generated by ΔKPQ Nav1.5 channels

PubMed Central

Du, Yi-mei; Xia, Cheng-kun; Zhao, Ning; Dong, Qian; Lei, Ming; Xia, Jia-hong

2012-01-01

Aim: To compare the effects of two stereoisomeric forms of glycyrrhetinic acid on different components of Na+ current, HERG and Kv1.5 channel currents. Methods: Wild-type (WT) and long QT syndrome type 3 (LQT-3) mutant ΔKPQ Nav1.5 channels, as well as HERG and Kv1.5 channels were expressed in Xenopus oocytes. In addition, isolated human atrial myocytes were used. Two-microelectrode voltage-clamp technique was used to record the voltage-activated currents. Results: Superfusion of 18β-glycyrrhetinic acid (18β-GA, 1–100 μmol/L) blocked both the peak current (INa,P) and late current (INa,L) generated by WT and ΔKPQ Nav1.5 channels in a concentration-dependent manner, while 18α-glycyrrhetinic acid (18α-GA) at the same concentrations had no effects. 18β-GA preferentially blocked INa,L (IC50=37.2±14.4 μmol/L) to INa,P (IC50=100.4±11.2 μmol/L) generated by ΔKPQ Nav1.5 channels. In human atrial myocytes, 18β-GA (30 μmol/L) inhibited 47% of INa,P and 87% of INa,L induced by Anemonia sulcata toxin (ATX-II, 30 nmol/L). Superfusion of 18β-GA (100 μmol/L) had no effects on HERG and Kv1.5 channel currents. Conclusion: 18β-GA preferentially blocked the late Na current without affecting HERG and Kv1.5 channels. PMID:22609834

Antifungal Susceptibility Testing of Aspergillus spp. by Using a Composite Correlation Index (CCI)-Based Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Method Appears To Not Offer Benefit over Traditional Broth Microdilution Testing

PubMed Central

Gitman, Melissa R.; McTaggart, Lisa; Spinato, Joanna; Poopalarajah, Rahgavi; Lister, Erin; Husain, Shahid

2017-01-01

ABSTRACT Aspergillus spp. cause serious invasive lung infections, and Aspergillus fumigatus is the most commonly encountered clinically significant species. Voriconazole is considered to be the drug of choice for treating A. fumigatus infections; however, rising resistance rates have been reported. We evaluated a matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS)-based method for the differentiation between wild-type and non-wild-type isolates of 20 Aspergillus spp. (including 2 isolates of Aspergillus ustus and 1 of Aspergillus calidoustus that were used as controls due their intrinsic low azole susceptibility with respect to the in vitro response to voriconazole). At 30 and 48 h of incubation, there was complete agreement between Cyp51A sequence analysis, broth microdilution, and MALDI-TOF MS classification of isolates as wild type or non-wild type. In this proof-of-concept study, we demonstrated that MALDI-TOF MS can be used to accurately detect A. fumigatus strains with reduced voriconazole susceptibility. However, rather than proving to be a rapid and simple method for antifungal susceptibility testing, this particular MS-based method showed no benefit over conventional testing methods. PMID:28404678

Antifungal Susceptibility Testing of Aspergillus spp. by Using a Composite Correlation Index (CCI)-Based Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Method Appears To Not Offer Benefit over Traditional Broth Microdilution Testing.

PubMed

Gitman, Melissa R; McTaggart, Lisa; Spinato, Joanna; Poopalarajah, Rahgavi; Lister, Erin; Husain, Shahid; Kus, Julianne V

2017-07-01

Aspergillus spp. cause serious invasive lung infections, and Aspergillus fumigatus is the most commonly encountered clinically significant species. Voriconazole is considered to be the drug of choice for treating A. fumigatus infections; however, rising resistance rates have been reported. We evaluated a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based method for the differentiation between wild-type and non-wild-type isolates of 20 Aspergillus spp. (including 2 isolates of Aspergillus ustus and 1 of Aspergillus calidoustus that were used as controls due their intrinsic low azole susceptibility with respect to the in vitro response to voriconazole). At 30 and 48 h of incubation, there was complete agreement between Cyp51A sequence analysis, broth microdilution, and MALDI-TOF MS classification of isolates as wild type or non-wild type. In this proof-of-concept study, we demonstrated that MALDI-TOF MS can be used to accurately detect A. fumigatus strains with reduced voriconazole susceptibility. However, rather than proving to be a rapid and simple method for antifungal susceptibility testing, this particular MS-based method showed no benefit over conventional testing methods. © Crown copyright 2017.

Coenzyme Q(1) as a probe for mitochondrial complex I activity in the intact perfused hyperoxia-exposed wild-type and Nqo1-null mouse lung.

PubMed

Bongard, Robert D; Myers, Charles R; Lindemer, Brian J; Baumgardt, Shelley; Gonzalez, Frank J; Merker, Marilyn P

2012-05-01

Previous studies showed that coenzyme Q(1) (CoQ(1)) reduction on passage through the rat pulmonary circulation was catalyzed by NAD(P)H:quinone oxidoreductase 1 (NQO1) and mitochondrial complex I, but that NQO1 genotype was not a factor in CoQ(1) reduction on passage through the mouse lung. The aim of the present study was to evaluate the complex I contribution to CoQ(1) reduction in the isolated perfused wild-type (NQO1(+/+)) and Nqo1-null (NQO1(-)/(-)) mouse lung. CoQ(1) reduction was measured as the steady-state pulmonary venous CoQ(1) hydroquinone (CoQ(1)H(2)) efflux rate during infusion of CoQ(1) into the pulmonary arterial inflow. CoQ(1)H(2) efflux rates during infusion of 50 μM CoQ(1) were not significantly different for NQO1(+/+) and NQO1(-/-) lungs (0.80 ± 0.03 and 0.68 ± 0.07 μmol·min(-1)·g lung dry wt(-1), respectively, P > 0.05). The mitochondrial complex I inhibitor rotenone depressed CoQ(1)H(2) efflux rates for both genotypes (0.19 ± 0.08 and 0.08 ± 0.04 μmol·min(-1)·g lung dry wt(-1) for NQO1(+/+) and NQO1(-/-), respectively, P < 0.05). Exposure of mice to 100% O(2) for 48 h also depressed CoQ(1)H(2) efflux rates in NQO1(+/+) and NQO1(-/-) lungs (0.43 ± 0.03 and 0.11 ± 0.04 μmol·min(-1)·g lung dry wt(-1), respectively, P < 0.05 by ANOVA). The impact of rotenone or hyperoxia on CoQ(1) redox metabolism could not be attributed to effects on lung wet-to-dry weight ratios, perfusion pressures, perfused surface areas, or total venous effluent CoQ(1) recoveries, the latter measured by spectrophotometry or mass spectrometry. Complex I activity in mitochondria-enriched lung fractions was depressed in hyperoxia-exposed lungs for both genotypes. This study provides new evidence for the potential utility of CoQ(1) as a nondestructive indicator of the impact of pharmacological or pathological exposures on complex I activity in the intact perfused mouse lung.

Disruption of Hypoxia-Inducible Transcription Factor-Prolyl Hydroxylase Domain-1 (PHD-1−/−) Attenuates Ex Vivo Myocardial Ischemia/Reperfusion Injury Through Hypoxia-Inducible Factor-1α Transcription Factor and Its Target Genes in Mice

PubMed Central

Adluri, Ram Sudheer; Thirunavukkarasu, Mahesh; Dunna, Nageswara Rao; Zhan, Lijun; Oriowo, Babatunde; Takeda, Kotaro; Sanchez, Juan A.; Otani, Hajime; Maulik, Gautam; Fong, Guo-Hua

2011-01-01

Abstract Hypoxia-inducible transcription factor (HIF)-prolyl hydroxylases domain (PHD-1–3) are oxygen sensors that regulate the stability of the HIFs in an oxygen-dependent manner. Suppression of PHD enzymes leads to stabilization of HIFs and offers a potential treatment option for many ischemic disorders, such as peripheral artery occlusive disease, myocardial infarction, and stroke. Here, we show that homozygous disruption of PHD-1 (PHD-1−/−) could facilitate HIF-1α-mediated cardioprotection in ischemia/reperfused (I/R) myocardium. Wild-type (WT) and PHD-1−/− mice were randomized into WT time-matched control (TMC), PHD-1−/− TMC (PHD1TMC), WT I/R, and PHD-1−/− I/R (PHD1IR). Isolated hearts from each group were subjected to 30 min of global ischemia followed by 2 h of reperfusion. TMC hearts were perfused for 2 h 30 min without ischemia. Decreased infarct size (35% ± 0.6% vs. 49% ± 0.4%) and apoptotic cardiomyocytes (106 ± 13 vs. 233 ± 21 counts/100 high-power field) were observed in PHD1IR compared to wild-type ischemia/reperfusion (WTIR). Protein expression of HIF-1α was significantly increased in PHD1IR compared to WTIR. mRNA expression of β-catenin (1.9-fold), endothelial nitric oxide synthase (1.9-fold), p65 (1.9-fold), and Bcl-2 (2.7-fold) were upregulated in the PHD1IR compared with WTIR, which was studied by real-time quantitative polymerase chain reaction. Further, gel-shift analysis showed increased DNA binding activity of HIF-1α and nuclear factor-kappaB in PHD1IR compared to WTIR. In addition, nuclear translocation of β-catenin was increased in PHD1IR compared with WTIR. These findings indicated that silencing of PHD-1 attenuates myocardial I/R injury probably by enhancing HIF-1α/β-catenin/endothelial nitric oxide synthase/nuclear factor-kappaB and Bcl-2 signaling pathway. Antioxid. Redox Signal. 15, 1789–1797. PMID:21083501

Kinetic and CD/MCD spectroscopic studies of the atypical, three-His-ligated, non-heme Fe2+ center in diketone dioxygenase: the role of hydrophilic outer shell residues in catalysis.

PubMed

Straganz, Grit D; Diebold, Adrienne R; Egger, Sigrid; Nidetzky, Bernd; Solomon, Edward I

2010-02-09

Diketone cleaving enzyme (Dke1) is a dioxygenase with an atypical, three-histidine-ligated, mononuclear non-heme Fe(2+) center. To assess the role in enzyme catalysis of the hydrophilic residues in the active site pocket, residues Glu98, Arg80, Tyr70, and Thr107 were subjected to mutational analysis. Steady state and pre-steady state kinetics indicated a role for Glu98 in promoting both substrate binding and O(2) reduction. Additionally, the Glu98 substitution eliminated the pH dependence of substrate binding (k(cat)(app)/K(M)(app)-pH profile) present in wild-type Dke1 (pK(a) = 6.3 +/- 0.4 and 8.4 +/- 0.4). MCD spectroscopy revealed that the Glu98 --> Gln mutation leads to the conversion of the six-coordinate (6C) resting Fe(2+) center present in the wild-type enzyme at pH 7.0 to a mixture of five-coordinate (5C) and 6C sites. The 6C geometry was restored with a pH shift to 9.5 which also resulted in ligand field (LF) energy splittings identical to that found for wild-type (WT) Dke1 at pH 9.5. In WT Dke1, these LF transitions are shifted up in energy by approximately 300 cm(-1) at pH 9.5 relative to pH 7.0. These data, combined with CD pH titrations which reveal a pK(a) of approximately 8.2 for resting WT Dke1 and the Glu98 --> Gln variant, indicate the deprotonation of a metal-ligated water. Together, the kinetic and spectroscopic data reveal a stabilizing effect of Glu98 on the 6C geometry of the metal center, priming it for substrate ligation. Arg80 and Tyr70 are shown to promote O(2) reduction, while Thr107 stabilizes the Fe(II) cofactor.

Virulence characterization of Campylobacter jejuni isolated from resident wild birds in Tokachi area, Japan.

PubMed

Shyaka, Anselme; Kusumoto, Akiko; Chaisowwong, Warangkhana; Okouchi, Yoshiki; Fukumoto, Shinya; Yoshimura, Aya; Kawamoto, Keiko

2015-08-01

The prevalence of Campylobacter jejuni in wild birds is a potential hazard for human and animal health. The aim of this study was to establish the prevalence of C. jejuni in wild birds in Tokachi area, Hokkaido, Japan and investigate their virulence in vitro. In total, 173 cloacal swabs from individual wild birds were collected for the detection of Campylobacter spp. Thirty four samples (19.7%) were positive for Campylobacter of which 94.1% (32/34 samples) were C. jejuni. Additionally, one C. coli and one C. fetus were isolated. Seven C. jejuni isolates (one from crows and the other from pigeons) had important virulence genes including all three CDT genes (cdtA, cdtB and cdtC) and flaA, flaB, ciaB and cadF, and the other isolates were lacking cdtA gene. Further studies on in vitro virulence-associated phenotypes, such as motility assay on soft agar and invasion assay in Caco-2 cells, were performed. The wild bird C. jejuni isolates adhered and invaded human cells. Although the numbers of viable intracellular bacteria of wild bird isolates were lower than a type strain NCTC11168, they persisted at 48-hr and underwent replication in host cells.

Virulence characterization of Campylobacter jejuni isolated from resident wild birds in Tokachi area, Japan

PubMed Central

SHYAKA, Anselme; KUSUMOTO, Akiko; CHAISOWWONG, Warangkhana; OKOUCHI, Yoshiki; FUKUMOTO, Shinya; YOSHIMURA, Aya; KAWAMOTO, Keiko

2015-01-01

The prevalence of Campylobacter jejuni in wild birds is a potential hazard for human and animal health. The aim of this study was to establish the prevalence of C. jejuni in wild birds in Tokachi area, Hokkaido, Japan and investigate their virulence in vitro. In total, 173 cloacal swabs from individual wild birds were collected for the detection of Campylobacter spp. Thirty four samples (19.7%) were positive for Campylobacter of which 94.1% (32/34 samples) were C. jejuni. Additionally, one C. coli and one C. fetus were isolated. Seven C. jejuni isolates (one from crows and the other from pigeons) had important virulence genes including all three CDT genes (cdtA, cdtB and cdtC) and flaA, flaB, ciaB and cadF, and the other isolates were lacking cdtA gene. Further studies on in vitro virulence-associated phenotypes, such as motility assay on soft agar and invasion assay in Caco-2 cells, were performed. The wild bird C. jejuni isolates adhered and invaded human cells. Although the numbers of viable intracellular bacteria of wild bird isolates were lower than a type strain NCTC11168, they persisted at 48-hr and underwent replication in host cells. PMID:25843040

Impact of human immunodeficiency virus type 1 resistance to protease inhibitors on evolution of resistance to the maturation inhibitor bevirimat (PA-457).

PubMed

Adamson, Catherine S; Waki, Kayoko; Ablan, Sherimay D; Salzwedel, Karl; Freed, Eric O

2009-05-01

The maturation inhibitor bevirimat [3-O-(3',3'dimethysuccinyl)betulinic acid; BVM; also known as PA-457 or DSB] potently inhibits human immunodeficiency virus type 1 (HIV-1) replication by blocking protease (PR)-mediated cleavage at the junction between capsid (CA) and spacer peptide 1 (SP1) in Gag. We previously isolated a panel of single-amino-acid substitutions that confer resistance to BVM in vitro (C. S. Adamson, S. D. Ablan, I. Boeras, R. Goila-Gaur, F. Soheilian, K. Nagashima, F. Li, K. Salzwedel, M. Sakalian, C. T. Wild, and E. O. Freed, J. Virol. 80:10957-10971, 2006). The BVM resistance mutations cluster at or near the CA-SP1 cleavage site. Because BVM likely will be used clinically in patients harboring viruses resistant to PR inhibitors (PIs), in this study we evaluated the interplay between a PI-resistant (PIR) PR and the BVM resistance mutations in Gag. As expected, the PIR mutations had no effect on inhibition by BVM; however, we observed general processing defects and a slight delay in viral replication in Jurkat T cells associated with the PIR mutations, even in the absence of compound. When combined, most BVM resistance and PIR mutations acted additively to impair viral replication, particularly in the presence of BVM. The BVM-resistant mutant SP1-A1V was an exception, as it supported robust replication in the context of either wild-type (WT) or PIR PR, even at high BVM concentrations. Significantly, the emergence of BVM resistance was delayed in the context of the PIR PR, and the SP1-A1V mutation was acquired most frequently with either WT or PIR PR. These results suggest that resistance to BVM is less likely to emerge in patients who have failed PIs than in patients who are PI naive. We predict that the SP1-A1V substitution is the most likely to emerge in vivo, as this mutant replicates robustly independently of PR mutations or BVM. These findings offer insights into the effect of PIR mutations on the evolution of BVM resistance in PI-experienced patients.

Probing the folded state and mechanical unfolding pathways of T4 lysozyme using all-atom and coarse-grained molecular simulation

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

Zheng, Wenjun, E-mail: wjzheng@buffalo.edu; Glenn, Paul

2015-01-21

The Bacteriophage T4 Lysozyme (T4L) is a prototype modular protein comprised of an N-terminal and a C-domain domain, which was extensively studied to understand the folding/unfolding mechanism of modular proteins. To offer detailed structural and dynamic insights to the folded-state stability and the mechanical unfolding behaviors of T4L, we have performed extensive equilibrium and steered molecular dynamics simulations of both the wild-type (WT) and a circular permutation (CP) variant of T4L using all-atom and coarse-grained force fields. Our all-atom and coarse-grained simulations of the folded state have consistently found greater stability of the C-domain than the N-domain in isolation, whichmore » is in agreement with past thermostatic studies of T4L. While the all-atom simulation cannot fully explain the mechanical unfolding behaviors of the WT and the CP variant observed in an optical tweezers study, the coarse-grained simulations based on the Go model or a modified elastic network model (mENM) are in qualitative agreement with the experimental finding of greater unfolding cooperativity in the WT than the CP variant. Interestingly, the two coarse-grained models predict different structural mechanisms for the observed change in cooperativity between the WT and the CP variant—while the Go model predicts minor modification of the unfolding pathways by circular permutation (i.e., preserving the general order that the N-domain unfolds before the C-domain), the mENM predicts a dramatic change in unfolding pathways (e.g., different order of N/C-domain unfolding in the WT and the CP variant). Based on our simulations, we have analyzed the limitations of and the key differences between these models and offered testable predictions for future experiments to resolve the structural mechanism for cooperative folding/unfolding of T4L.« less

AZT-induced mitochondrial toxicity: an epigenetic paradigm for dysregulation of gene expression through mitochondrial oxidative stress.

PubMed

Koczor, Christopher A; Jiao, Zhe; Fields, Earl; Russ, Rodney; Ludaway, Tomika; Lewis, William

2015-10-01

Mitochondrial dysfunction causes oxidative stress and cardiomyopathy. Oxidative stress also is a side effect of dideoxynucleoside antiretrovirals (NRTI) and is observed in NRTI-induced cardiomyopathy. We show here that treatment with the NRTI AZT {1-[(2R,4S,5S)-4-azido-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione} modulates cardiac gene expression epigenetically through production of mitochondrially derived reactive oxygen species. Transgenic mice with ubiquitous expression of mitochondrially targeted catalase (MCAT) and C57Bl/6 wild-type mice littermates (WT) were administered AZT (0.22 mg/day po, 35 days), and cardiac DNA and mRNA were isolated. In AZT-treated WT, 95 cardiac genes were differentially expressed compared with vehicle-treated WTs. When MCAT mice were treated with AZT, each of those 95 genes reverted toward the expression of vehicle-treated WTs. In AZT-treated WT hearts, Mthfr [5,10-methylenetetrahydrofolate reductase; a critical enzyme in synthesis of methionine cycle intermediates including S-adenosylmethionine (SAM)], was overexpressed. Steady-state abundance of SAM in cardiac extracts from AZT-treated MCAT mice increased 60% above that of vehicle-treated MCAT. No such change occurred in WT. AZT caused hypermethylation (47%) and hypomethylation (53%) of differentially methylated DNA regions in WT cardiac DNA. AZT-treated MCAT heart DNA exhibited greater hypermethylation (91%) and less hypomethylation (9%) compared with vehicle-treated MCAT controls. The gene encoding protein kinase C-α displayed multifocal epigenetic regulation caused by oxidative stress. Results show that mitochondrially derived oxidative stress in the heart hinders cardiac DNA methylation, alters steady-state abundance of SAM, alters cardiac gene expression, and promotes characteristic pathophysiological changes of cardiomyopathy. This mechanism for NRTI toxicity offers insight into long-term side effects from these commonly used antiviral agents. Copyright © 2015 the American Physiological Society.

Embryonic catalase protects against ethanol embryopathies in acatalasemic mice and transgenic human catalase-expressing mice in embryo culture

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

Miller-Pinsler, Lutfiya; Wells, Peter G., E-mail: pg.wells@utoronto.ca; Department of Pharmacology and Toxicology, Faculty of Medicine, University of Toronto, Toronto, Ontario

Reactive oxygen species (ROS) have been implicated in the mechanism of ethanol (EtOH) teratogenicity, but the protective role of the embryonic antioxidative enzyme catalase is unclear, as embryonic activity is only about 5% of maternal levels. We addressed this question in a whole embryo culture model. C57BL/6 mouse embryos expressing human catalase (hCat) or their wild-type (C57BL/6 WT) controls, and C3Ga.Cg-Cat{sup b}/J catalase-deficient, acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug = GD 1), exposed for 24 h to 2 or 4 mg/mL EtOH or vehicle, and evaluated formore » functional and morphological changes. hCat and C57BL/6 WT vehicle-exposed embryos developed normally, while EtOH was embryopathic in C57BL/6 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed, turning and head length, whereas hCat embryos were protected (p < 0.001). Maternal pretreatment of C57BL/6 WT dams with 50 kU/kg PEG-catalase (PEG-cat) 8 h prior to embryo culture, which increases embryonic catalase activity, blocked all EtOH embryopathies (p < 0.001). Vehicle-exposed aCat mouse embryos had lower yolk sac diameters compared to WT controls, suggesting that endogenous ROS are embryopathic. EtOH was more embryopathic in aCat embryos than WT controls, evidenced by reduced head length and somite development (p < 0.01), and trends for reduced anterior neuropore closure, turning and crown–rump length. Maternal pretreatment of aCat dams with PEG-Cat blocked all EtOH embryopathies (p < 0.05). These data suggest that embryonic catalase is a determinant of risk for EtOH embryopathies. - Highlights: • Ethanol (EtOH) exposure causes structural embryopathies in embryo culture. • Genetically enhanced catalase (hCat) protects against EtOH embryopathies. • Genetically deficient catalase (aCat) exacerbates EtOH embryopathies. • Embryonic catalase is developmentally important. • EtOH developmental toxicity involves reactive oxygen species formation.« less

p53 Regulates Bone Differentiation and Osteosarcoma Formation | Center for Cancer Research

Cancer.gov

Osteosarcoma is an uncommon cancer that usually begins in the large bones of the arm or leg, but is the second leading cause of cancer-related death in children and young adults. The tumor suppressor protein, p53, appears to be an important player in osteosarcomagenesis in part because these cancers are one of the most common to develop in patients with Li-Fraumeni syndrome, which is caused by an inherited mutation in p53. However, the precise role of p53 in osteosarcoma development has not been established. To begin investigating its importance to the formation of normal bone and osteosarcomas, Jing Huang, Ph.D., of CCR’s Laboratory of Cancer Biology and Genetics, and his colleagues, isolated bone marrow-derived mesenchymal stem cells (BMSCs) from p53 wild type (WT) and knock out (KO) mice using a recently validated approach. Because BMSCs are one of the cells-of-origin of osteosarcoma, they serve as a useful model system. BMSCs contain a subset of multipotent stem cells that can differentiate into several cell types, including osteoblasts, and are important mediators of bone homeostasis.

Genetic characterization of measles viruses isolated in Turkey during 2000 and 2001

PubMed Central

Korukluoglu, Gulay; Liffick, Stephanie; Guris, Dalya; Kobune, Fumio; Rota, Paul A; Bellini, William J; Ceylan, Ali; Ertem, Meliksah

2005-01-01

Background Molecular epidemiologic studies have made significant contributions to measles surveillance activities by helping to identify source and transmission pathways of the virus. This report describes the genetic characterization of wild-type measles viruses isolated in Turkey in 2000 and 2001. Results Wild-type measles viruses were isolated from 24 cases from five provinces in Turkey during 2001. The viruses were analyzed using the standard genotyping protocols. All isolates were classified as genotype D6, the same genotype that was identified in Turkey in previous outbreaks during 1998. Conclusion Turkey has begun implementation of a national program to eliminate measles by 2010. Therefore, this baseline genotype data will provide a means to monitor the success of the elimination program. PMID:16029506

A different role of angiotensin II type 1a receptor in the development and hypertrophy of plantaris muscle in mice.

PubMed

Zempo, Hirofumi; Suzuki, Jun-Ichi; Ogawa, Masahito; Watanabe, Ryo; Isobe, Mitsuaki

2016-02-01

The role of angiotensin II type 1 (AT1) receptors in muscle development and hypertrophy remains unclear. This study was designed to reveal the effects that a loss of AT1 receptors has on skeletal muscle development and hypertrophy in mice. Eight-week-old male AT1a receptor knockout (AT1a(-/-)) mice were used for this experiment. The plantaris muscle to body weight ratio, muscle fiber cross-sectional area, and number of muscle fibers of AT1a(-/-) mice was significantly greater than wild type (WT) mice in the non-intervention condition. Next, the functional overload (OL) model was used to induce plantaris muscle hypertrophy by surgically removing the two triceps muscles consisting of the calf, soleus, and gastrocnemius muscles in mice. After 14 days of OL intervention, the plantaris muscle weight, the amount of fiber, and the fiber area increased. However, the magnitude of the increment of plantaris weight was not different between the two strains. Agtr1a mRNA expression did not change after OL in WT muscle. Actually, the Agt mRNA expression level of WT-OL was lower than WT-Control (C) muscle. An atrophy-related gene, atrogin-1 mRNA expression levels of AT1a(-/-)-C, WT-OL, and AT1a(-/-)-OL muscle were lower than that of WT-C muscle. Our findings suggest that AT1 receptor contributes to plantaris muscle development via atrogin-1 in mice.

Circulation of Endemic Type 2 Vaccine-Derived Poliovirus in Egypt from 1983 to 1993

PubMed Central

Yang, Chen-Fu; Naguib, Tary; Yang, Su-Ju; Nasr, Eman; Jorba, Jaume; Ahmed, Nahed; Campagnoli, Ray; van der Avoort, Harrie; Shimizu, Hiroyuki; Yoneyama, Tetsuo; Miyamura, Tatsuo; Pallansch, Mark; Kew, Olen

2003-01-01

From 1988 to 1993, 30 cases of poliomyelitis associated with poliovirus type 2 were found in seven governorates of Egypt. Because many of the cases were geographically and temporally clustered and because the case isolates differed antigenically from the vaccine strain, it was initially assumed that the cases signaled the continued circulation of wild type 2 poliovirus. However, comparison of sequences encoding the major capsid protein, VP1 (903 nucleotides), revealed that the isolates were related (93 to 97% nucleotide sequence identity) to the Sabin type 2 oral poliovirus vaccine (OPV) strain and unrelated (<82% nucleotide sequence identity) to the wild type 2 polioviruses previously indigenous to Egypt (last known isolate: 1979) or to any contemporary wild type 2 polioviruses found elsewhere. The rate and pattern of VP1 divergence among the circulating vaccine-derived poliovirus (cVDPV) isolates suggested that all lineages were derived from a single OPV infection that occurred around 1983 and that progeny from the initiating infection circulated for approximately a decade within Egypt along several independent chains of transmission. Complete genomic sequences of an early (1988) and a late (1993) cVDPV isolate revealed that their 5′ untranslated region (5′ UTR) and noncapsid- 3′ UTR sequences were derived from other species C enteroviruses. Circulation of type 2 cVDPVs occurred at a time of low OPV coverage in the affected communities and ceased when OPV coverage rates increased. The potential for cVDPVs to circulate in populations with low immunity to poliovirus has important implications for current and future strategies to eradicate polio worldwide. PMID:12857906

Circulation of endemic type 2 vaccine-derived poliovirus in Egypt from 1983 to 1993.

PubMed

Yang, Chen-Fu; Naguib, Tary; Yang, Su-Ju; Nasr, Eman; Jorba, Jaume; Ahmed, Nahed; Campagnoli, Ray; van der Avoort, Harrie; Shimizu, Hiroyuki; Yoneyama, Tetsuo; Miyamura, Tatsuo; Pallansch, Mark; Kew, Olen

2003-08-01

From 1988 to 1993, 30 cases of poliomyelitis associated with poliovirus type 2 were found in seven governorates of Egypt. Because many of the cases were geographically and temporally clustered and because the case isolates differed antigenically from the vaccine strain, it was initially assumed that the cases signaled the continued circulation of wild type 2 poliovirus. However, comparison of sequences encoding the major capsid protein, VP1 (903 nucleotides), revealed that the isolates were related (93 to 97% nucleotide sequence identity) to the Sabin type 2 oral poliovirus vaccine (OPV) strain and unrelated (<82% nucleotide sequence identity) to the wild type 2 polioviruses previously indigenous to Egypt (last known isolate: 1979) or to any contemporary wild type 2 polioviruses found elsewhere. The rate and pattern of VP1 divergence among the circulating vaccine-derived poliovirus (cVDPV) isolates suggested that all lineages were derived from a single OPV infection that occurred around 1983 and that progeny from the initiating infection circulated for approximately a decade within Egypt along several independent chains of transmission. Complete genomic sequences of an early (1988) and a late (1993) cVDPV isolate revealed that their 5' untranslated region (5' UTR) and noncapsid- 3' UTR sequences were derived from other species C enteroviruses. Circulation of type 2 cVDPVs occurred at a time of low OPV coverage in the affected communities and ceased when OPV coverage rates increased. The potential for cVDPVs to circulate in populations with low immunity to poliovirus has important implications for current and future strategies to eradicate polio worldwide.

Identification of European-type hepatitis E virus subtype 3e isolates in Japanese wild boars: molecular tracing of HEV from swine to wild boars.

PubMed

Nakano, Tatsunori; Takahashi, Kazuaki; Arai, Masahiro; Okano, Hiroshi; Kato, Hideaki; Ayada, Minoru; Okamoto, Hiroaki; Mishiro, Shunji

2013-08-01

Nucleotide sequences of hepatitis E virus (HEV) isolates infecting wild boars in Mie prefecture, which is located in the central region of Japan and is far from the most prevalent regions of HEV infection in Japan, were determined and characterised. Among 144 serum samples of wild boars captured in Mie prefecture, 7 were positive for HEV-RNA. The nucleotide sequence of nearly the entire genome was determined for 4 of the 7 positive samples. Phylogenetic tree analyses indicated that 6 samples were subtype 3e and 1 was subtype 3a among the 7 isolates. We identified the indigenization of subtype 3e isolates in Japanese wild boars. Furthermore, 5 subtype 3e isolates were closely related and were located in the peripheral branch of subtype 3e isolates from European countries in the phylogenetic tree. The structure indicated that the ancestor of the 5 subtype 3e isolates originated in Europe. The phylogenetic structure and coalescent analyses suggested that the subtype 3e isolates entered Japan from Europe by importation of large-race pigs around 1966. The results also indicated that several lineages of subtype 3e expanded to a wide area of Japan around 1992 and 1 of the lineages was indigenized in wild boars in Mie prefecture between 1992 and 2009. The appearance of a wild boar cluster in the peripheral branch in the phylogenetic lineage may indicate the direction of gene flow of HEV subtype 3e from swine to wild boars. Clarification of the transmission direction or route should be helpful to prevent a future endemic or epidemic of HEV infection. Copyright © 2013 Elsevier B.V. All rights reserved.

Multidrug-resistant pathogenic Escherichia coli isolated from wild birds in a veterinary hospital.

PubMed

Borges, C A; Beraldo, L G; Maluta, R P; Cardozo, M V; Barboza, K B; Guastalli, E A L; Kariyawasam, S; DebRoy, C; Ávila, F A

2017-02-01

Wild birds are carriers of Escherichia coli. However, little is known about their role as reservoirs for extra-intestinal pathogenic E. coli (ExPEC). In this work we investigated E. coli strains carrying virulence genes related to human and animal ExPEC isolated from free-living wild birds treated in a veterinary hospital. Multidrug resistance was found in 47.4% of the strains, but none of them were extended-spectrum beta-lactamase producers. Not only the virulence genes, but also the serogroups (e.g. O1 and O2) detected in the isolates of E. coli have already been implicated in human and bird diseases. The sequence types detected were also found in wild, companion and food animals, environmental and human clinical isolates in different countries. Furthermore, from the 19 isolates, 17 (89.5%) showed a degree of pathogenicity on an in vivo infection model. The isolates showed high heterogeneity by pulsed-field gel electrophoresis indicating that E. coli from these birds are clonally diverse. Overall, the results showed that wild birds can be reservoirs and/or vectors of highly pathogenic and multidrug-resistant E. coli that have the potential to cause disease in humans and poultry.

Profiling of the Terpene Metabolome in Carrot Fruits of Wild ( Daucus carota L. ssp. carota) Accessions and Characterization of a Geraniol Synthase.

PubMed

Yahyaa, Mosaab; Ibdah, Muhammad; Marzouk, Sally; Ibdah, Mwafaq

2018-03-14

Fruits from wild carrot ( Daucus carota L. ssp. carota) have been used for medicinal purposes since ancient times. The oil of its seeds, with their abundant monoterpenes and sesquiterpenes, has drawn attention in recent years because of its potential pharmaceutical application. A combined chemical, biochemical, and molecular study was conducted to evaluate the differential accumulation of terpene volatiles in carrot fruits of wild accessions. This work reports a similarity-based cloning strategy identification and functional characterization of one carrot monoterpene terpene synthase, WtDcTPS1. Recombinant WtDcTPS1 protein produces mainly geraniol, the predominant monoterpene in carrot seeds of wild accession 23727. The results suggest a role for the WtDcTPS1 gene in the biosynthesis of carrot fruit aroma and flavor compounds.

Comparison of Toxic Metal Distribution Characteristics and Health Risk between Cultured and Wild Fish Captured from Honghu City, China.

PubMed

Zhang, Jingdong; Zhu, Liyun; Li, Fei; Liu, Chaoyang; Qiu, Zhenzhen; Xiao, Minsi; Cai, Ying

2018-02-14

Honghu Lake, which listed in the "Ramsar Convention", is the seventh largest freshwater lake in China and is regarded as one of the biggest freshwater product output areas in China. The toxic element distribution in cultured and wild fish and the corresponding health risks through fish consumption from Honghu area were investigated. The mean concentration in the muscle of cultured and wild fish ( Carassius auratus and Ctenopharyngodon idellus ) decreased in the order: Zn (18.94) > Cu (0.8489) > Cr (0.2840) > Pb (0.2052) and Zn (16.30) > Cr (1.947) > Cu (0.4166) > Pb (0.0525) > Cd (0.0060) (mean; mg/kg, wet weight). Scales (Multi factor pollution index (MPI) = 3.342) and the liver (MPI = 1.276) were regarded as the main accumulation tissues for cultured fish, and the bladder (MPI = 0.640) and intestine (MPI = 0.477) were regarded as the main accumulation tissues for wild fish. There were no obvious health risks associated with the consumption of cultured and wild fish based on the calculated results of the target hazard quotient (THQ), carcinogenic risk (CR), and estimated weekly intake (EWI). Pb and Cr were recognized as the major health risk contributors for inhabitants through wild and cultured fish consumption. Cultured fish had a greater health risk than wild fish based on the calculation results of THQ and CR. Muscle consumption resulted in more health risks than mixed edible tissues for cultured fish, but for wild fish, the conclusion was the opposite. Mixed fish (cultured:wild = 1:1) muscle consumption had relatively lower risks than the consumption of cultured or wild fish muscle separately. Consuming no more than 465 g/day (wet wt) of cultured fish muscle, 68 g/day (wet wt) of wild fish muscle, 452 g/day (wet wt) of mixed cultured fish edible tissues or 186 g/day (wet wt) of mixed wild fish edible tissues from the Honghu area can assure human health.

Molecular characterization of Belgian pseudorabies virus isolates from domestic swine and wild boar.

PubMed

Verpoest, Sara; Cay, Ann Brigitte; De Regge, Nick

2014-08-06

Aujeszky's disease is an economically important disease in domestic swine caused by suid herpesvirus 1, also called pseudorabies virus (PRV). In several European countries, including Belgium, the virus has successfully been eradicated from the domestic swine population. The presence of PRV in the wild boar population however poses a risk for possible reintroduction of the virus into the domestic pig population. It is therefore important to assess the genetic relatedness between circulating strains and possible epidemiological links. In this study, nine historical Belgian domestic swine isolates that circulated before 1990 and five recent wild boar isolates obtained since 2006 from Belgium and the Grand Duchy of Luxembourg were genetically characterized by restriction fragment length polymorphism (RFLP) analysis and phylogenetic analysis. While all wild boar isolates were characterized as type I RFLP genotypes, the RFLP patterns of the domestic swine isolates suggest that a shift from genotype I to genotype II might have occurred in the 1980s in the domestic population. By phylogenetic analysis, Belgian wild boar isolates belonging to both clade A and B were observed, while all domestic swine isolates clustered within clade A. The joint phylogenetic analysis of both wild boar and domestic swine strains showed that some isolates with identical sequences were present within both populations, raising the question whether these strains represent an increased risk for reintroduction of the virus into the domestic population. Copyright © 2014 Elsevier B.V. All rights reserved.

Type 5 and 8 capsular polysaccharides are expressed by Staphylococcus aureus isolates from rabbits, poultry, pigs, and horses.

PubMed Central

Poutrel, B; Sutra, L

1993-01-01

A total of 103 Staphylococcus aureus isolates from rabbits (n = 37), poultry (n = 33), pigs (n = 27), and horses (n = 6) and 14 Staphylococcus intermedius isolates from wild animals were serotyped for capsular polysaccharide types 5 and 8 by an enzyme-linked immunosorbent assay using polyclonal rabbit antibodies. About 98% of the S. aureus isolates were typeable. Type 5 was predominant in the poultry (75.8%) and pig (66.7%) isolates, whereas type 8 was more frequent among the isolates from rabbits (59.5%) and horses (83.3%). By contrast, none of the 14 S. intermedius isolates was typeable. PMID:8432841

Potent hydroxyl radical-scavenging activity of drought-induced type-2 metallothionein in wild watermelon.

PubMed

Akashi, Kinya; Nishimura, Noriyuki; Ishida, Yoshinori; Yokota, Akiho

2004-10-08

Wild watermelon (Citrullus lanatus sp.) has the ability to tolerate severe drought/high light stress conditions despite carrying out normal C3-type photosynthesis. Here, mRNA differential display was employed to isolate drought-responsive genes in the leaves of wild watermelon. One of the isolated genes, CLMT2, shared significant homology with type-2 metallothionein (MT) sequences from other plants. The second-order rate constant for the reaction between a recombinant CLMT2 protein and hydroxyl radicals was estimated to be 1.2 x 10(11) M(-1) s(-1), demonstrating that CLMT2 had an extraordinary high activity for detoxifying hydroxyl radicals. Moreover, hydroxyl radical-catalyzed degradation of watermelon genomic DNA was effectively suppressed by CLMT2 in vitro. This is the first demonstration of a plant MT with antioxidant properties. The results suggest that CLMT2 induction contributes to the survival of wild watermelon under severe drought/high light stress conditions. Copyright 2004 Elsevier Inc.

Wild-Type Measles Viruses with Non-Standard Genome Lengths

PubMed Central

Bankamp, Bettina; Liu, Chunyu; Rivailler, Pierre; Bera, Jayati; Shrivastava, Susmita; Kirkness, Ewen F.; Bellini, William J.; Rota, Paul A.

2014-01-01

The length of the single stranded, negative sense RNA genome of measles virus (MeV) is highly conserved at 15,894 nucleotides (nt). MeVs can be grouped into 24 genotypes based on the highly variable 450 nucleotides coding for the carboxyl-terminus of the nucleocapsid protein (N-450). Here, we report the genomic sequences of 2 wild-type viral isolates of genotype D4 with genome lengths of 15,900 nt. Both genomes had a 7 nt insertion in the 3′ untranslated region (UTR) of the matrix (M) gene and a 1 nt deletion in the 5′ UTR of the fusion (F) gene. The net gain of 6 nt complies with the rule-of-six required for replication competency of the genomes of morbilliviruses. The insertions and deletion (indels) were confirmed in a patient sample that was the source of one of the viral isolates. The positions of the indels were identical in both viral isolates, even though epidemiological data and the 3 nt differences in N-450 between the two genomes suggested that the viruses represented separate chains of transmission. Identical indels were found in the M-F intergenic regions of 14 additional genotype D4 viral isolates that were imported into the US during 2007–2010. Viral isolates with and without indels produced plaques of similar size and replicated efficiently in A549/hSLAM and Vero/hSLAM cells. This is the first report of wild-type MeVs with genome lengths other than 15,894 nt and demonstrates that the length of the M-F UTR of wild-type MeVs is flexible. PMID:24748123

FGFR3 Heterodimerization in Achondroplasia, the Most Common Form of Human Dwarfism*

PubMed Central

He, Lijuan; Shobnam, Nadia; Wimley, William C.; Hristova, Kalina

2011-01-01

The G380R mutation in the transmembrane domain of fibroblast growth factor receptor 3 (FGFR3) causes achondroplasia, the most common form of human dwarfism. Achondroplasia is a heterozygous disorder, and thus the affected individuals express both wild-type and mutant FGFR3. Yet heterodimerization in achondroplasia has not been characterized thus far. To investigate the formation of FGFR3 heterodimers in cellular membranes, we designed an FGFR3 construct that lacks the kinase domain, and we monitored the formation of inactive heterodimers between this construct and wild-type and mutant FGFR3. The formation of the inactive heterodimers depleted the pool of full-length receptors capable of forming active homodimers and ultimately reduced their phosphorylation. By analyzing the effect of the truncated FGFR3 on full-length receptor phosphorylation, we demonstrated that FGFR3 WT/G380R heterodimers form with lower probability than wild-type FGFR3 homodimers at low ligand concentration. These results further our knowledge of FGFR3-associated bone disorders. PMID:21324899

Decreased store operated Ca2+ entry in dendritic cells isolated from mice expressing PKB/SGK-resistant GSK3.

PubMed

Schmid, Evi; Yan, Jing; Nurbaeva, Meerim K; Russo, Antonella; Yang, Wenting; Faggio, Caterina; Shumilina, Ekaterina; Lang, Florian

2014-01-01

Dendritic cells (DCs), key players of immunity, are regulated by glycogen synthase kinase GSK3. GSK3 activity is suppressed by PKB/Akt and SGK isoforms, which are in turn stimulated by the PI3K pathway. Exposure to bacterial lipopolysaccharides increases cytosolic Ca(2+)-concentration ([Ca(2+)]i), an effect augmented in DCs isolated from mutant mice expressing PKB/SGK-resistant GSK3α,β (gsk3(KI) ). Factors affecting [Ca(2+)]i include Ca(2+)-release from intracellular stores (CRIS), store-operated Ca(2+)-entry (SOCE) through STIM1/STIM2-regulated Orai1, K(+)-dependent Na(+)/Ca(2+)-exchangers (NCKX), K(+)-independent Na(+)/Ca(2+)-exchangers (NCX) and calbindin-D28k. The present study explored whether PKB/SGK-dependent GSK3α, β-activity impacts on CRIS, SOCE, NCKX, NCX or calbindin. DCs were isolated from gsk3(KI) mice and respective wild-type mice (gsk3(WT) ), [Ca(2+)]i estimated from Fura2 fluorescence, Orai1, STIM1, STIM2 as well as calbindin-D28k protein abundance determined by Western blotting and mRNA levels quantified by real time PCR. As a result, thapsigargin-induced CRIS and SOCE were significantly blunted by GSK3-inhibitors SB216763 (1-10 µM, 30 min) or GSK-XIII (10 µM, 30 min) but were significantly lower in gsk3(WT) than in gsk3(KI) DCs. Orai1, STIM1 and STIM2 protein abundance was significantly lower and calbindin-D28k abundance significantly higher in gsk3(KI) than in gsk3(WT) DCs. Activity of NCKX and NCX was significantly higher in gsk3(KI) than in gsk3(WT) DCs and was significantly increased by SB216763 (1 µM, 30 min) or GSK-XIII (10 µM, 30 min). Treatment of gsk3(WT) DCs with SB216763 (1 µM, 4-24 h) or GSK-XIII (10 µM, 4-24 h) did not significantly modify the protein abundance of Orai1, STIM1 and STIM2. The present observations point to a dual role of GSK3 in the regulation of Ca(2+) in DCs. Acute inhibition of GSK3 blunted the increase of [Ca(2+)]i following CRIS and SOCE and stimulated NCKX/NCX activity. However, expression of PKB/SGK-resistant GSK3α, β downregulated the increase of [Ca(2+)]i following CRIS and SOCE, an effect at least partially due to downregulation of Orai1, STIM1 and STIM2 expression as well as upregulation of Na(+)/Ca(2+)-exchanger activity and calbindin D28k expression.

Impaired revascularization in a mouse model of type 2 diabetes is associated with dysregulation of a complex angiogenic-regulatory network.

PubMed

Schiekofer, Stephan; Galasso, Gennaro; Sato, Kaori; Kraus, Benjamin J; Walsh, Kenneth

2005-08-01

Diabetes is a risk factor for the development of cardiovascular diseases associated with impaired angiogenesis or increased endothelial cell apoptosis. Here it is shown that angiogenic repair of ischemic hindlimbs was impaired in Lepr(db/db) mice, a leptin receptor-deficient model of diabetes, compared with wild-type (WT) C57BL/6 mice, as evaluated by laser Doppler flow and capillary density analyses. To identify molecular targets associated with this disease process, hindlimb cDNA expression profiles were created from adductor muscle of Lepr(db/db) and WT mice before and after hindlimb ischemia using Affymetrix GeneChip Mouse Expression Set microarrays. The expression patterns of numerous angiogenesis-related proteins were altered in Lepr(db/db) versus WT mice after ischemic injury. These transcripts included neuropilin-1, vascular endothelial growth factor-A, placental growth factor, elastin, and matrix metalloproteinases implicated in blood vessel growth and maintenance of vessel wall integrity. These data illustrate that impaired ischemia-induced neovascularization in type 2 diabetes is associated with the dysregulation of a complex angiogenesis-regulatory network.

Constitutive glycogen synthase kinase-3α/β activity protects against chronic β-adrenergic remodelling of the heart

PubMed Central

Webb, Ian G.; Nishino, Yasuhiro; Clark, James E.; Murdoch, Colin; Walker, Simon J.; Makowski, Marcus R.; Botnar, Rene M.; Redwood, Simon R.; Shah, Ajay M.; Marber, Michael S.

2010-01-01

Aims Glycogen synthase kinase 3 (GSK-3) signalling is implicated in the growth of the heart during development and in response to stress. However, its precise role remains unclear. We set out to characterize developmental growth and response to chronic isoproterenol (ISO) stress in knockin (KI) mice lacking the critical N-terminal serines, 21 of GSK-3α and 9 of GSK-3β respectively, required for inactivation by upstream kinases. Methods and results Between 5 and 15 weeks, KI mice grew more rapidly, but normalized heart weight and contractile performance were similar to wild-type (WT) mice. Isolated hearts of both genotypes responded comparably to acute ISO infusion with increases in heart rate and contractility. In WT mice, chronic subcutaneous ISO infusion over 14 days resulted in cardiac hypertrophy, interstitial fibrosis, and impaired contractility, accompanied by foetal gene reactivation. These effects were all significantly attenuated in KI mice. Indeed, ISO-treated KI hearts demonstrated reversible physiological remodelling traits with increased stroke volume and a preserved contractile response to acute adrenergic stimulation. Furthermore, simultaneous pharmacological inhibition of GSK-3 in KI mice treated with chronic subcutaneous ISO recapitulated the adverse remodelling phenotype seen in WT hearts. Conclusion Expression of inactivation-resistant GSK-3α/β does not affect eutrophic myocardial growth but protects against pathological hypertrophy induced by chronic adrenergic stimulation, maintaining cardiac function and attenuating interstitial fibrosis. Accordingly, strategies to prevent phosphorylation of Ser-21/9, and consequent inactivation of GSK-3α/β, may enable a sustained cardiac response to chronic β-agonist stimulation while preventing pathological remodelling. PMID:20299330

Genetic Resistance to Scrapie Infection in Experimentally Challenged Goats

PubMed Central

Lacroux, Caroline; Perrin-Chauvineau, Cécile; Corbière, Fabien; Aron, Naima; Aguilar-Calvo, Patricia; Torres, Juan Maria; Costes, Pierrette; Brémaud, Isabelle; Lugan, Séverine; Schelcher, François; Barillet, Francis

2014-01-01

In goats, several field studies have identified coding mutations of the gene encoding the prion protein (I/M142, N/D146, S/D146, R/Q211, and Q/K222) that are associated with a lower risk of developing classical scrapie. However, the data related to the levels of resistance to transmissible spongiform encephalopathies (TSEs) of these different PRNP gene mutations are still considered insufficient for developing large-scale genetic selection against scrapie in this species. In this study, we inoculated wild-type (WT) PRNP (I142R154R211Q222) goats and homozygous and/or heterozygous I/M142, R/H154, R/Q211, and Q/K222 goats with a goat natural scrapie isolate by either the oral or the intracerebral (i.c.) route. Our results indicate that the I/M142 PRNP polymorphism does not provide substantial resistance to scrapie infection following intracerebral or oral inoculation. They also demonstrate that H154, Q211, and K222 PRNP allele carriers are all resistant to scrapie infection following oral exposure. However, in comparison to WT animals, the H154 and Q211 allele carriers displayed only moderate increases in the incubation period following i.c. challenge. After i.c. challenge, heterozygous K222 and a small proportion of homozygous K222 goats also developed the disease, but with incubation periods that were 4 to 5 times longer than those in WT animals. These results support the contention that the K222 goat prion protein variant provides a strong but not absolutely protective effect against classical scrapie. PMID:24284317

NKG2D is Required for Regulation of Lung Pathology and Dendritic Cell Function Following RSV Infection.

PubMed

Liu, Huan; Osterburg, Andrew R; Flury, Jennifer; Huang, Shuo; McCormack, Francis X; Cormier, Stephania A; Borchers, Michael T

2018-03-15

Respiratory syncytial virus (RSV) is a common cause of respiratory tract infection in vulnerable populations. Natural killer (NK) cells and dendritic cells (DC) are important for the effector functions of both cell types following infection. Wild type and NKG2D deficient mice were infected with RSV. Lung pathology, was assessed by histology. DC function and phenotype was evaluated by ELISA and flow cytometry. The expression of NKG2D ligands on lung and lymph node DCs was measured by immunostaining and flow cytometry. Adoptive transfer experiments were performed to assess the importance of NKG2D dependent DC function in RSV infection. NKG2D deficient mice exhibited greater lung pathology, marked by the accumulation of DCs following RSV infection.  DCs isolated from NKG2D deficient mice had impaired responses towards TLR ligands. DCs expressed NKG2D ligands on their surface, which was further increased in NKG2D deficient mice and during RSV infection. Adoptive transfer of DCs isolated from WT mice into the airways of NKG2D deficient mice ameliorated the enhanced inflammation in NKG2D deficient mice after RSV infection. NKG2D-dependent interactions with DCs control the phenotype and function of DCs and play a critical role in pulmonary host defenses against RSV infection.

HSP27 Alleviates Cardiac Aging in Mice via a Mechanism Involving Antioxidation and Mitophagy Activation.

PubMed

Lin, Shenglan; Wang, Yana; Zhang, Xiaojin; Kong, Qiuyue; Li, Chuanfu; Li, Yuehua; Ding, Zhengnian; Liu, Li

2016-01-01

Aging-induced cardiac dysfunction is a prominent feature of cardiac aging. Heat shock protein 27 (HSP27) protects cardiac function against ischemia or chemical challenge. We hypothesized that HSP27 attenuates cardiac aging. Transgenic (Tg) mice with cardiac-specific expression of the HSP27 gene and wild-type (WT) littermates were employed in the experiments. Echocardiography revealed a significant decline in the cardiac function of old WT mice compared with young WT mice. In striking contrast, the aging-induced impairment of cardiac function was attenuated in old Tg mice compared with old WT mice. Levels of cardiac aging markers were lower in old Tg mouse hearts than in old WT mouse hearts. Less interstitial fibrosis and lower contents of reactive oxygen species and ubiquitin-conjugated proteins were detected in old Tg hearts than in old WT hearts. Furthermore, old Tg hearts demonstrated lower accumulation of LC3-II and p62 than old WT hearts. Levels of Atg13, Vps34, and Rab7 were also higher in old Tg hearts than in old WT hearts. Additionally, old Tg hearts had higher levels of PINK1 and Parkin than old WT hearts, suggesting that mitophagy was activated in old Tg hearts. Taken together, HSP27 alleviated cardiac aging and this action involved antioxidation and mitophagy activation.

Live Cell Imaging Reveals Differential Modifications to Cytoplasmic Dynein Properties by Phospho- and Dephospho-mimic Mutations of the Intermediate Chain 2C S84

PubMed Central

Blasier, Kiev R.; Humsi, Michael K.; Ha, Junghoon; Ross, Mitchell W.; Smiley, W. Russell; Inamdar, Nirja A.; Mitchell, David J.; Lo, Kevin W.-H.; Pfister, K. Kevin

2014-01-01

Cytoplasmic dynein is a multi-subunit motor protein responsible for intracellular cargo transport toward microtubule minus ends. There are multiple isoforms of the dynein intermediate chain (DYNC1I, IC) which is encoded by two genes. One way to regulate cytoplasmic dynein is by IC phosphorylation. The IC-2C isoform is expressed in all cells and the functional significance of phosphorylation on IC-2C serine 84 was investigated using live cell imaging of fluorescent protein-tagged wild type IC-2C (WT) and phospho- and dephospho-mimic mutant isoforms in axonal transport model systems. Both mutations modulated dynein functional properties. The dephospho-mimic mutant IC-2C S84A had greater co-localization with mitochondria than IC-2C wild-type (WT) or the phospho-mimic mutant IC-2C S84D. The dephospho-mimic mutant IC-2C S84A was also more likely to be motile than the phospho-mimic mutant IC-2C S84D or IC-2C WT. In contrast, the phospho-mimic mutant IC-2C S84D mutant was more likely to move in the retrograde direction than was the IC-2C S84A mutant. The phospho-mimic IC-2C S84D was also as likely as IC-2C WT to co-localize with mitochondria. Both the S84D phospho- and S84A, dephospho-mimic mutants were found to be capable of microtubule minus end directed (retrograde) movement in axons. They were also observed to be passively transported in the anterograde direction. These data suggest that the IC-2C S84 has a role in modulating dynein properties. PMID:24798412

Cost-minimization analysis of panitumumab compared with cetuximab for first-line treatment of patients with wild-type RAS metastatic colorectal cancer.

PubMed

Graham, Christopher N; Hechmati, Guy; Fakih, Marwan G; Knox, Hediyyih N; Maglinte, Gregory A; Hjelmgren, Jonas; Barber, Beth; Schwartzberg, Lee S

2015-01-01

To compare the costs of first-line treatment with panitumumab + FOLFOX in comparison to cetuximab + FOLFIRI among patients with wild-type (WT) RAS metastatic colorectal cancer (mCRC) in the US. A cost-minimization model was developed assuming similar treatment efficacy between both regimens. The model estimated the costs associated with drug acquisition, treatment administration frequency (every 2 weeks for panitumumab, weekly for cetuximab), and incidence of infusion reactions. Average anti-EGFR doses were calculated from the ASPECCT clinical trial, and average doses of chemotherapy regimens were based on product labels. Using the medical component of the consumer price index, adverse event costs were inflated to 2014 US dollars, and all other costs were reported in 2014 US dollars. The time horizon for the model was based on average first-line progression-free survival of a WT RAS patient, estimated from parametric survival analyses of PRIME clinical trial data. Relative to cetuximab + FOLFIRI in the first-line treatment of WT RAS mCRC, the cost-minimization model demonstrated lower projected drug acquisition, administration, and adverse event costs for patients who received panitumumab + FOLFOX. The overall cost per patient for first-line treatment was $179,219 for panitumumab + FOLFOX vs $202,344 for cetuximab + FOLFIRI, resulting in a per-patient saving of $23,125 (11.4%) in favor of panitumumab + FOLFOX. From a value perspective, the cost-minimization model supports panitumumab + FOLFOX instead of cetuximab + FOLFIRI as the preferred first-line treatment of WT RAS mCRC patients requiring systemic therapy.

Temporal Resolution of Autophosphorylation for Normal and Oncogenic Forms of EGFR and Differential Effects of Gefitinib†

PubMed Central

Kim, Youngjoo; Li, Zhimin; Apetri, Mihaela; Luo, BeiBei; Settleman, Jeffrey E.; Anderson, Karen S.

2012-01-01

Epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases (RTK). EGFR overexpression or mutation in many different forms of cancers has highlighted its role as an important therapeutic target. Gefitinib, the first small molecule inhibitor of EGFR kinase function to be approved for the treatment of non-small cell lung cancer (NSCLC) by the FDA, demonstrates clinical activity primarily in patients with tumors that harbor somatic kinase domain mutations in EGFR. Here, we compare wild-type EGFR autophosphorylation kinetics to the L834R (also called L858R) EGFR form, one of the most common mutations in lung cancer patients. Using rapid chemical quench, time resolved electrospray mass spectrometry (ESI-MS) and western blot analyses, we examined the order of autophosphorylation in wild-type (WT) and L834R EGFR and the effect of gefitinib (Iressa ™) on the phosphorylation of individual tyrosines. These studies establish that there is a temporal order of autophosphorylation of key tyrosines involved in downstream signaling for WT EGFR and a loss of order for the oncogenic L834R mutant. These studies also reveal unique signature patterns of drug sensitivity for inhibition of tyrosine autophosphorylation by gefitinib; distinct for WT and oncogenic L834R mutant forms of EGFR. Fluorescence studies show that for WT EGFR, the binding affinity for gefitinib is weaker for the phosphorylated protein while for the oncogenic mutant, L834R EGFR, the binding affinity of gefitinib is substantially enhanced and likely contributes to the efficacy observed clinically. This mechanistic information is important in understanding the molecular details underpinning clinical observations as well as to aid in the design of more potent and selective EGFR inhibitors. PMID:22657099

Phenformin enhances the therapeutic effect of selumetinib in KRAS-mutant non-small cell lung cancer irrespective of LKB1 status.

PubMed

Zhang, Jun; Nannapaneni, Sreenivas; Wang, Dongsheng; Liu, Fakeng; Wang, Xu; Jin, Rui; Liu, Xiuju; Rahman, Mohammad Aminur; Peng, Xianghong; Qian, Guoqing; Chen, Zhuo G; Wong, Kwok-Kin; Khuri, Fadlo R; Zhou, Wei; Shin, Dong M

2017-08-29

MEK inhibition is potentially valuable in targeting KRAS-mutant non-small cell lung cancer (NSCLC). Here, we analyzed whether concomitant LKB1 mutation alters sensitivity to the MEK inhibitor selumetinib, and whether the metabolism drug phenformin can enhance the therapeutic effect of selumetinib in isogenic cell lines with different LKB1 status. Isogenic pairs of KRAS-mutant NSCLC cell lines A549, H460 and H157, each with wild-type and null LKB1, as well as genetically engineered mouse-derived cell lines 634 ( kras G12D/wt /p53 -/- /lkb1 wt/wt ) and t2 ( kras G12D/wt /p53 -/- / lkb1 -/- ) were used in vitro to analyze the activities of selumetinib, phenformin and their combination. Synergy was measured and potential mechanisms investigated. The in vitro findings were then confirmed in vivo using xenograft models. The re-expression of wild type LKB1 increased phospho-ERK level, suggesting that restored dependency on MEK->ERK->MAPK signaling might have contributed to the enhanced sensitivity to selumetinib. In contrast, the loss of LKB1 sensitized cells to phenformin. At certain combination ratios, phenformin and selumetinib showed synergistic activity regardless of LKB1 status. Their combination reduced phospho-ERK and S6 levels and induced potent apoptosis, but was likely through different mechanisms in cells with different LKB1 status. Finally, in xenograft models bearing isogenic A549 cells, we confirmed that loss of LKB1 confers resistance to selumetinib, and phenformin significantly enhances the therapeutic effect of selumetinib. Irrespective of LKB1 status, phenformin may enhance the anti-tumor effect of selumetinib in KRAS-mutant NSCLC. The dual targeting of MEK and cancer metabolism may provide a useful strategy to treat this subset of lung cancer.

Plant height revertants of Dominant Semidwarf mutant rice created by low-energy ion irradiation

NASA Astrophysics Data System (ADS)

Liu, Binmei; Wu, Yuejin; Xu, Xue; Song, M.; Zhao, M.; Fu, X. D.

2008-04-01

Dominant Semidwarf mutant rice (Sdd) was obtained from its wild type (WT) by irradiation with a low-energy ion beam. Six tall revertants of Sdd were induced by irradiation. The revertants restored the plant height to that of WT plants. Investigation of the agronomic character and genetic analysis indicate that the revertants are similar to WT plants with putative different inherited mutations. The revertants were checked for DNA differences using the simple sequence repeat technique. Among 408 such primers used, only 2 primers detected mutation sites in the revertants, which provided the molecular evidence for the revertants induced from Sdd. This study indicates that ion irradiation may be used as a mutagen to create revertants for plant architecture studies and could be a new application.

Modeling and Validation of the Ecological Behavior of Wild-Type Listeria monocytogenes and Stress-Resistant Variants.

PubMed

Metselaar, Karin I; Abee, Tjakko; Zwietering, Marcel H; den Besten, Heidy M W

2016-09-01

Listeria monocytogenes exhibits a heterogeneous response upon stress exposure which can be partially attributed to the presence of stable stress-resistant variants. This study aimed to evaluate the impact of the presence of stress-resistant variants of Listeria monocytogenes and their corresponding trade-offs on population composition under different environmental conditions. A set of stress robustness and growth parameters of the wild type (WT) and an rpsU deletion variant was obtained and used to model their growth behavior under combined mild stress conditions and to model their kinetics under single- and mixed-strain conditions in a simulated food chain. Growth predictions for the WT and the rpsU deletion variant matched the experimental data generally well, although some deviations from the predictions were observed. The data highlighted the influence of the environmental conditions on the ratio between the WT and variant. Prediction of performance in the simulated food chain proved to be challenging. The trend of faster growth and lower stress robustness for the WT than for the rpsU variant in the different steps of the chain was confirmed, but especially for the inactivation steps and the time needed to resume growth after an inactivation step, the experimental data deviated from the model predictions. This report provides insights into the conditions which can select for stress-resistant variants in industrial settings and discusses their potential persistence in food processing environments. Listeria monocytogenes exhibits a heterogeneous stress response which can partially be attributed to the presence of genetic variants. These stress-resistant variants survive better under severe conditions but have, on the other hand, a reduced growth rate. To date, the ecological behavior and potential impact of the presence of stress-resistant variants is not fully understood. In this study, we quantitatively assessed growth and inactivation behavior of wild-type L. monocytogenes and its stress-resistant variants. Predictions were validated under different conditions, as well as along a model food chain. This work illustrates the effects of environmental factors on population dynamics of L. monocytogenes and is a first step in evaluating the impact of population diversity on food safety. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Continuous in vivo infusion of interferon-gamma (IFN-γ) enhances engraftment of syngeneic wild-type cells in Fanca–/– and Fancg–/– mice

PubMed Central

Si, Yue; Ciccone, Samantha; Yang, Feng-Chun; Yuan, Jin; Zeng, Daisy; Chen, Shi; van de Vrugt, Henri J.; Critser, John; Arwert, Fre; Haneline, Laura S.; Clapp, D. Wade

2006-01-01

Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by bone marrow (BM) failure and cancer susceptibility. Identification of the cDNAs of FA complementation types allows the potential of using gene transfer technology to introduce functional cDNAs as transgenes into autologous stem cells and provide a cure for the BM failure in FA patients. However, strategies to enhance the mobilization, transduction, and engraftment of exogenous stem cells are required to optimize efficacy prior to widespread clinical use. Hypersensitivity of Fancc–/– cells to interferon-gamma (IFN-γ), a nongenotoxic immune-regulatory cytokine, enhances engraftment of syngeneic wild-type (WT) cells in Fancc–/– mice. However, whether this phenotype is of broad relevance in other FA complementation groups is unresolved. Here we show that primitive and mature myeloid progenitors in Fanca–/– and Fancg–/– mice are hypersensitive to IFN-γ and that in vivo infusion of IFN-γ at clinically relevant concentrations was sufficient to allow consistent long-term engraftment of isogenic WT repopulating stem cells. Given that FANCA, FANCC, and FANCG complementation groups account for more than 90% of all FA patients, these data provide evidence that IFN-γ conditioning may be a useful nongenotoxic strategy for myelopreparation in FA patients. PMID:16946306

Continuous in vivo infusion of interferon-gamma (IFN-gamma) enhances engraftment of syngeneic wild-type cells in Fanca-/- and Fancg-/- mice.

PubMed

Si, Yue; Ciccone, Samantha; Yang, Feng-Chun; Yuan, Jin; Zeng, Daisy; Chen, Shi; van de Vrugt, Henri J; Critser, John; Arwert, Fre; Haneline, Laura S; Clapp, D Wade

2006-12-15

Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by bone marrow (BM) failure and cancer susceptibility. Identification of the cDNAs of FA complementation types allows the potential of using gene transfer technology to introduce functional cDNAs as transgenes into autologous stem cells and provide a cure for the BM failure in FA patients. However, strategies to enhance the mobilization, transduction, and engraftment of exogenous stem cells are required to optimize efficacy prior to widespread clinical use. Hypersensitivity of Fancc-/- cells to interferon-gamma (IFN-gamma), a nongenotoxic immune-regulatory cytokine, enhances engraftment of syngeneic wild-type (WT) cells in Fancc-/- mice. However, whether this phenotype is of broad relevance in other FA complementation groups is unresolved. Here we show that primitive and mature myeloid progenitors in Fanca-/- and Fancg-/- mice are hypersensitive to IFN-gamma and that in vivo infusion of IFN-gamma at clinically relevant concentrations was sufficient to allow consistent long-term engraftment of isogenic WT repopulating stem cells. Given that FANCA, FANCC, and FANCG complementation groups account for more than 90% of all FA patients, these data provide evidence that IFN-gamma conditioning may be a useful nongenotoxic strategy for myelopreparation in FA patients.

Latency of Varicella Zoster Virus in Dorsal Root, Cranial, and Enteric Ganglia in Vaccinated Children

PubMed Central

Gershon, Anne A.; Chen, Jason; Davis, Larry; Krinsky, Clarissa; Cowles, Robert; Reichard, Ross; Gershon, Michael

2012-01-01

Despite vaccination, varicella-zoster virus (VZV) remains an important pathogen. We investigated VZV latency in autopsy specimens from vaccinees, in gastrointestinal tissue removed surgically, and in a guinea pig model. We propose that retrograde transport from infected skin and viremia deliver VZV to neurons in which it becomes latent. Wild type (WT) VZV was found to be latent in many ganglia of vaccinated children with no history of varicella, suggesting that subclinical infection with WT-VZV occurs with subsequent viremic dissemination. The 30% to 40% rate of WT-VZV zoster reported in vaccinees and occasional trigeminal zoster due to vaccine type VZV (vOka) are consistent with viremic delivery of VZV to multiple ganglia. Most human intestinal specimens contained latent VZV within neurons of the enteric nervous system (ENS). Induction of viremia in guinea pigs led to VZV latency throughout the ENS. The possibility VZV reactivation in the ENS is an unsuspected cause of gastrointestinal disease requires future investigation. PMID:23303966

Microscopic and Spectroscopic Analyses of Chlorhexidine Tolerance in Delftia acidovorans Biofilms

PubMed Central

Rema, Tara; Lawrence, John R.; Dynes, James J.; Hitchcock, Adam P.

2014-01-01

The physicochemical responses of Delftia acidovorans biofilms exposed to the commonly used antimicrobial chlorhexidine (CHX) were examined in this study. A CHX-sensitive mutant (MIC, 1.0 μg ml−1) was derived from a CHX-tolerant (MIC, 15.0 μg ml−1) D. acidovorans parent strain using transposon mutagenesis. D. acidovorans mutant (MT51) and wild-type (WT15) strain biofilms were cultivated in flow cells and then treated with CHX at sub-MIC and inhibitory concentrations and examined by confocal laser scanning microscopy (CLSM), scanning transmission X-ray microscopy (STXM), and infrared (IR) spectroscopy. Specific morphological, structural, and chemical compositional differences between the CHX-treated and -untreated biofilms of both strains were observed. Apart from architectural differences, CLSM revealed a negative effect of CHX on biofilm thickness in the CHX-sensitive MT51 biofilms relative to those of the WT15 strain. STXM analyses showed that the WT15 biofilms contained two morphochemical cell variants, whereas only one type was detected in the MT51 biofilms. The cells in the MT51 biofilms bioaccumulated CHX to a similar extent as one of the cell types found in the WT15 biofilms, whereas the other cell type in the WT15 biofilms did not bioaccumulate CHX. STXM and IR spectral analyses revealed that CHX-sensitive MT51 cells accumulated the highest levels of CHX. Pretreating biofilms with EDTA promoted the accumulation of CHX in all cells. Thus, it is suggested that a subpopulation of cells that do not accumulate CHX appear to be responsible for greater CHX resistance in D. acidovorans WT15 biofilm in conjunction with the possible involvement of bacterial membrane stability. PMID:25022584

Multiplexed Elimination of Wild-Type DNA and High-Resolution Melting Prior to Targeted Resequencing of Liquid Biopsies.

PubMed

Ladas, Ioannis; Fitarelli-Kiehl, Mariana; Song, Chen; Adalsteinsson, Viktor A; Parsons, Heather A; Lin, Nancy U; Wagle, Nikhil; Makrigiorgos, G Mike

2017-10-01

The use of clinical samples and circulating cell-free DNA (cfDNA) collected from liquid biopsies for diagnostic and prognostic applications in cancer is burgeoning, and improved methods that reduce the influence of excess wild-type (WT) portion of the sample are desirable. Here we present enrichment of mutation-containing sequences using enzymatic degradation of WT DNA. Mutation enrichment is combined with high-resolution melting (HRM) performed in multiplexed closed-tube reactions as a rapid, cost-effective screening tool before targeted resequencing. We developed a homogeneous, closed-tube approach to use a double-stranded DNA-specific nuclease for degradation of WT DNA at multiple targets simultaneously. The No Denaturation Nuclease-assisted Minor Allele Enrichment with Probe Overlap (ND-NaME-PrO) uses WT oligonucleotides overlapping both strands on putative DNA targets. Under conditions of partial denaturation (DNA breathing), the oligonucleotide probes enhance double-stranded DNA-specific nuclease digestion at the selected targets, with high preference toward WT over mutant DNA. To validate ND-NaME-PrO, we used multiplexed HRM, digital PCR, and MiSeq targeted resequencing of mutated genomic DNA and cfDNA. Serial dilution of KRAS mutation-containing DNA shows mutation enrichment by 10- to 120-fold and detection of allelic fractions down to 0.01%. Multiplexed ND-NaME-PrO combined with multiplexed PCR-HRM showed mutation scanning of 10-20 DNA amplicons simultaneously. ND-NaME-PrO applied on cfDNA from clinical samples enables mutation enrichment and HRM scanning over 10 DNA targets. cfDNA mutations were enriched up to approximately 100-fold (average approximately 25-fold) and identified via targeted resequencing. Closed-tube homogeneous ND-NaME-PrO combined with multiplexed HRM is a convenient approach to efficiently enrich for mutations on multiple DNA targets and to enable prescreening before targeted resequencing. © 2017 American Association for Clinical Chemistry.

Mouse model of human RPE65 P25L hypomorph resembles wild type under normal light rearing but is fully resistant to acute light damage.

PubMed

Li, Yan; Yu, Shirley; Duncan, Todd; Li, Yichao; Liu, Pinghu; Gene, Erelda; Cortes-Pena, Yoel; Qian, Haohua; Dong, Lijin; Redmond, T Michael

2015-08-01

Human RPE65 mutations cause a spectrum of blinding retinal dystrophies from severe early-onset disease to milder manifestations. The RPE65 P25L missense mutation, though having <10% of wild-type (WT) activity, causes relatively mild retinal degeneration. To better understand these mild forms of RPE65-related retinal degeneration, and their effect on cone photoreceptor survival, we generated an Rpe65/P25L knock-in (KI/KI) mouse model. We found that, when subject to the low-light regime (∼100 lux) of regular mouse housing, homozygous Rpe65/P25L KI/KI mice are morphologically and functionally very similar to WT siblings. While mutant protein expression is decreased by over 80%, KI/KI mice retinae retain comparable 11-cis-retinal levels with WT. Consistently, the scotopic and photopic electroretinographic (ERG) responses to single-flash stimuli also show no difference between KI/KI and WT mice. However, the recovery of a-wave response following moderate visual pigment bleach is delayed in KI/KI mice. Importantly, KI/KI mice show significantly increased resistance to high-intensity (20 000 lux for 30 min) light-induced retinal damage (LIRD) as compared with WT, indicating impaired rhodopsin regeneration in KI/KI. Taken together, the Rpe65/P25L mutant produces sufficient chromophore under normal conditions to keep opsins replete and thus manifests a minimal phenotype. Only when exposed to intensive light is this hypomorphic mutation manifested physiologically, as its reduced expression and catalytic activity protects against the successive cycles of opsin regeneration underlying LIRD. These data also help define minimal requirements of chromophore for photoreceptor survival in vivo and may be useful in assessing a beneficial therapeutic dose for RPE65 gene therapy in humans. Published by Oxford University Press 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Histone acetylation rescues contextual fear conditioning in nNOS KO mice and accelerates extinction of cued fear conditioning in wild type mice.

PubMed

Itzhak, Yossef; Anderson, Karen L; Kelley, Jonathan B; Petkov, Martin

2012-05-01

Epigenetic regulation of chromatin structure is an essential molecular mechanism that contributes to the formation of synaptic plasticity and long-term memory (LTM). An important regulatory process of chromatin structure is acetylation and deacetylation of histone proteins. Inhibition of histone deacetylase (HDAC) increases acetylation of histone proteins and facilitate learning and memory. Nitric oxide (NO) signaling pathway has a role in synaptic plasticity, LTM and regulation of histone acetylation. We have previously shown that NO signaling pathway is required for contextual fear conditioning. The present study investigated the effects of systemic administration of the HDAC inhibitor sodium butyrate (NaB) on fear conditioning in neuronal nitric oxide synthase (nNOS) knockout (KO) and wild type (WT) mice. The effect of single administration of NaB on total H3 and H4 histone acetylation in hippocampus and amygdala was also investigated. A single administration of NaB prior to fear conditioning (a) rescued contextual fear conditioning of nNOS KO mice and (b) had long-term (weeks) facilitatory effect on the extinction of cued fear memory of WT mice. The facilitatory effect of NaB on extinction of cued fear memory of WT mice was confirmed in a study whereupon NaB was administered during extinction. Results suggest that (a) the rescue of contextual fear conditioning in nNOS KO mice is associated with NaB-induced increase in H3 histone acetylation and (b) the accelerated extinction of cued fear memory in WT mice is associated with NaB-induced increase in H4 histone acetylation. Hence, a single administration of HDAC inhibitor may rescue NO-dependent cognitive deficits and afford a long-term accelerating effect on extinction of fear memory of WT mice. Copyright © 2012 Elsevier Inc. All rights reserved.

A heterozygous IDH1R132H/WT mutation induces genome-wide alterations in DNA methylation.

PubMed

Duncan, Christopher G; Barwick, Benjamin G; Jin, Genglin; Rago, Carlo; Kapoor-Vazirani, Priya; Powell, Doris R; Chi, Jen-Tsan; Bigner, Darell D; Vertino, Paula M; Yan, Hai

2012-12-01

Monoallelic point mutations of the NADP(+)-dependent isocitrate dehydrogenases IDH1 and IDH2 occur frequently in gliomas, acute myeloid leukemias, and chondromas, and display robust association with specific DNA hypermethylation signatures. Here we show that heterozygous expression of the IDH1(R132H) allele is sufficient to induce the genome-wide alterations in DNA methylation characteristic of these tumors. Using a gene-targeting approach, we knocked-in a single copy of the most frequently observed IDH1 mutation, R132H, into a human cancer cell line and profiled changes in DNA methylation at over 27,000 CpG dinucleotides relative to wild-type parental cells. We find that IDH1(R132H/WT) mutation induces widespread alterations in DNA methylation, including hypermethylation of 2010 and hypomethylation of 842 CpG loci. We demonstrate that many of these alterations are consistent with those observed in IDH1-mutant and G-CIMP+ primary gliomas and can segregate IDH wild-type and mutated tumors as well as those exhibiting the G-CIMP phenotype in unsupervised analysis of two primary glioma cohorts. Further, we show that the direction of IDH1(R132H/WT)-mediated DNA methylation change is largely dependent upon preexisting DNA methylation levels, resulting in depletion of moderately methylated loci. Additionally, whereas the levels of multiple histone H3 and H4 methylation modifications were globally increased, consistent with broad inhibition of histone demethylation, hypermethylation at H3K9 in particular accompanied locus-specific DNA hypermethylation at several genes down-regulated in IDH1(R132H/WT) knock-in cells. These data provide insight on epigenetic alterations induced by IDH1 mutations and support a causal role for IDH1(R132H/WT) mutants in driving epigenetic instability in human cancer cells.

Real-time reverse transcription-polymerase chain reaction assays for identification of wild poliovirus 1 & 3.

PubMed

Sharma, Deepa K; Nalavade, Uma P; Deshpande, Jagadish M

2015-10-01

The poliovirus serotype identification and intratypic differentiation by real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay is suitable for serotype mixtures but not for intratypic mixtures of wild and vaccine poliovirus strains. This study was undertaken to develop wild poliovirus 1 and 3 (WPV1 and WPV3) specific rRT-PCR assays for use. Specific primers and probes for rRT-PCR were designed based on VP1 sequences of WPV1 and WPV3 isolated in India since 2000. The specificity of the rRT-PCR assays was evaluated using WPV1 and WPV3 of different genetic lineages, non-polio enteroviruses (NPEVs) and mixtures of wild/wild and wild/Sabin vaccine strains. The sensitivity of the assays was determined by testing serial 10-fold dilutions of wild poliovirus 1 and 3 stock suspensions of known titre. No cross-reactivity with Sabin strains, intertypic wild poliovirus isolates or 27 types of NPEVs across all the four Enterovirus species was found for both the wild poliovirus 1 and 3 rRT-PCR assays. All WPV1 and WPV3 strains isolated since 2000 were successfully amplified. The rRT-PCR assays detected 10 4.40 CCID 50 /ml of WPV1 and 10 4.00 CCID 50 /ml of WPV3, respectively either as single isolate or mixture with Sabin vaccine strains or intertypic wild poliovirus. rRT-PCR assays for WPV1 and WPV3 have been validated to detect all the genetic variations of the WPV1 and WPV3 isolated in India for the last decade. When used in combination with the current rRT-PCR assay testing was complete for confirmation of the presence of wild poliovirus in intratypic mixtures.

The Macrophage Galactose-Type Lectin-1 (MGL1) Recognizes Taenia crassiceps Antigens, Triggers Intracellular Signaling, and Is Critical for Resistance to This Infection

PubMed Central

Montero-Barrera, Daniel; Valderrama-Carvajal, Héctor; Terrazas, César A.; Rojas-Hernández, Saúl; Ledesma-Soto, Yadira; Vera-Arias, Laura; Carrasco-Yépez, Maricela; Gómez-García, Lorena; Martínez-Saucedo, Diana; Becerra-Díaz, Mireya; Terrazas, Luis I.

2015-01-01

C-type lectins are multifunctional sugar-binding molecules expressed on dendritic cells (DCs) and macrophages that internalize antigens for processing and presentation. Macrophage galactose-type lectin 1 (MGL1) recognizes glycoconjugates expressing Lewis X structures which contain galactose residues, and it is selectively expressed on immature DCs and macrophages. Helminth parasites contain large amounts of glycosylated components, which play a role in the immune regulation induced by such infections. Macrophages from MGL1−/− mice showed less binding ability toward parasite antigens than their wild-type (WT) counterparts. Exposure of WT macrophages to T. crassiceps antigens triggered tyrosine phosphorylation signaling activity, which was diminished in MGL1−/− macrophages. Following T. crassiceps infection, MGL1−/− mice failed to produce significant levels of inflammatory cytokines early in the infection compared to WT mice. In contrast, MGL1−/− mice developed a Th2-dominant immune response that was associated with significantly higher parasite loads, whereas WT mice were resistant. Flow cytometry and RT-PCR analyses showed overexpression of the mannose receptors, IL-4Rα, PDL2, arginase-1, Ym1, and RELM-α on MGL1−/− macrophages. These studies indicate that MGL1 is involved in T. crassiceps recognition and subsequent innate immune activation and resistance. PMID:25664320

Knockout of toll-like receptor-2 attenuates both the proinflammatory state of diabetes and incipient diabetic nephropathy.

PubMed

Devaraj, Sridevi; Tobias, Peter; Kasinath, Balakuntalam S; Ramsamooj, Rajendra; Afify, Alaa; Jialal, Ishwarlal

2011-08-01

Type 1 diabetes (T1DM) is a proinflammatory state and confers an increased risk for vascular complications. Toll-like receptors (TLR) could participate in diabetic vasculopathies. Whether TLR activation contributes to the proinflammatory state of T1DM and the pathogenesis of diabetic nephropathy remains unknown. We induced T1DM in TLR2 knockout mice (TLR2-/-) and wild-type littermates (C57BL/6J-WT) using streptozotocin (STZ). Fasting blood, peritoneal macrophages, and kidneys were obtained for flow cytometry, Western blot, microscopy, and cytokine assays at 6 and 14 weeks after induction of diabetes. Macrophage TLR2 expression and MyD88-dependent signaling were increased in diabetic mice (WT+STZ) compared with nondiabetic WT mice. These biomarkers were attenuated in diabetic TLR2-/- macrophages. WT+STZ mice showed increased kidney:body weight ratio due to cell hypertrophy, increased albuminuria, decreased kidney nephrin, podocin, and podocyte number and increased transforming growth factor-β and laminin compared with WT mice. Nephrin, podocin, and podocyte number and effacement were restored, and transforming growth factor-β and laminin levels were decreased in TLR2-/-+ STZ mice kidneys versus WT+STZ. Peritoneal and kidney macrophages were predominantly M1 phenotype in WT+STZ mice; this was attenuated in TLR2-/-+STZ mice. These data support a role for TLR2 in promoting inflammation and early changes of incipient diabetic nephropathy, in addition to albuminuria and podocyte loss.

Identification and Characterization of Small-Molecule Inhibitors of the R132H/R132H Mutant Isocitrate Dehydrogenase 1 Homodimer and R132H/Wild-Type Heterodimer.

PubMed

Brooks, Eric; Wu, Xiang; Hanel, Art; Nguyen, Shaun; Wang, Jing; Zhang, Jeffrey H; Harrison, Amanda; Zhang, Wentao

2014-09-01

Recurrent genetic mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) have been identified in multiple tumor types. The most frequent mutation, IDH1 R132H, is a gain-of-function mutation resulting in an enzyme-catalyzing conversion of α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG). A high-throughput assay quantifying consumption of NADPH by IDH1 R132H has been optimized and implemented to screen 3 million compounds in 1536-well formats. The primary high-throughput screening hits were further characterized by RapidFire-mass spectrometry measuring 2-HG directly. Multiple distinct chemotypes were identified with nanomolar potencies (6-300 nM). All inhibitors were found to be inactive against the wild-type IDH1 homodimers. An IDH1 heterodimer between wild-type and R132H mutant is capable of catalyzing conversion of α-KG to 2-HG and isocitrate to α-KG. Interestingly, one of the inhibitors, EXEL-9324, was found to inhibit both conversions by the IDH1 heterodimer. This indicates the R132H/WT heterodimer may adopt conformations distinct from that of the R132H/R132H homodimer. Further enzymatic studies support this conclusion as the heterodimer exhibited a significantly lower apparent Michaelis-Menten constant for α-KG (K(m)=110 µM) compared with the R132H homodimer (K(m)= 1200 µM). The enhanced apparent affinity for α-KG suggests R132H/WT heterodimeric IDH1 can produce 2-HG more efficiently at normal intracellular levels of α-KG (approximately 100 µM). © 2014 Society for Laboratory Automation and Screening.

Contribution of Bordetella bronchiseptica Filamentous Hemagglutinin and Pertactin to Respiratory Disease in Swine▿ †

PubMed Central

Nicholson, Tracy L.; Brockmeier, Susan L.; Loving, Crystal L.

2009-01-01

Bordetella bronchiseptica is pervasive in swine populations and plays multiple roles in respiratory disease. Most studies addressing virulence factors of B. bronchiseptica are based on isolates derived from hosts other than pigs. Two well-studied virulence factors implicated in the adhesion process are filamentous hemagglutinin (FHA) and pertactin (PRN). We hypothesized that both FHA and PRN would serve critical roles in the adhesion process and be necessary for colonization of the swine respiratory tract. To investigate the role of FHA and PRN in Bordetella pathogenesis in swine, we constructed mutants containing an in-frame deletion of the FHA or the PRN structural gene in a virulent B. bronchiseptica swine isolate. Both mutants were compared to the wild-type swine isolate for their ability to colonize and cause disease in swine. Colonization of the FHA mutant was lower than that of the wild type at all respiratory tract sites and time points examined and caused limited to no disease. In contrast, the PRN mutant caused similar disease severity relative to the wild type; however, colonization of the PRN mutant was reduced relative to the wild type during early and late infection and induced higher anti-Bordetella antibody titers. Together, our results indicate that despite inducing different pathologies and antibody responses, both FHA and PRN are necessary for optimal colonization of the swine respiratory tract. PMID:19237531

The wild type as concept and in experimental practice: A history of its role in classical genetics and evolutionary theory.

PubMed

Holmes, Tarquin

2017-06-01

Wild types in genetics are specialised strains of laboratory experimental organism which principally serve as standards against which variation is measured. As selectively inbred lineages highly isolated from ancestral wild populations, there appears to be little wild or typical about them. I will nonetheless argue that they have historically been successfully used as stand-ins for nature, allowing knowledge produced in the laboratory to be extrapolated to the natural world. In this paper, I will explore the 19th century origins of the wild type concept, the theoretical and experimental innovations which allowed concepts and organisms to move from wild nature to laboratory domestication c. 1900 (resulting in the production of standardised lab strains), and the conflict among early geneticists between interactionist and atomist accounts of wild type, which would eventually lead to the conceptual disintegration of wild types and the triumph of genocentrism and population genetics. I conclude by discussing how the strategy of using wild type strains to represent nature in the lab has nonetheless survived the downfall of the wild type concept and continues to provide, significant limitations acknowledged, an epistemically productive means of investigating heredity and evolutionary variation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Piper betle induces phase I & II genes through Nrf2/ARE signaling pathway in mouse embryonic fibroblasts derived from wild type and Nrf2 knockout cells.

PubMed

Wan Hasan, Wan Nuraini; Kwak, Mi-Kyoung; Makpol, Suzana; Wan Ngah, Wan Zurinah; Mohd Yusof, Yasmin Anum

2014-02-23

Nuclear factor-erythroid 2 p45 related factor 2 (Nrf2) is a primary transcription factor, protecting cells from oxidative stress by regulating a number of antioxidants and phase II detoxifying enzymes. Dietary components such as sulforaphane in broccoli and quercetin in onions have been shown to be inducers of Nrf2. Piper betle (PB) grows well in tropical climate and the leaves are used in a number of traditional remedies for the treatment of stomach ailments and infections among Asians. The aim of this study was to elucidate the effect of Piper betle (PB) leaves extract in Nrf2 signaling pathway by using 2 types of cells; mouse embryonic fibroblasts (MEFs) derived from wild-type (WT) and Nrf2 knockout (N0) mice. WT and N0 cells were treated with 5 and 10 μg/ml of PB for 10 and 12-h for the determination of nuclear translocation of Nrf2 protein. Luciferase reporter gene activity was performed to evaluate the antioxidant response element (ARE)-induction by PB. Real-time PCR and Western blot were conducted on both WT and N0 cells after PB treatment for the determination of antioxidant enzymes [superoxide dismutase (SOD1) and heme-oxygenase (HO-1)], phase I oxidoreductase enzymes [ quinone oxidoreductase (NQO1)] and phase II detoxifying enzyme [glutathione S-transferase (GST)]. Nuclear translocation of Nrf2 by PB in WT cells was better after 10 h incubation compared to 12 h. Real time PCR and Western blot analysis showed increased expressions of Nrf2, NQO1 and GSTA1 genes with corresponding increases in glutathione, NQO1 and HO-1 proteins in WT cells. Reporter gene ARE was stimulated by PB as shown by ARE/luciferase assay. Interestingly, PB induced SOD1 gene and protein expressions in N0 cells but not in WT cells. The results of this study confirmed that PB activated Nrf2-ARE signaling pathway which subsequently induced some phase I oxidoreductase, phase II detoxifying and antioxidant genes expression via ARE reporter gene involved in the Nrf2 pathway with the exception of SOD1 which may not be dependent on this pathway.

Piper betle induces phase I & II genes through Nrf2/ARE signaling pathway in mouse embryonic fibroblasts derived from wild type and Nrf2 knockout cells

PubMed Central

2014-01-01

Background Nuclear factor-erythroid 2 p45 related factor 2 (Nrf2) is a primary transcription factor, protecting cells from oxidative stress by regulating a number of antioxidants and phase II detoxifying enzymes. Dietary components such as sulforaphane in broccoli and quercetin in onions have been shown to be inducers of Nrf2. Piper betle (PB) grows well in tropical climate and the leaves are used in a number of traditional remedies for the treatment of stomach ailments and infections among Asians. The aim of this study was to elucidate the effect of Piper betle (PB) leaves extract in Nrf2 signaling pathway by using 2 types of cells; mouse embryonic fibroblasts (MEFs) derived from wild-type (WT) and Nrf2 knockout (N0) mice. Methods WT and N0 cells were treated with 5 and 10 μg/ml of PB for 10 and 12-h for the determination of nuclear translocation of Nrf2 protein. Luciferase reporter gene activity was performed to evaluate the antioxidant response element (ARE)-induction by PB. Real-time PCR and Western blot were conducted on both WT and N0 cells after PB treatment for the determination of antioxidant enzymes [superoxide dismutase (SOD1) and heme-oxygenase (HO-1)], phase I oxidoreductase enzymes [NAD(P)H: quinone oxidoreductase (NQO1)] and phase II detoxifying enzyme [glutathione S-transferase (GST)]. Results Nuclear translocation of Nrf2 by PB in WT cells was better after 10 h incubation compared to 12 h. Real time PCR and Western blot analysis showed increased expressions of Nrf2, NQO1 and GSTA1 genes with corresponding increases in glutathione, NQO1 and HO-1 proteins in WT cells. Reporter gene ARE was stimulated by PB as shown by ARE/luciferase assay. Interestingly, PB induced SOD1 gene and protein expressions in N0 cells but not in WT cells. Conclusion The results of this study confirmed that PB activated Nrf2-ARE signaling pathway which subsequently induced some phase I oxidoreductase, phase II detoxifying and antioxidant genes expression via ARE reporter gene involved in the Nrf2 pathway with the exception of SOD1 which may not be dependent on this pathway. PMID:24559113

Prevalence of infectious salmon anaemia virus (ISAV) in wild salmonids in western Norway.

PubMed

Plarre, H; Devold, M; Snow, M; Nylund, A

2005-08-09

Studies of infectious salmon anaemia virus (ISAV), an important pathogen of farmed salmon in Norway, Scotland, the Faeroe Islands, Ireland, Canada, the USA and Chile, suggest that natural reservoirs for this virus can be found on both sides of the North Atlantic. Based on existing information about ISAV it is believed to be maintained in wild populations of trout and salmon in Europe. It has further been suggested that ISAV is transmitted between wild hosts, mainly during their freshwater spawning phase in rivers, and that wild salmonids, mainly trout, are possible carriers of benign wild-type variants of ISAV. Change in virulence is probably a result of deletions of amino acid segments from the highly polymorphic region (HPR) of benign wild-type isolates after transmission to farmed salmon. Hence, it has been suggested that the frequency of new outbreaks of ISA in farmed salmon could partly reflect natural variation in the prevalence of ISAV in wild populations of salmonids. The aims of the present study were to screen for ISAV in wild salmonids during spawning in rivers and to determine the pathogenicity of resultant isolates from wild fish. Tissues from wild salmonids were screened by RT-PCR and real-time PCR. The prevalence of ISAV in wild trout Salmo trutta varied from 62 to 100% between tested rivers in 2001. The prevalence dropped in 2002, ranging from 13 to 36% in the same rivers and to only 6% in 2003. All ISAV were nonpathogenic when injected into disease-free Atlantic salmon, but were capable of propagation, as indicated by subsequent viral recovery. However, non-pathogenic ISAV has also been found in farmed salmon, where a prevalence as high as 60% has been registered, but with no mortalities occurring. Based on the results of the present and other studies, it must be concluded that vital information about the importance of wild and man-made reservoirs for the emergence of ISA in salmon farming is still lacking. This information can only be gained by further screening of possible reservoirs, combined with the development of a molecular tool for typing virulence and the geographical origin of the virus isolates.

[Expression of matrix metalloproteinase-19 in the human cornea. Wound healing in the MMP-19 knock-out mouse model].

PubMed

Treumer, F; Flöhr, C; Klettner, A; Nölle, B; Roider, J

2010-07-01

At present there are no data in the literature on the expression of matrix metalloprotein-19 in the human cornea. The aim of this study was to analyze the expression of matrix metalloproteinase-19 in the human cornea and to investigate its potential role in corneal wound healing using a MMP-19 knock-out mouse model. A method with Western blotting and immunohistological staining for MMP-19 was performed using paraffin embedded human corneas. Excimer laser keratectomy was performed in wild type (wt) and MMP-19 knock-out (ko) mice and the rate of re-epithelialization was analyzed after 8 h and 18 h. MMP-19 was strongly expressed in the human corneal epithelium mainly in the basal cell layer. MMP-19 was not expressed in the corneal stroma. In the mouse model the size of the corneal lesion after 8 h was 83% (wt) and 89.9% (ko) of the initial area (p=0.09). After 18 h the lesion was 17% (wt) and 13.3% (ko) of the initial area (p=0.01). Laminin-5 was expressed in the migrating epithelial cells with no differences between wild type and knock-out mouse. MMP-19 showed a strong expression in the basal cells of the human corneal epithelium. Corneal re-epithelialization was slightly faster in the MMP-19 knock-out mouse. No differences in the expression of laminin-5 could be detected.

Murray's Law in elastin haploinsufficient (Eln+/-) and wild-type (WT) mice.

PubMed

Sather, Bradley A; Hageman, Daniel; Wagenseil, Jessica E

2012-12-01

Using either the principle of minimum energy or constant shear stress, a relation can be derived that predicts the diameters of branching vessels at a bifurcation. This relation, known as Murray's Law, has been shown to predict vessel diameters in a variety of cardiovascular systems from adult humans to developing chicks. The goal of this study is to investigate Murray's Law in vessels from mice that are haploinsufficient for the elastin protein (Eln+/-). Elastin is one of the major proteins in the blood vessel wall and is organized in concentric rings, known as lamellae, with smooth muscle cells (SMCs) around the vessel lumen. Eln+/- mice have an increased number of lamellae, as well as smaller, thinner vessels. It is possible that due to decreased amounts of elastin available for vessel wall remodeling during development and in adulthood, Eln+/- vessels would not follow Murray's Law. We examined vessel bifurcations in six different physiologic regions, including the brain, heart, epidermis, ceocum (or cecum), testes, and intestines, in Eln+/- mice and wild-type (WT) littermates. All vessels were between 40 and 300 μm in diameter. We found that the diameters of both Eln+/- and WT vessels have an average of 13% error from the diameters predicted by Murray's Law, with no significant differences between genotypes or physiologic regions. The data suggest that vessels are optimized to follow Murray's Law, despite limitations on the proteins available for growth and remodeling of the vessel wall.

The Role of Dipeptidyl Peptidase IV in Lung Metastasis of Breast Cancer Cells

DTIC Science & Technology

1999-05-01

Our studies focused on (1) cloning and sequencing of wild-type endothelial DPP IV (wtDPP IV) and preparation of truncated DPP IV ( tDPP IV); (2...that was identical to hepatic DPP IV. Acid extraction of rat lung yielded a tDPP IV, which was an effective inhibitor of breast cancer cell adhesion to

An evaluation of the basis and consequences of a stay-green mutation in the navel negra (nan) citrus mutant using transcriptomic and proteomic profiling and metabolite analysis

USDA-ARS?s Scientific Manuscript database

A Citrus sinensis spontaneous mutant, navel negra (nan), produces fruit with an abnormal brown colored flavedo during ripening. Analysis of pigment composition in the wild type (WT) and nan flavedo suggested that typical ripening-related chlorophyll (Chl) degradation, but not carotenoid biosynthesis...

DEPENDENCE OF ECDYSTEROID METABOLISM AND DEVELOPMENT IN HOST LARVAE ON THE TIME OF BACULOVIRUS INFECTION AND THE ACTIVITY OF THE UDP-GLUCOSYL TRANSFERASE GENE.

EPA Science Inventory

Infection of fourth-instar gypsy moth (Lymantria dispar, Lepidoptera: Lymantriidae) larvae with the wild-type (Wt) gypsy moth baculovirus, LdNPV on the first day post-molt, or infection of fifth instars on the fifth day post-molt, results in elevated ecdysteroid levels in both he...

Kernel Composition, Starch Structure, and Enzyme Digestibility of Opaque-2 Maize and Quality Protein Maize

USDA-ARS?s Scientific Manuscript database

Objectives of this study were to understand how opaque-2 (o2) mutation and quality protein maize (QPM) affect maize kernel composition and starch structure, property, and enzyme digestibility. Kernels of o2 maize contained less protein (9.6−12.5%) than those of the wild-type (WT) counterparts (12...

Female mice lacking active nadph-oxidase enzymes are protected against “western diet”--induced obesity and metabolic syndrome

USDA-ARS?s Scientific Manuscript database

NADPH oxidase (Nox) enzymes have been implicated in regulation of adipocyte differentiation and inflammation in a variety of tissues. We examined the effects of feeding AIN-93G or a “Western diet” (WD) (45% fat, 0.5% cholesterol) on development of obesity and “metabolic syndrome” in wild type (WT) m...

Wild-type bone marrow transplant partially reverses neuroinflammation in progranulin-deficient mice.

PubMed

Yang, Yue; Aloi, Macarena S; Cudaback, Eiron; Josephsen, Samuel R; Rice, Samantha J; Jorstad, Nikolas L; Keene, C Dirk; Montine, Thomas J

2014-11-01

Frontotemporal dementia (FTD) is a neurodegenerative disease with devastating changes in behavioral performance and social function. Mutations in the progranulin gene (GRN) are one of the most common causes of inherited FTD due to reduced progranulin expression or activity, including in brain where it is expressed primarily by neurons and microglia. Thus, efforts aimed at enhancing progranulin levels might be a promising therapeutic strategy. Bone marrow (BM)-derived cells are able to engraft in the brain and adopt a microglial phenotype under myeloablative irradiation conditioning. This ability makes BM-derived cells a potential cellular vehicle for transferring therapeutic molecules to the central nervous system. Here, we utilized BM cells from Grn(+/+) (wild type or wt) mice labeled with green fluorescence protein for delivery of progranulin to progranulin-deficient (Grn(-/-)) mice. Our results showed that wt bone marrow transplantation (BMT) partially reconstituted progranulin in the periphery and in cerebral cortex of Grn(-/-) mice. We demonstrated a pro-inflammatory effect in vivo and in ex vivo preparations of cerebral cortex of Grn(-/-) mice that was partially to fully reversed 5 months after BMT. Our findings suggest that BMT can be administered as a stem cell-based approach to prevent or to treat neurodegenerative diseases.

Benzo[a]pyrene (BP) DNA adduct formation in DNA repair–deficient p53 haploinsufficient [Xpa(−/−)p53(+/−)] and wild-type mice fed BP and BP plus chlorophyllin for 28 days

PubMed Central

Poirier, Miriam C.

2012-01-01

We have evaluated DNA damage (DNA adduct formation) after feeding benzo[a]pyrene (BP) to wild-type (WT) and cancer-susceptible Xpa(−/−)p53(+/−) mice deficient in nucleotide excision repair and haploinsufficient for the tumor suppressor p53. DNA damage was evaluated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ES-MS/MS), which measures r7,t8,t9-trihydroxy-c-10-(N 2-deoxyguanosyl)-7,8,9,10-tetrahydrobenzo[a]pyrene (BPdG), and a chemiluminescence immunoassay (CIA), using anti-r7,t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)–DNA antiserum, which measures both BPdG and the other stable BP-DNA adducts. When mice were fed 100 ppm BP for 28 days, BP-induced DNA damage measured in esophagus, liver and lung was typically higher in Xpa(−/−)p53(+/−) mice, compared with WT mice. This result is consistent with the previously observed tumor susceptibility of Xpa(−/−)p53(+/−) mice. BPdG, the major DNA adduct associated with tumorigenicity, was the primary DNA adduct formed in esophagus (a target tissue in the mouse), whereas total BP-DNA adducts predominated in higher levels in the liver (a non-target tissue in the mouse). In an attempt to lower BP-induced DNA damage, we fed the WT and Xpa(−/−)p53(+/−) mice 0.3% chlorophyllin (CHL) in the BP-containing diet for 28 days. The addition of CHL resulted in an increase of BP–DNA adducts in esophagus, liver and lung of WT mice, a lowering of BPdG in esophagi of WT mice and livers of Xpa(−/−)p53(+/−) mice and an increase of BPdG in livers of WT mice. Therefore, the addition of CHL to a BP-containing diet showed a lack of consistent chemoprotective effect, indicating that oral CHL administration may not reduce PAH–DNA adduct levels consistently in human organs. PMID:22828138

Expression of Interleukin-4 by Recombinant Respiratory Syncytial Virus Is Associated with Accelerated Inflammation and a Nonfunctional Cytotoxic T-Lymphocyte Response following Primary Infection but Not following Challenge with Wild-Type Virus

PubMed Central

Bukreyev, Alexander; Belyakov, Igor M.; Prince, Gregory A.; Yim, Kevin C.; Harris, Katie K.; Berzofsky, Jay A.; Collins, Peter L.

2005-01-01

The outcome of a viral infection or of immunization with a vaccine can be influenced by the local cytokine environment. In studies of experimental vaccines against respiratory syncytial virus (RSV), an increased stimulation of Th2 (T helper 2) lymphocytes was associated with increased immunopathology upon subsequent RSV infection. For this study, we investigated the effect of increased local expression of the Th2 cytokine interleukin-4 (IL-4) from the genome of a recombinant RSV following primary infection and after a challenge with wild-type (wt) RSV. Mice infected with RSV/IL-4 exhibited an accelerated pulmonary inflammatory response compared to those infected with wt RSV, although the wt RSV group caught up by day 8. In the first few days postinfection, RSV/IL-4 was associated with a small but significant acceleration in the expansion of pulmonary T lymphocytes specific for an RSV CD8+ cytotoxic T-lymphocyte (CTL) epitope presented as a major histocompatibility complex class I tetramer. However, by day 7 the response of tetramer-positive T lymphocytes in the wt RSV group caught up and exceeded that of the RSV/IL-4 group. At all times, the CTL response of the RSV/IL-4 group was deficient in the production of gamma interferon and was nonfunctional for in vitro cell killing. The accelerated inflammatory response coincided with an accelerated accumulation and activation of pulmonary dendritic cells early in infection, but thereafter the dendritic cells were deficient in the expression of B7-1, which governs the acquisition of cytolytic activity by CTL. Following a challenge with wt RSV, there was an increase in Th2 cytokines in the animals that had previously been infected with RSV/IL-4 compared to those previously infected with wt RSV, but the CD8+ CTL response and the amount of pulmonary inflammation were not significantly different. Thus, a strong Th2 environment during primary pulmonary immunization with live RSV resulted in early inflammation and a largely nonfunctional primary CTL response but had a minimal effect on the secondary response. PMID:16014914

[Analysis of genetic characteristics of type II non-wild poliovirus in mainland China, 2010].

PubMed

Jiang, Hua-Fang; Yan, Dong-Mei; Zhu, Shuang-Li; Wang, Dong-Yan; Zhang, Yong; Zhu, Hui; An, Hong-Qiu; Xu, Wen-Bo; Kong, Xiao-Hui

2012-03-01

To study the genetic characteristics of 123 type II non-wild polioviruses isolated from acute flaccid paralysis (AFP) cases in mainland China in 2010, provide the scientific basis for maintaining the "polio-free" status, and the switching use of polio vaccine for China. VP1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and the PCR products were then sequenced. The sequence results were analyzed with Sequencher 4.8, BioEdit 7.0.9 and MEGA 5.0. Of 65 strains, nt2909 was found to be a mutation hotspot, and also a neurovirulence determinant in VP1 region. During 2010, two vaccine-derived polioviruses (VDPVs) were isolated from Yunnan province, China and no wild poliovirus (WPV) was isolated. The epidemiological studies and laboratory results of the two VDPVs showed that they were newly discovered VDPVs because of the genetic difference from other VDPVs strains isolated in the world, implying the sensitive poliovirus surveillance network could timely detect the transmission of VDPVs and the importation of WPV.

Multicenter Study of Epidemiological Cutoff Values and Detection of Resistance in Candida spp. to Anidulafungin, Caspofungin, and Micafungin Using the Sensititre YeastOne Colorimetric Method

PubMed Central

Alvarez-Fernandez, M.; Cantón, E.; Carver, P. L.; Chen, S. C.-A.; Eschenauer, G.; Getsinger, D. L.; Gonzalez, G. M.; Grancini, A.; Hanson, K. E.; Kidd, S. E.; Klinker, K.; Kubin, C. J.; Kus, J. V.; Lockhart, S. R.; Meletiadis, J.; Morris, A. J.; Pelaez, T.; Rodriguez-Iglesias, M.; Sánchez-Reus, F.; Shoham, S.; Wengenack, N. L.; Borrell Solé, N.; Echeverria, J.; Esperalba, J.; Gómez-G. de la Pedrosa, E.; García García, I.; Linares, M. J.; Marco, F.; Merino, P.; Pemán, J.; Pérez del Molino, L.; Roselló Mayans, E.; Rubio Calvo, C.; Ruiz Pérez de Pipaon, M.; Yagüe, G.; Garcia-Effron, G.; Perlin, D. S.; Sanguinetti, M.; Shields, R.; Turnidge, J.

2015-01-01

Neither breakpoints (BPs) nor epidemiological cutoff values (ECVs) have been established for Candida spp. with anidulafungin, caspofungin, and micafungin when using the Sensititre YeastOne (SYO) broth dilution colorimetric method. In addition, reference caspofungin MICs have so far proven to be unreliable. Candida species wild-type (WT) MIC distributions (for microorganisms in a species/drug combination with no detectable phenotypic resistance) were established for 6,007 Candida albicans, 186 C. dubliniensis, 3,188 C. glabrata complex, 119 C. guilliermondii, 493 C. krusei, 205 C. lusitaniae, 3,136 C. parapsilosis complex, and 1,016 C. tropicalis isolates. SYO MIC data gathered from 38 laboratories in Australia, Canada, Europe, Mexico, New Zealand, South Africa, and the United States were pooled to statistically define SYO ECVs. ECVs for anidulafungin, caspofungin, and micafungin encompassing ≥97.5% of the statistically modeled population were, respectively, 0.12, 0.25, and 0.06 μg/ml for C. albicans, 0.12, 0.25, and 0.03 μg/ml for C. glabrata complex, 4, 2, and 4 μg/ml for C. parapsilosis complex, 0.5, 0.25, and 0.06 μg/ml for C. tropicalis, 0.25, 1, and 0.25 μg/ml for C. krusei, 0.25, 1, and 0.12 μg/ml for C. lusitaniae, 4, 2, and 2 μg/ml for C. guilliermondii, and 0.25, 0.25, and 0.12 μg/ml for C. dubliniensis. Species-specific SYO ECVs for anidulafungin, caspofungin, and micafungin correctly classified 72 (88.9%), 74 (91.4%), 76 (93.8%), respectively, of 81 Candida isolates with identified fks mutations. SYO ECVs may aid in detecting non-WT isolates with reduced susceptibility to anidulafungin, micafungin, and especially caspofungin, since testing the susceptibilities of Candida spp. to caspofungin by reference methodologies is not recommended. PMID:26282428

In Situ Detection and Quantification of AR-V7, AR-FL, PSA, and KRAS Point Mutations in Circulating Tumor Cells.

PubMed

El-Heliebi, Amin; Hille, Claudia; Laxman, Navya; Svedlund, Jessica; Haudum, Christoph; Ercan, Erkan; Kroneis, Thomas; Chen, Shukun; Smolle, Maria; Rossmann, Christopher; Krzywkowski, Tomasz; Ahlford, Annika; Darai, Evangelia; von Amsberg, Gunhild; Alsdorf, Winfried; König, Frank; Löhr, Matthias; de Kruijff, Inge; Riethdorf, Sabine; Gorges, Tobias M; Pantel, Klaus; Bauernhofer, Thomas; Nilsson, Mats; Sedlmayr, Peter

2018-03-01

Liquid biopsies can be used in castration-resistant prostate cancer (CRPC) to detect androgen receptor splice variant 7 (AR-V7), a splicing product of the androgen receptor. Patients with AR-V7-positive circulating tumor cells (CTCs) have greater benefit of taxane chemotherapy compared with novel hormonal therapies, indicating a treatment-selection biomarker. Likewise, in those with pancreatic cancer (PaCa), KRAS mutations act as prognostic biomarkers. Thus, there is an urgent need for technology investigating the expression and mutation status of CTCs. Here, we report an approach that adds AR-V7 or KRAS status to CTC enumeration, compatible with multiple CTC-isolation platforms. We studied 3 independent CTC-isolation devices (CellCollector, Parsortix, CellSearch) for the evaluation of AR-V7 or KRAS status of CTCs with in situ padlock probe technology. Padlock probes allow highly specific detection and visualization of transcripts on a cellular level. We applied padlock probes for detecting AR-V7, androgen receptor full length (AR-FL), and prostate-specific antigen (PSA) in CRPC and KRAS wild-type (wt) and mutant (mut) transcripts in PaCa in CTCs from 46 patients. In situ analysis showed that 71% (22 of 31) of CRPC patients had detectable AR-V7 expression ranging from low to high expression [1-76 rolling circle products (RCPs)/CTC]. In PaCa patients, 40% (6 of 15) had KRAS mut expressing CTCs with 1 to 8 RCPs/CTC. In situ padlock probe analysis revealed CTCs with no detectable cytokeratin expression but positivity for AR-V7 or KRAS mut transcripts. Padlock probe technology enables quantification of AR-V7, AR-FL, PSA, and KRAS mut/wt transcripts in CTCs. The technology is easily applicable in routine laboratories and compatible with multiple CTC-isolation devices. © 2017 American Association for Clinical Chemistry.

Multicenter Study of Method-Dependent Epidemiological Cutoff Values for Detection of Resistance in Candida spp. and Aspergillus spp. to Amphotericin B and Echinocandins for the Etest Agar Diffusion Method.

PubMed

Espinel-Ingroff, A; Arendrup, M; Cantón, E; Cordoba, S; Dannaoui, E; García-Rodríguez, J; Gonzalez, G M; Govender, N P; Martin-Mazuelos, E; Lackner, M; Lass-Flörl, C; Linares Sicilia, M J; Rodriguez-Iglesias, M A; Pelaez, T; Shields, R K; Garcia-Effron, G; Guinea, J; Sanguinetti, M; Turnidge, J

2017-01-01

Method-dependent Etest epidemiological cutoff values (ECVs) are not available for susceptibility testing of either Candida or Aspergillus species with amphotericin B or echinocandins. In addition, reference caspofungin MICs for Candida spp. are unreliable. Candida and Aspergillus species wild-type (WT) Etest MIC distributions (microorganisms in a species-drug combination with no detectable phenotypic resistance) were established for 4,341 Candida albicans, 113 C. dubliniensis, 1,683 C. glabrata species complex (SC), 709 C. krusei, 767 C. parapsilosis SC, 796 C. tropicalis, 1,637 Aspergillus fumigatus SC, 238 A. flavus SC, 321 A. niger SC, and 247 A. terreus SC isolates. Etest MICs from 15 laboratories (in Argentina, Europe, Mexico, South Africa, and the United States) were pooled to establish Etest ECVs. Anidulafungin, caspofungin, micafungin, and amphotericin B ECVs (in micrograms per milliliter) encompassing ≥97.5% of the statistically modeled population were 0.016, 0.5, 0.03, and 1 for C. albicans; 0.03, 1, 0.03, and 2 for C. glabrata SC; 0.06, 1, 0.25, and 4 for C. krusei; 8, 4, 2, and 2 for C. parapsilosis SC; and 0.03, 1, 0.12, and 2 for C. tropicalis The amphotericin B ECV was 0.25 μg/ml for C. dubliniensis and 2, 8, 2, and 16 μg/ml for the complexes of A. fumigatus, A. flavus, A. niger, and A. terreus, respectively. While anidulafungin Etest ECVs classified 92% of the Candida fks mutants evaluated as non-WT, the performance was lower for caspofungin (75%) and micafungin (84%) cutoffs. Finally, although anidulafungin (as an echinocandin surrogate susceptibility marker) and amphotericin B ECVs should identify Candida and Aspergillus isolates with reduced susceptibility to these agents using the Etest, these ECVs will not categorize a fungal isolate as susceptible or resistant, as breakpoints do. Copyright © 2016 American Society for Microbiology.

Lack of Angiopoietin‐Like‐2 Expression Limits the Metabolic Stress Induced by a High‐Fat Diet and Maintains Endothelial Function in Mice

PubMed Central

Yu, Carol; Luo, Xiaoyan; Farhat, Nada; Daneault, Caroline; Duquette, Natacha; Martel, Cécile; Lambert, Jean; Thorin‐Trescases, Nathalie; Rosiers, Christine Des; Thorin, Éric

2014-01-01

Background Angiopoietin‐like‐2 (angptl2) is produced by several cell types including endothelial cells, adipocytes and macrophages, and contributes to the inflammatory process in cardiovascular diseases. We hypothesized that angptl2 impairs endothelial function, and that lowering angptl2 levels protects the endothelium against high‐fat diet (HFD)‐induced fat accumulation and hypercholesterolemia. Methods and Results Acute recombinant angptl2 reduced (P<0.05) acetylcholine‐mediated vasodilation of isolated wild‐type (WT) mouse femoral artery, an effect reversed (P<0.05) by the antioxidant N‐acetylcysteine. Accordingly, in angptl2 knockdown (KD) mice, ACh‐mediated endothelium‐dependent vasodilation was greater (P<0.05) than in WT mice. In arteries from KD mice, prostacyclin contributed to the overall dilation unlike in WT mice. After a 3‐month HFD, overall vasodilation was not altered, but dissecting out the endothelial intrinsic pathways revealed that NO production was reduced in arteries isolated from HFD‐fed WT mice (P<0.05), while NO release was maintained in KD mice. Similarly, endothelium‐derived hyperpolarizing factor (EDHF) was preserved in mesenteric arteries from HFD‐fed KD mice but not in those from WT mice. Finally, the HFD increased (P<0.05) total cholesterol–to–high‐density lipoprotein ratios, low‐density lipoprotein–to–high‐density lipoprotein ratios, and leptin levels in WT mice only, while glycemia remained similar in the 2 strains. KD mice displayed less triglyceride accumulation in the liver (P<0.05 versus WT), and adipocyte diameters in mesenteric and epididymal white adipose tissues were smaller (P<0.05) in KD than in WT fed an HFD, while inflammatory gene expression increased (P<0.05) in the fat of WT mice only. Conclusions Lack of angptl2 expression limits the metabolic stress induced by an HFD and maintains endothelial function in mice. PMID:25128474

University of California San Francisco (UCSF-2): Gene Expression Profiling of Normal Mouse Skin, Hras WT and Hras -/- | Office of Cancer Genomics

Cancer.gov

University of California San Francisco (UCSF-2): Gene Expression Profiling of Normal Mouse Skin, Hras WT and Hras -/- This data set contains the transcriptional profiles of 20 dorsal skin samples from eight-week-old mice. Mice were generated by crossing FVB/N to Mus spretus mice to generate F1 mice, and then crossing F1 mice back to the FVB/N strain. 10  FVB/N mice lacking Hras1 (aka HrasKO, Hras-/-) and 10  FVB/N mice with wild-type Hras1 were generated. Read the abstract.

The Brown Midrib Leaf (bml) Mutation in Rice (Oryza sativa L.) Causes Premature Leaf Senescence and the Induction of Defense Responses.

PubMed

Akhter, Delara; Qin, Ran; Nath, Ujjal Kumar; Alamin, Md; Jin, Xiaoli; Shi, Chunhai

2018-04-09

Isolating and characterizing mutants with altered senescence phenotypes is one of the ways to understand the molecular basis of leaf aging. Using ethyl methane sulfonate mutagenesis, a new rice ( Oryza sativa ) mutant, brown midrib leaf ( bml ), was isolated from the indica cultivar 'Zhenong34'. The bml mutants had brown midribs in their leaves and initiated senescence prematurely, at the onset of heading. The mutants had abnormal cells with degraded chloroplasts and contained less chlorophyll compared to the wild type (WT). The bml mutant showed excessive accumulation of reactive oxygen species (ROS), increased activities of superoxide dismutase, catalase, and malondialdehyde, upregulation of senescence-induced STAY-GREEN genes and senescence-related transcription factors, and down regulation of photosynthesis-related genes. The levels of abscisic acid (ABA) and jasmonic acid (JA) were increased in bml with the upregulation of some ABA and JA biosynthetic genes. In pathogen response, bml demonstrated higher resistance against Xanthomonas oryzae pv. oryzae and upregulation of four pathogenesis-related genes compared to the WT. A genetic study confirmed that the bml trait was caused by a single recessive nuclear gene ( BML ). A map-based cloning using insertion/deletion markers confirmed that BML was located in the 57.32kb interval between the L5IS7 and L5IS11 markers on the short arm of chromosome 5. A sequence analysis of the candidate region identified a 1 bp substitution (G to A) in the 5'-UTR (+98) of bml . BML is a candidate gene associated with leaf senescence, ROS regulation, and disease response, also involved in hormone signaling in rice. Therefore, this gene might be useful in marker-assisted backcrossing/gene editing to improve rice cultivars.

The nuclear form of glutathione peroxidase 4 is associated with sperm nuclear matrix and is required for proper paternal chromatin decondensation at fertilization.

PubMed

Puglisi, Rossella; Maccari, Irene; Pipolo, Simona; Conrad, Marcus; Mangia, Franco; Boitani, Carla

2012-04-01

The nuclear isoform of the selenoprotein Phospholipid Hydroperoxide Glutathione Peroxidase (nGPx4) is expressed in haploid male germ cells, contains several cysteines and is able to oxidize protein thiols, besides glutathione. In this study we have investigated the subnuclear localization of this isoform in isolated mouse male germ cells at different steps of maturation. Immunoblotting and confocal microscopy analyses of subnuclear fractions showed that nGPx4 is localized to the nuclear matrix together with well known markers of this subnuclear compartment like lamin B and topoisomerase IIβ at all stages of germ cell differentiation. The peculiar nGPx4 distribution was confirmed by both biochemical and morphological analyses of COS-1 cells overexpressing Flag-tagged nGPx4. To test the functional role of nGPx4 in the process of chromatin assembly, sperm isolated from the caput and the cauda epididymides of wild-type (WT) and genetically deficient in nGPx4 (nGPx4-KO) mice were analyzed in an in vitro chromatin decondensation assay. Results showed that sperm from nGPx4-KO mice were more prone to decondense than those from WT mice at all stages of epididymal maturation, providing conclusive evidence that nGPx4 is required for a correct sperm chromatin compaction. We next addressed the issue of whether the lack of nGPx4 impacts on early events occurring at fertilization. Indeed, in vitro fertilization experiments showed an acceleration of sperm chromatin dispersion in oocytes fertilized by nGpx4-KO sperm compared with control. Overall these data indicate that the absence of nGPx4 leads to sperm nuclear matrix/chromatin instability that may negatively affect the embryo development. Copyright © 2011 Wiley Periodicals, Inc.

A Novel Binary Mixture of Helicoverpa armigera Single Nucleopolyhedrovirus Genotypic Variants Has Improved Insecticidal Characteristics for Control of Cotton Bollworms

PubMed Central

Arrizubieta, Maite; Simón, Oihane; Williams, Trevor

2015-01-01

The genotypic diversity of two Spanish isolates of Helicoverpa armigera single nucleopolyhedrovirus (HearSNPV) was evaluated with the aim of identifying mixtures of genotypes with improved insecticidal characteristics for control of the cotton bollworm. Two genotypic variants, HearSP1A and HearSP1B, were cloned in vitro from the most pathogenic wild-type isolate of the Iberian Peninsula, HearSNPV-SP1 (HearSP1-wt). Similarly, six genotypic variants (HearLB1 to -6) were obtained by endpoint dilution from larvae collected from cotton crops in southern Spain that died from virus disease during laboratory rearing. Variants differed significantly in their insecticidal properties, pathogenicity, speed of kill, and occlusion body (OB) production (OBs/larva). HearSP1B was ∼3-fold more pathogenic than HearSP1-wt and the other variants. HearLB1, HearLB2, HeaLB5, and HearLB6 were the fastest-killing variants. Moreover, although highly virulent, HearLB1, HearLB4, and HearLB5 produced more OBs/larva than did the other variants. The co-occluded HearSP1B:LB6 mixture at a 1:1 proportion was 1.7- to 2.8-fold more pathogenic than any single variant and other mixtures tested and also killed larvae as fast as the most virulent genotypes. Serial passage resulted in modified proportions of the component variants of the HearSP1B:LB6 co-occluded mixture, suggesting that transmissibility could be further improved by this process. We conclude that the improved insecticidal phenotype of the HearSP1B:LB6 co-occluded mixture underlines the utility of the genotypic variant dissection and reassociation approach for the development of effective virus-based insecticides. PMID:25841011

Two barcodes encoded by the type-1 PDZ and by phospho-Ser312 regulate retromer/WASH-mediated sorting of the ß1-adrenergic receptor from endosomes to the plasma membrane.

PubMed

Nooh, Mohammed M; Bahouth, Suleiman W

2017-01-01

Recycling of the majority of agonist-internalized GPCR is dependent on a type I-PDZ "barcode" in their C-tail. The recycling of wild-type (WT) ß 1 -AR is also dependent on its default "type-1 PDZ barcode", but trafficking of the ß 1 -AR is inhibited when PKA or its substrate serine at position 312 (Ser 312 ) are inactivated. We tested the hypothesis that phospho-Ser 312 provided a second barcode for ß 1 -AR sorting from endosomes to the plasma membrane by determining the role of retromer/WASH complexes in ß 1 -AR trafficking. Recycling of WT ß 1 -AR or WT ß 2 -AR was dependent on targeting the retromer to endosomal membranes via SNX3 and rab7a, and on complexing the retromer to the WASH pentamer via the C-tail of FAM21 (FAM21 C ). These maneuvers however, did not inhibit the recycling of a phospho-Ser 312 ß 1 -AR mimic ((S312D) ß 1 -AR). Knockdown of the trans-acting PDZ protein sorting nexin27 (SNX27) inhibited the recycling of WT ß 1 -AR and WT ß 2 -AR, but had no effect on (S312D) ß 1 -AR∆PDZ or on phosphorylation of WT ß 1 -AR by PKA at Ser 312 . However, depletion of FKBP15, a FAM21 C -binding endosomal protein, selectively inhibited WT ß 1 -AR but not ß 2 -AR recycling, suggesting divergence might exist in GPCR trafficking roadmaps. These results indicate that two barcodes are involved in sorting WT ß 1 -AR out of early endosomes. The first and antecedent "barcode" was the "type-1 PDZ", followed by a second reversible "phospho-Ser 312 " verification "barcode". This organization allows tight regulation of ß 1 -AR density to signaling intensity in conditions associated with aberrant ß 1 -AR signaling such as in hypertension and heart failure. Copyright © 2016 Elsevier Inc. All rights reserved.

Cardiac Function in Young and Old Little Mice

PubMed Central

Reddy, Anilkumar K.; Amador-Noguez, Daniel; Darlington, Gretchen J.; Scholz, Beth A.; Michael, Lloyd H.; Hartley, Craig J.; Entman, Mark L.; Taffet, George E.

2009-01-01

We studied cardiac function in young and old, wild-type (WT), and longer-living Little mice using cardiac flow velocities, echocardiographic measurements, and left ventricular (LV) pressure (P) to determine if enhanced reserves were in part responsible for longevity in these mice. Resting/baseline cardiac function, as measured by velocities, LV dimensions, +dP/dtmax, and −dP/dtmax, was significantly lower in young Little mice versus young WT mice. Fractional shortening (FS) increased significantly, and neither +dP/dtmax nor −dP/dtmax declined with age in Little mice. In contrast, old WT mice had no change in FS but had significantly lower +dP/dtmax and −dP/dtmax versus young WT mice. Significant decreases were observed in the velocity indices of old Little mice versus old WT mice, but other parameters were unchanged. The magnitude of dobutamine stress response remained unchanged with age in Little mice, while that in WT mice decreased. These data suggest that while resting cardiac function in Little mice versus WT mice is lower at young age, it is relatively unaltered with aging. Additionally, cardiac function in response to stress was maintained with age in Little mice but not in their WT counterparts. Thus, some mouse models of increased longevity may not be associated with enhanced reserves. PMID:18166681

The human clone ST22 SCCmec IV methicillin-resistant Staphylococcus aureus isolated from swine herds and wild primates in Nepal: is man the common source?

PubMed

Roberts, Marilyn C; Joshi, Prabhu Raj; Greninger, Alexander L; Melendez, Daira; Paudel, Saroj; Acharya, Mahesh; Bimali, Nabin Kishor; Koju, Narayan P; No, David; Chalise, Mukesh; Kyes, Randall C

2018-05-01

Swine nasal samples [n = 282] were collected from 12 randomly selected farms around Kathmandu, Nepal, from healthy animals. In addition, wild monkey (Macaca mulatta) saliva samples [n = 59] were collected near temples areas in Kathmandu using a non-invasive sampling technique. All samples were processed for MRSA using standardized selective media and conventional biochemical tests. MRSA verification was done and isolates characterized by SCCmec, multilocus sequence typing, whole genome sequencing [WGS] and antibiotic susceptibilities. Six (2.1%) swine MRSA were isolated from five of the different swine herds tested, five were ST22 type IV and one ST88 type V. Four (6.8%) macaques MRSA were isolated, with three ST22 SCCmec type IV and one ST239 type III. WGS sequencing showed that the eight ciprofloxacin resistant ST22 isolates carried gyrA mutation [S84L]. Six isolates carried the erm(C) genes, five isolates carried aacC-aphD genes and four isolates carried blaZ genes. The swine linezolid resistant ST22 did not carry any known acquired linezolid resistance genes but had a mutation in ribosomal protein L22 [A29V] and an insertion in L4 [68KG69], both previously associated with linezolid resistance. Multiple virulence factors were also identified. This is the first time MRSA ST22 SCCmec IV has been isolated from livestock or primates.

Characterization of Clade 2.3.2.1 H5N1 Highly Pathogenic Avian Influenza Viruses Isolated from Wild Birds (Mandarin Duck and Eurasian Eagle Owl) in 2010 in Korea

PubMed Central

Choi, Jun-Gu; Kang, Hyun-Mi; Jeon, Woo-Jin; Choi, Kang-Seuk; Kim, Kwang-Il; Song, Byung Min; Lee, Hee-Soo; Kim, Jae-Hong; Lee, Youn-Jeong

2013-01-01

Starting in late November 2010, the H5N1 highly pathogenic avian influenza (HPAI) virus was isolated from many types of wild ducks and raptors and was subsequently isolated from poultry in Korea. We assessed the genetic and pathogenic properties of the HPAI viruses isolated from a fecal sample from a mandarin duck and a dead Eurasian eagle owl, the most affected wild bird species during the 2010/2011 HPAI outbreak in Korea. These viruses have similar genetic backgrounds and exhibited the highest genetic similarity with recent Eurasian clade 2.3.2.1 HPAI viruses. In animal inoculation experiments, regardless of their originating hosts, the two Korean isolates produced highly pathogenic characteristics in chickens, ducks and mice without pre-adaptation. These results raise concerns about veterinary and public health. Surveillance of wild birds could provide a good early warning signal for possible HPAI infection in poultry as well as in humans. PMID:23611846

Root hairs increase root exudation and rhizosphere extension

NASA Astrophysics Data System (ADS)

Holz, Maire; Zarebandanadkouki, Mohsen; Kuzyakov, Yakov; Carmintati, Andrea

2017-04-01

Plant roots employ various mechanisms to increase their access to limited soil resources. An example of such strategies is the production of root hairs. Root hairs extend the root surface and therefore increase the access to nutrients. Additionally, carbon release from root hairs might facilitate nutrient uptake by spreading of carbon in the rhizosphere and enhancing microbial activity. The aim of this study was to test: i) how root hairs change the allocation of carbon in the soil-plant system; ii) whether root hairs exude carbon into the soil and iii) how differences in C release between plants with and without root hairs affect rhizosphere extension. We grew barley plants with and without root hairs (wild type: WT, bald root barley: brb) in rhizoboxes filled with a sandy soil. Root elongation was monitored over time. After 4 weeks of growth, plants were labelled with 14CO2. A filter paper was placed on the soil surface before labelling and was removed after 36 h. 14C imaging of the soil surface and of the filter paper was used to quantify the allocation of 14C into the roots and the exudation of 14C, respectively. Plants were sampled destructively one day after labeling to quantify 14C in the plant-soil system. 14CO2 release from soil over time (17 d) was quantified by trapping CO2 in NaOH with an additional subset of plants. WT and brb plants had a similar aboveground biomass and allocated similar amounts of 14C into shoots (170 KBq for WT; 152 KBq for brb) and roots one day after labelling. Biomass of root, rhizosphere soil as well as root elongation were lower for brb compared to the wild type. WT plants transported more C from the shoots to the roots (22.8% for WT; 13.8% for brb) and from the root into the rhizosphere (8.8% for WT 3.5% for brb). Yet lower amounts of 14CO2 were released from soil over time for WT. Radial and longitudinal rhizosphere extension was increased for WT compared to brb (4.7 vs. 2.6 mm; 5.6 vs. 3.1 cm). The total exudation which was estimated based on the grey values of the filter paper images was 1.6 times higher for WT compared to brb. After one month, brb plants performed as good as WT plants, presumably because nutrients and water were not limiting for young plants. Under nutrient limiting conditions higher C release as well as increased longitudinal and radial rhizosphere extension for WT may maintain higher nutrient accessibility compared to root hair free plants.

Differential proteomic responses of selectively bred and wild-type Sydney rock oyster populations exposed to elevated CO2.

PubMed

Thompson, E L; O'Connor, W; Parker, L; Ross, P; Raftos, D A

2015-03-01

Previous work suggests that larvae from Sydney rock oysters that have been selectively bred for fast growth and disease resistance are more resilient to the impacts of ocean acidification than nonselected, wild-type oysters. In this study, we used proteomics to investigate the molecular differences between oyster populations in adult Sydney rock oysters and to identify whether these form the basis for observations seen in larvae. Adult oysters from a selective breeding line (B2) and nonselected wild types (WT) were exposed for 4 weeks to elevated pCO2 (856 μatm) before their proteomes were compared to those of oysters held under ambient conditions (375 μatm pCO2 ). Exposure to elevated pCO2 resulted in substantial changes in the proteomes of oysters from both the selectively bred and wild-type populations. When biological functions were assigned, these differential proteins fell into five broad, potentially interrelated categories of subcellular functions, in both oyster populations. These functional categories were energy production, cellular stress responses, the cytoskeleton, protein synthesis and cell signalling. In the wild-type population, proteins were predominantly upregulated. However, unexpectedly, these cellular systems were downregulated in the selectively bred oyster population, indicating cellular dysfunction. We argue that this reflects a trade-off, whereby an adaptive capacity for enhanced mitochondrial energy production in the selectively bred population may help to protect larvae from the effects of elevated CO2 , whilst being deleterious to adult oysters. © 2015 John Wiley & Sons Ltd.

Antimicrobial susceptibility of porcine Brachyspira hyodysenteriae and Brachyspira pilosicoli isolated in Sweden between 1990 and 2010.

PubMed

Pringle, Märit; Landén, Annica; Unnerstad, Helle Ericsson; Molander, Benedicta; Bengtsson, Björn

2012-09-21

The anaerobic spirochetes Brachyspira hyodysenteriae and Brachyspira pilosicoli cause diarrheal diseases in pigs. Their fastidious nature has hampered standardization of methods for antimicrobial susceptibility testing. For monitoring of antimicrobial susceptibility wild type cutoff values are needed to define where the wild type distribution of MICs ends and no approved cutoffs are available for Brachyspira spp. In this study antimicrobial susceptibility data for both species (in total 906 isolates) were compiled and analyzed and wild type cut off values for B. hyodysenteriae proposed. The MICs of tiamulin, valnemulin, tylosin, tylvalosin, doxycycline and lincomycin were determined by broth dilution in brain heart infusion broth supplemented with 10% fetal calf serum. The compiled MICs from the broth dilution tests of the B. hyodysenteriae type strain, B78T (ATCC® 27164T), showed that the method yields reproducible results. In an international perspective the frequencies of isolates with decreased antimicrobial susceptibility were low among both B. hyodysenteriae and B. pilosicoli. However, in B. pilosicoli a constant level of 10-15% isolates with tiamulin MICs >4 μg/ml was detected between 2002 and 2010 and in B. hyodysenteriae a gradual increase in tiamulin MICs was seen between 1990 and 2003 although this increase has ceased during the last years. The wild type cutoff values proposed for B. hyodysenteriae are: tiamulin >0.25 μg/ml, valnemulin >0.125 μg/ml, tylosin >16 μg/ml, tylvalosin >1 μg/ml, lincomycin >1 μg/ml and doxycycline >0.5 μg/ml. The broth dilution method used in this study has over the years generated tightly grouped MIC populations for the field isolates and reproducible results for the control strain B78T and is therefore a suitable antimicrobial susceptibility test method for monitoring of Brachyspira spp. Here we propose wild type cutoff values for six antimicrobial agents for B. hyodysenteriae tested by broth dilution based on MIC distributions and the current knowledge on mechanisms of resistance in this species. There are few studies on antimicrobial resistance mechanisms and MIC distributions in B. pilosicoli but to some extent the cutoff values proposed for B. hyodysenteriae may be applicable also for monitoring of antimicrobial susceptibility in B. pilosicoli.

Antimicrobial susceptibility of porcine Brachyspira hyodysenteriae and Brachyspira pilosicoli isolated in Sweden between 1990 and 2010

PubMed Central

2012-01-01

Background The anaerobic spirochetes Brachyspira hyodysenteriae and Brachyspira pilosicoli cause diarrheal diseases in pigs. Their fastidious nature has hampered standardization of methods for antimicrobial susceptibility testing. For monitoring of antimicrobial susceptibility wild type cutoff values are needed to define where the wild type distribution of MICs ends and no approved cutoffs are available for Brachyspira spp. In this study antimicrobial susceptibility data for both species (in total 906 isolates) were compiled and analyzed and wild type cut off values for B. hyodysenteriae proposed. Methods The MICs of tiamulin, valnemulin, tylosin, tylvalosin, doxycycline and lincomycin were determined by broth dilution in brain heart infusion broth supplemented with 10% fetal calf serum. Results The compiled MICs from the broth dilution tests of the B. hyodysenteriae type strain, B78T (ATCC® 27164T), showed that the method yields reproducible results. In an international perspective the frequencies of isolates with decreased antimicrobial susceptibility were low among both B. hyodysenteriae and B. pilosicoli. However, in B. pilosicoli a constant level of 10-15% isolates with tiamulin MICs >4 μg/ml was detected between 2002 and 2010 and in B. hyodysenteriae a gradual increase in tiamulin MICs was seen between 1990 and 2003 although this increase has ceased during the last years. The wild type cutoff values proposed for B. hyodysenteriae are: tiamulin >0.25 μg/ml, valnemulin >0.125 μg/ml, tylosin >16 μg/ml, tylvalosin >1 μg/ml, lincomycin >1 μg/ml and doxycycline >0.5 μg/ml. Conclusions The broth dilution method used in this study has over the years generated tightly grouped MIC populations for the field isolates and reproducible results for the control strain B78T and is therefore a suitable antimicrobial susceptibility test method for monitoring of Brachyspira spp. Here we propose wild type cutoff values for six antimicrobial agents for B. hyodysenteriae tested by broth dilution based on MIC distributions and the current knowledge on mechanisms of resistance in this species. There are few studies on antimicrobial resistance mechanisms and MIC distributions in B. pilosicoli but to some extent the cutoff values proposed for B. hyodysenteriae may be applicable also for monitoring of antimicrobial susceptibility in B. pilosicoli. PMID:22998753

Mitochondrial catalase suppresses naturally occurring lung cancer in old mice.

PubMed

Ge, Xuang; Pettan-Brewer, Christina; Morton, John; Carter, Katrina; Fatemi, Sy; Rabinovitch, Peter; Ladiges, Warren C

2015-01-01

Lung cancer is generally difficult to detect until the late stages of disease, when it is much more difficult to treat because of the more aggressive and invasive behavior. Advanced lung cancer is much more common in older adults making it even more challenging to treat. Adenocarcinoma belongs to a category of non-small cell lung cancers, which comprise up to 40% of all lung cancers, and about half of these have an activating K-ras mutation. Because treatment relapses are common, more effective unconventional treatment and prevention methods are needed. In this regard, the antioxidant enzyme catalase targeted to mitochondria (mCAT) has been shown to delay aging and cancer in mice, and the progression of transgenic oncogene and syngeneic tumors was suppressed, helping support the notion that attenuation of mitochondria-generated hydrogen peroxide signaling is associated with an antitumor effect. In order to determine if mCAT has any effect on naturally occurring lung cancer of the adenocarcinoma type in old mice, the tumor incidence and progression were examined in the lungs of old mCAT transgenic and wild-type (WT) mice with a CB6F1 (Balb/c X C57BL/6) background. CB6F1 mice with a WT genotype were found to have a high incidence of adenomas at 24 months of age, which progressed to adenocarcinomas at 32 months of age. CB6F1 mice with the mCAT genotype had significantly reduced incidence and severity of lung tumors at both ages. Fibroblasts isolated from the lungs of old mCAT mice, but not WT mice, were shown to secrete soluble factors that inhibited lung tumor cell growth suggesting that stromal fibroblasts play a role in mediating the antitumor effects of mCAT. The aged CB6F1 mouse, with its high incidence of K-ras mutant lung cancer, is an excellent model to further study the anticancer potential of mitochondria-targeted therapy.

Mitochondrial catalase suppresses naturally occurring lung cancer in old mice

PubMed Central

Ge, Xuang; Pettan-Brewer, Christina; Morton, John; Carter, Katrina; Fatemi, Sy; Rabinovitch, Peter; Ladiges, Warren C.

2015-01-01

Lung cancer is generally difficult to detect until the late stages of disease, when it is much more difficult to treat because of the more aggressive and invasive behavior. Advanced lung cancer is much more common in older adults making it even more challenging to treat. Adenocarcinoma belongs to a category of non-small cell lung cancers, which comprise up to 40% of all lung cancers, and about half of these have an activating K-ras mutation. Because treatment relapses are common, more effective unconventional treatment and prevention methods are needed. In this regard, the antioxidant enzyme catalase targeted to mitochondria (mCAT) has been shown to delay aging and cancer in mice, and the progression of transgenic oncogene and syngeneic tumors was suppressed, helping support the notion that attenuation of mitochondria-generated hydrogen peroxide signaling is associated with an antitumor effect. In order to determine if mCAT has any effect on naturally occurring lung cancer of the adenocarcinoma type in old mice, the tumor incidence and progression were examined in the lungs of old mCAT transgenic and wild-type (WT) mice with a CB6F1 (Balb/c X C57BL/6) background. CB6F1 mice with a WT genotype were found to have a high incidence of adenomas at 24 months of age, which progressed to adenocarcinomas at 32 months of age. CB6F1 mice with the mCAT genotype had significantly reduced incidence and severity of lung tumors at both ages. Fibroblasts isolated from the lungs of old mCAT mice, but not WT mice, were shown to secrete soluble factors that inhibited lung tumor cell growth suggesting that stromal fibroblasts play a role in mediating the antitumor effects of mCAT. The aged CB6F1 mouse, with its high incidence of K-ras mutant lung cancer, is an excellent model to further study the anticancer potential of mitochondria-targeted therapy. PMID:26400209

FNDC5 attenuates adipose tissue inflammation and insulin resistance via AMPK-mediated macrophage polarization in obesity.

PubMed

Xiong, Xiao-Qing; Geng, Zhi; Zhou, Bing; Zhang, Feng; Han, Ying; Zhou, Ye-Bo; Wang, Jue-Jin; Gao, Xing-Ya; Chen, Qi; Li, Yue-Hua; Kang, Yu-Ming; Zhu, Guo-Qing

2018-06-01

Obesity-induced chronic inflammation is critical in the pathogenesis of insulin resistance, and the recruitment and proinflammatory activation of adipose tissue macrophages (ATMs) is important for the development of this process. Here, we examined the effects of fibronectin type III domain-containing 5 (FNDC5) on inflammation and insulin resistance in high-fat diet-induced obese mice. Male wild-type (WT) and FNDC5 -/- mice were fed with standard chow (Ctrl) or high fat diet (HFD) for 20 weeks to induce obesity and insulin resistance. Firstly, effects of FNDC5 gene deletion on obesity, insulin resistance, macrophage accumulation and polarization and adipose tissue inflammation were determined in mice. Secondly, the macrophage polarity shift was further examined with flow cytometry in isolated stromal vascular fraction (SVF). Thirdly, the effects of exogenous FNDC5 on lipopolysaccharide (LPS)-induced macrophage polarization, inflammation and the underlying signaling mechanism were investigated in RAW264.7 macrophages and primary mouse peritoneal cavity macrophages (PMs). Finally, the therapeutic effects of FNDC5 overexpression were examined in HFD-induced obese WT and FNDC5 -/- mice. FNDC5 gene deletion aggravated obesity, insulin resistance, fat accumulation and inflammation accompanied with enhanced AMPK inhibition, macrophages recruitment and M1 polarization in mice fed with HFD. Exogenous FNDC5 inhibited LPS-induced M1 macrophage polarization and inflammatory cytokine production via AMPK phosphorylation in both RAW264.7 macrophages and PMs. FNDC5 overexpression attenuated insulin resistance, AMPK inhibition, M1 macrophage polarization and inflammatory cytokine production in adipose tissue of obese WT and FNDC5 -/- mice. FNDC5 attenuates adipose tissue inflammation and insulin resistance via AMPK-mediated macrophage polarization in HFD-induced obesity. FNDC5 plays several beneficial roles in obesity and may be used as a therapeutic regimen for preventing inflammation and insulin resistance in obesity and diabetes. Copyright © 2018 Elsevier Inc. All rights reserved.

Loss of T cells influences sex differences in behavior and brain structure.

PubMed

Rilett, Kelly C; Friedel, Miriam; Ellegood, Jacob; MacKenzie, Robyn N; Lerch, Jason P; Foster, Jane A

2015-05-01

Clinical and animal studies demonstrate that immune-brain communication influences behavior and brain function. Mice lacking T cell receptor β and δ chains were tested in the elevated plus maze, open field, and light-dark test and showed reduced anxiety-like behavior compared to wild type. Interestingly sex differences were observed in the behavioural phenotype of TCRβ-/-δ- mice. Specifically, female TCRβ-/-δ- mice spent more time in the light chamber compared to wild type females, whereas male TCRβ-/-δ- spent more time in the center of the open field compared to wild type males. In addition, TCRβ-/-δ- mice did not show sex differences in activity-related behaviors observed in WT mice. Ex vivo brain imaging (7 Tesla MRI) revealed volume changes in hippocampus, hypothalamus, amygdala, periaqueductal gray, and dorsal raphe and other brain regions between wild type and T cell receptor knockout mice. There was also a loss of sexual dimorphism in brain volume in the bed nucleus of the stria terminalis, normally the most sexually dimorphic region in the brain, in immune compromised mice. These data demonstrate the presence of T cells is important in the development of sex differences in CNS circuitry and behavior. Copyright © 2015 Elsevier Inc. All rights reserved.

Differential expression of the TWEAK receptor Fn14 in IDH1 wild-type and mutant gliomas.

PubMed

Hersh, David S; Peng, Sen; Dancy, Jimena G; Galisteo, Rebeca; Eschbacher, Jennifer M; Castellani, Rudy J; Heath, Jonathan E; Legesse, Teklu; Kim, Anthony J; Woodworth, Graeme F; Tran, Nhan L; Winkles, Jeffrey A

2018-06-01

The TNF receptor superfamily member Fn14 is overexpressed by many solid tumor types, including glioblastoma (GBM), the most common and lethal form of adult brain cancer. GBM is notable for a highly infiltrative growth pattern and several groups have reported that high Fn14 expression levels can increase tumor cell invasiveness. We reported previously that the mesenchymal and proneural GBM transcriptomic subtypes expressed the highest and lowest levels of Fn14 mRNA, respectively. Given the recent histopathological re-classification of human gliomas by the World Health Organization based on isocitrate dehydrogenase 1 (IDH1) gene mutation status, we extended this work by comparing Fn14 gene expression in IDH1 wild-type (WT) and mutant (R132H) gliomas and in cell lines engineered to overexpress the IDH1 R132H enzyme. We found that both low-grade and high-grade (i.e., GBM) IDH1 R132H gliomas exhibit low Fn14 mRNA and protein levels compared to IDH1 WT gliomas. Forced overexpression of the IDH1 R132H protein in glioma cells reduced Fn14 expression, while treatment of IDH1 R132H-overexpressing cells with the IDH1 R132H inhibitor AGI-5198 or the DNA demethylating agent 5-aza-2'-deoxycytidine increased Fn14 expression. These results support a role for Fn14 in the more aggressive and invasive phenotype associated with IDH1 WT tumors and indicate that the low levels of Fn14 gene expression noted in IDH1 R132H mutant gliomas may be due to epigenetic regulation via changes in DNA methylation.

Loss of monocyte chemoattractant protein-1 alters macrophage polarization and reduces NFκB activation in the foreign body response.

PubMed

Moore, Laura Beth; Sawyer, Andrew J; Charokopos, Antonios; Skokos, Eleni A; Kyriakides, Themis R

2015-01-01

Implantation of biomaterials elicits a foreign body response characterized by fusion of macrophages to form foreign body giant cells and fibrotic encapsulation. Studies of the macrophage polarization involved in this response have suggested that alternative (M2) activation is associated with more favorable outcomes. Here we investigated this process in vivo by implanting mixed cellulose ester filters or polydimethylsiloxane disks in the peritoneal cavity of wild-type (WT) and monocyte chemoattractant protein-1 (MCP-1) knockout mice. We analyzed classical (M1) and alternative (M2) gene expression via quantitative polymerase chain reaction, immunohistochemistry and enzyme-linked immunosorbent assay in both non-adherent cells isolated by lavage and implant-adherent cells. Our results show that macrophages undergo unique activation that displays features of both M1 and M2 polarization including induction of tumor necrosis factor α (TNF), which induces the expression and nuclear translocation of p50 and RelA determined by immunofluorescence and Western blot. Both processes were compromised in fusion-deficient MCP-1 KO macrophages in vitro and in vivo. Furthermore, inclusion of BAY 11-7028, an inhibitor of NFκB activation, reduced nuclear translocation of RelA and fusion in WT macrophages. Our studies suggest that peritoneal implants elicit a unique macrophage polarization phenotype leading to induction of TNF and activation of the NFκB pathway. Published by Elsevier Ltd.

Introduction of Pea DNA Helicase 45 Into Sugarcane (Saccharum spp. Hybrid) Enhances Cell Membrane Thermostability And Upregulation Of Stress-responsive Genes Leads To Abiotic Stress Tolerance.

PubMed

Augustine, Sruthy Maria; Ashwin Narayan, J; Syamaladevi, Divya P; Appunu, C; Chakravarthi, M; Ravichandran, V; Tuteja, Narendra; Subramonian, N

2015-05-01

DNA helicases are motor proteins that play an essential role in nucleic acid metabolism, by providing a duplex-unwinding function. To improve the drought and salinity tolerance of sugarcane, a DEAD-box helicase gene isolated from pea with a constitutive promoter, Port Ubi 2.3 was transformed into the commercial sugarcane variety Co 86032 through Agrobacterium-mediated transformation, and the transgenics were screened for tolerance to soil moisture stress and salinity. The transgene integration was confirmed through polymerase chain reaction, and the V 0 transgenic events showed significantly higher cell membrane thermostability under normal irrigated conditions. The V 1 transgenic events were screened for tolerance to soil moisture stress and exhibited significantly higher cell membrane thermostability, transgene expression, relative water content, gas exchange parameters, chlorophyll content, and photosynthetic efficiency under soil moisture stress compared to wild-type (WT). The overexpression of PDH45 transgenic sugarcane also led to the upregulation of DREB2-induced downstream stress-related genes. The transgenic events demonstrated higher germination ability and better chlorophyll retention than WT under salinity stress. Our results suggest the possibility for development of increased abiotic stress tolerant sugarcane cultivars through overexpression of PDH45 gene. Perhaps this is the first report, which provides evidence for increased drought and salinity tolerance in sugarcane through overexpression of PDH45.

SlCOR413IM1: A novel cold-regulation gene from tomato, enhances drought stress tolerance in tobacco.

PubMed

Ma, Xiaocui; Wang, Guodong; Zhao, Weiyang; Yang, Minmin; Ma, Nana; Kong, Fanying; Dong, Xinchun; Meng, Qingwei

2017-09-01

Drought stress adversely affects plant growth, development, and productivity. Genes functioning in plant response to drought stress are essential for drought tolerance. In this study, SlCOR413IM1, a cold-regulated gene isolated from Solanum lycopersium, was transferred to Nicotiana tabacum to investigate its function under drought stress. The subcellular localisation of SlCOR413IM1-GFP fusion protein in Arabidopsis protoplasts suggested that SlCOR413IM1 is a chloroplast protein. Expression analyses revealed that SlCOR413IM1 responded to drought and cold stresses. Under drought stress, transgenic plants maintained the high maximum photochemical efficiency, net photosynthetic rate (Pn) and D1 protein content of photosystem II (PSII). Compared with wild-type (WT) plants, transgenic plants showed higher superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities and proline and soluble sugar content, which reduced reactive oxygen species (ROS) generation. However, the high SOD and APX activities in transgenic plants were independent of their transcription levels. Moreover, the transgenic plants exhibited better seed germination, water status and survival, as well as lower malondialdehyde (MDA) content and relative electrical conductivity (REC) than WT plants under drought stress. Taken together, these data demonstrated that overexpression of SlCOR413IM1 enhanced drought stress tolerance in transgenic tobacco. Copyright © 2017. Published by Elsevier GmbH.

Role of Cbl-PI3K Interaction during Skeletal Remodeling in a Murine Model of Bone Repair.

PubMed

Scanlon, Vanessa; Soung, Do Yu; Adapala, Naga Suresh; Morgan, Elise; Hansen, Marc F; Drissi, Hicham; Sanjay, Archana

2015-01-01

Mice in which Cbl is unable to bind PI3K (YF mice) display increased bone volume due to enhanced bone formation and repressed bone resorption during normal bone homeostasis. We investigated the effects of disrupted Cbl-PI3K interaction on fracture healing to determine whether this interaction has an effect on bone repair. Mid-diaphyseal femoral fractures induced in wild type (WT) and YF mice were temporally evaluated via micro-computed tomography scans, biomechanical testing, histological and histomorphometric analyses. Imaging analyses revealed no change in soft callus formation, increased bony callus formation, and delayed callus remodeling in YF mice compared to WT mice. Histomorphometric analyses showed significantly increased osteoblast surface per bone surface and osteoclast numbers in the calluses of YF fractured mice, as well as increased incorporation of dynamic bone labels. Furthermore, using laser capture micro-dissection of the fracture callus we found that cells lacking Cbl-PI3K interaction have higher expression of Osterix, TRAP, and Cathepsin K. We also found increased expression of genes involved in propagating PI3K signaling in cells isolated from the YF fracture callus, suggesting that the lack of Cbl-PI3K interaction perhaps results in enhanced PI3K signaling, leading to increased bone formation, but delayed remodeling in the healing femora.

The membrane tethered transcription factor EcbZIP17 from finger millet promotes plant growth and enhances tolerance to abiotic stresses.

PubMed

Ramakrishna, Chopperla; Singh, Sonam; Raghavendrarao, Sangala; Padaria, Jasdeep C; Mohanty, Sasmita; Sharma, Tilak Raj; Solanke, Amolkumar U

2018-02-01

The occurrence of various stresses, as the outcome of global climate change, results in the yield losses of crop plants. Prospecting of genes in stress tolerant plant species may help to protect and improve their agronomic performance. Finger millet (Eleusine coracana L.) is a valuable source of superior genes and alleles for stress tolerance. In this study, we isolated a novel endoplasmic reticulum (ER) membrane tethered bZIP transcription factor from finger millet, EcbZIP17. Transgenic tobacco plants overexpressing this gene showed better vegetative growth and seed yield compared with wild type (WT) plants under optimal growth conditions and confirmed upregulation of brassinosteroid signalling genes. Under various abiotic stresses, such as 250 mM NaCl, 10% PEG6000, 400 mM mannitol, water withdrawal, and heat stress, the transgenic plants showed higher germination rate, biomass, primary and secondary root formation, and recovery rate, compared with WT plants. The transgenic plants exposed to an ER stress inducer resulted in greater leaf diameter and plant height as well as higher expression of the ER stress-responsive genes BiP, PDIL, and CRT1. Overall, our results indicated that EcbZIP17 improves plant growth at optimal conditions through brassinosteroid signalling and provide tolerance to various environmental stresses via ER signalling pathways.

Cloning and expression of cyclodextrin glycosyltransferase gene from Paenibacillus sp. T16 isolated from hot spring soil in northern Thailand.

PubMed

Charoensakdi, Ratiya; Murakami, Shuichiro; Aoki, Kenji; Rimphanitchayakit, Vichien; Limpaseni, Tipaporn

2007-05-31

Gene encoding cyclodextrin glycosyltransferase (CGTase), from thermotolerant Paenibacillus sp. T16 isolated from hot spring area in northern Thailand, was cloned and expressed in E. coli (JM109). The nucleotide sequences of both wild type and transformed CGTases consisted of 2139 bp open reading frame, 713 deduced amino acids residues with difference of 4 amino acid residues. The recombinant cells required 24 h culture time and a neutral pH for culture medium to produce compatible amount of CGTase compared to 72 h culture time and pH 10 for wild type. The recombinant and wild-type CGTases were purified by starch adsorption and phenyl sepharose column chromatography and characterized in parallel. Both enzymes showed molecular weight of 77 kDa and similar optimum pHs and temperatures with recombinant enzyme showing broader range. There were some significant difference in pH, temperature stability and kinetic parameters. The presence of high starch concentration resulted in higher thermostability in recombinant enzyme than the wild type. The recombinant enzyme was more stable at higher temperature and lower pH, with lower K(m) for coupling reaction using cellobiose and cyclodextrins as substrates.

Photo- and heterotrophic nitrogenase activity by the cyano-bacterium Nostoc in symbiosis with the bryophyte Anthoceros

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

Steinberg, N.A.; Meeks, J.C.

1987-04-01

In symbiosis with Anthoceros, Nostoc is thought to do little or no photosynthesis. However, light-dependent /sup 14/CO/sub 2/ fixation by symbiotic Nostoc, freshly isolated from pure cultures of the reconstituted Anthoceros-Nostoc association, was 16% of that by free-living Nostoc. A DCMU-resistant mutant of Nostoc was isolated that fixed CO/sub 2/ at rates comparable to wild-type in both symbiotic and free-living growth states. To determine if symbiotic Nostoc can use its photosynthate directly to fix nitrogen, acetylene reduction by Anthoceros associations reconstituted with wild-type Nostoc was compared to associations with the DCMU-resistant mutant. In wild-type Anthoceros-Nostoc acetylene reduction was inhibited 97%more » by 5 ..mu..M DCMU, while inhibition of the DCMU-resistant Nostoc association was only 63%. Additions of glucose, fructose, maltose or sucrose to wild-type associations completely restored DCMU-inhibited acetylene reduction in the light. Acetylene reduction in the dark was stimulated by glucose, attaining 84% of the uninhibited light-dependent value. The authors conclude that symbiotic Nostoc maintains a pool of photosynthate which supports nitrogenase activity. The pool can also be supplemented from plant sources.« less

Use of Murine Bioassay to Resolve Ovine Transmissible Spongiform Encephalopathy Cases Showing a Bovine Spongiform Encephalopathy Molecular Profile

PubMed Central

Beck, Katy E; Sallis, Rosemary E; Lockey, Richard; Vickery, Christopher M; Béringue, Vincent; Laude, Hubert; Holder, Thomas M; Thorne, Leigh; Terry, Linda A; Tout, Anna C; Jayasena, Dhanushka; Griffiths, Peter C; Cawthraw, Saira; Ellis, Richard; Balkema-Buschmann, Anne; Groschup, Martin H; Simmons, Marion M; Spiropoulos, John

2012-01-01

Two cases of unusual transmissible spongiform encephalopathy (TSE) were diagnosed on the same farm in ARQ/ARQ PrP sheep showing attributes of both bovine spongiform encephalopathy (BSE) and scrapie. These cases, UK-1 and UK-2, were investigated further by transmissions to wild-type and ovine transgenic mice. Lesion profiles (LP) on primary isolation and subpassage, incubation period (IP) of disease, PrPSc immunohistochemical (IHC) deposition pattern and Western blot profiles were used to characterize the prions causing disease in these sheep. Results showed that both cases were compatible with scrapie. The presence of BSE was contraindicated by the following: LP on primary isolation in RIII and/or MR (modified RIII) mice; IP and LP after serial passage in wild-type mice; PrPSc deposition pattern in wild-type mice; and IP and Western blot data in transgenic mice. Furthermore, immunohistochemistry (IHC) revealed that each case generated two distinct PrPSc deposition patterns in both wild-type and transgenic mice, suggesting that two scrapie strains coexisted in the ovine hosts. Critically, these data confirmed the original differential IHC categorization that these UK-1 and UK-2 cases were not compatible with BSE. PMID:21919992

Comparison of the hepatic and thyroid gland effects of sodium phenobarbital in wild type and constitutive androstane receptor (CAR) knockout rats and pregnenolone-16α-carbonitrile in wild type and pregnane X receptor (PXR) knockout rats.

PubMed

Haines, Corinne; Chatham, Lynsey R; Vardy, Audrey; Elcombe, Clifford R; Foster, John R; Lake, Brian G

2018-05-01

A number of chemicals produce liver and thyroid gland tumours in rodents by nongenotoxic modes of action (MOAs). In this study the hepatic and thyroid gland effects of the constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) were examined in male Sprague-Dawley wild type (WT) rats and in CAR knockout (CAR KO) rats and the effects of the pregnane X receptor (PXR) activator pregnenolone-16α-carbonitrile (PCN) were examined in WT and PXR knockout (PXR KO) rats. Rats were either fed diets containing 0 (control) or 500 ppm NaPB or were dosed with 0 (control) or 100 mg/kg/day PCN orally for 7 days. The treatment of WT rats with NaPB and PCN for 7 days resulted in increased relative liver weight, increased hepatocyte replicative DNA synthesis (RDS) and the induction of cytochrome P450 CYP2B and CYP3A subfamily enzyme, mRNA and protein levels. In marked contrast, the treatment of CAR KO rats with NaPB and PXR KO rats with PCN did not result in any increases in liver weight and induction of CYP2B and CYP3A enzymes. The treatment of CAR KO rats with NaPB had no effect on hepatocyte RDS, while PCN produced only a small increase in hepatocyte RDS in PXR KO rats. Treatment with NaPB had no effect on thyroid gland weight in WT and CAR KO rats, whereas treatment with PCN resulted in an increase in relative thyroid gland weight in WT, but not in PXR KO, rats. Thyroid gland follicular cell RDS was increased by the treatment of WT rats with NaPB and PCN, with NaPB also producing a small increase in thyroid gland follicular cell RDS in CAR KO rats. Overall, the present study with CAR KO rats demonstrates that a functional CAR is required for NaPB-mediated increases in liver weight, stimulation of hepatocyte RDS and induction of hepatic CYP enzymes. The studies with PXR KO rats demonstrate that a functional PXR is required for PCN-mediated increases in liver weight and induction of hepatic CYP enzymes; with induction of hepatocyte RDS also being largely mediated through PXR. The hepatic effects of NaPB in CAR KO rats and of PCN in PXR KO rats are in agreement with those observed in other recent literature studies. These results suggest that CAR KO and PXR KO rats are useful experimental models for liver MOA studies with rodent CAR and PXR activators and may also be useful for thyroid gland MOA studies. Copyright © 2018 Elsevier B.V. All rights reserved.

PHEX Mimetic (SPR4-Peptide) Corrects and Improves HYP and Wild Type Mice Energy-Metabolism

PubMed Central

Zelenchuk, Lesya V.; Hedge, Anne-Marie; Rowe, Peter S. N.

2014-01-01

Context PHEX or DMP1 mutations cause hypophosphatemic-rickets and altered energy metabolism. PHEX binds to DMP1-ASARM-motif to form a complex with α5β3 integrin that suppresses FGF23 expression. ASARM-peptides increase FGF23 by disrupting the PHEX-DMP1-Integrin complex. We used a 4.2 kDa peptide (SPR4) that binds to ASARM-peptide/motif to study the DMP1-PHEX interaction and to assess SPR4 for the treatment of energy metabolism defects in HYP and potentially other bone-mineral disorders. Design Subcutaneously transplanted osmotic pumps were used to infuse SPR4-peptide or vehicle (VE) into wild-type mice (WT) and HYP-mice (PHEX mutation) for 4 weeks. Results SPR4 partially corrected HYP mice hypophosphatemia and increased serum 1.25(OH)2D3. Serum FGF23 remained high and PTH was unaffected. WT-SPR4 mice developed hypophosphatemia and hypercalcemia with increased PTH, FGF23 and 1.25(OH)2D3. SPR4 increased GAPDH HYP-bone expression 60× and corrected HYP-mice hyperglycemia and hypoinsulinemia. HYP-VE serum uric-acid (UA) levels were reduced and SPR4 infusion suppressed UA levels in WT-mice but not HYP-mice. SPR4 altered leptin, adiponectin, and sympathetic-tone and increased the fat mass/weight ratio for HYP and WT mice. Expression of perlipin-2 a gene involved in obesity was reduced in HYP-VE and WT-SPR4 mice but increased in HYP-SPR4 mice. Also, increased expression of two genes that inhibit insulin-signaling, ENPP1 and ESP, occurred with HYP-VE mice. In contrast, SPR4 reduced expression of both ENPP1 and ESP in WT mice and suppressed ENPP1 in HYP mice. Increased expression of FAM20C and sclerostin occurred with HYP-VE mice. SPR4 suppressed expression of FAM20C and sclerostin in HYP and WT mice. Conclusions ASARM peptides and motifs are physiological substrates for PHEX and modulate osteocyte PHEX-DMP1-α5β3-integrin interactions and thereby FGF23 expression. These interactions also provide a nexus that regulates bone and energy metabolism. SPR4 suppression of sclerostin and/or sequestration of ASARM-peptides improves energy metabolism and may have utility for treating familial rickets, osteoporosis, obesity and diabetes. PMID:24839967

Laboratory and field evaluations for efficacy of a fast-killing baculovirus isolate from Spodoptera frugiperda

USDA-ARS?s Scientific Manuscript database

Three biopesticide parameters were evaluated for a fast-killing isolate (3AP2) Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) and a wild-type isolate (Sf3) of the same baculovirus. Both isolates were evaluated for virus production using in vivo methods, for speed of kill based on bioas...

Genetic conversion of a fungal plant pathogen to a non-pathogenic, endophytic mutualist

USGS Publications Warehouse

Freeman, Stanley; Rodriguez, Rusty J.

1993-01-01

The filamentous fungal ascomycete Colletotrichum magna causes anthracnose in cucurbit plants. Isolation of a nonpathogenic mutant of this species (path-1) resulted in maintained wild-type levels of in vitro sporulation, spore adhesion, appressorial formation, and infection. Path-1 grew throughout host tissues as an endophyte and retained the wild-type host range, which indicates that the genetics involved in pathogenicity and host specificity are distinct. Prior infection with path-1 protected plants from disease caused by Colletotrichum and Fusarium.Genetic analysis of a cross between path-1 and wild-type strains indicated mutation of a single locus.

Overexpression of Thioredoxin in Transgenic Mice Attenuates Focal Ischemic Brain Damage

NASA Astrophysics Data System (ADS)

Takagi, Yasushi; Mitsui, Akira; Nishiyama, Akira; Nozaki, Kazuhiko; Sono, Hiroshi; Gon, Yasuhiro; Hashimoto, Nobuo; Yodoi, Junji

1999-03-01

Thioredoxin (TRX) plays important biological roles both in intra- and extracellular compartments, including in regulation of various intracellular molecules via thiol redox control. We produced TRX overexpressing mice and confirmed that there were no anatomical and physiological differences between wild-type (WT) mice and TRX transgenic (Tg) mice. In the present study we subjected mice to focal brain ischemia to shed light on the role of TRX in brain ischemic injury. At 24 hr after middle cerebral artery occlusion, infarct areas and volume were significantly smaller in Tg mice than in WT mice. Moreover neurological deficit was ameliorated in Tg mice compared with WT mice. Protein carbonyl content, a marker of cellular protein oxidation, in Tg mice showed less increase than did that of WT mice after the ischemic insult. Furthermore, c-fos expression in Tg mice was stronger than in WT mice 1 hr after ischemia. Our results suggest that transgene expression of TRX decreased ischemic neuronal injury and that TRX and the redox state modified by TRX play a crucial role in brain damage during stroke.

Respiration accumulates Calvin cycle intermediates for the rapid start of photosynthesis in Synechocystis sp. PCC 6803.

PubMed

Shimakawa, Ginga; Hasunuma, Tomohisa; Kondo, Akihiko; Matsuda, Mami; Makino, Amane; Miyake, Chikahiro

2014-01-01

We tested the hypothesis that inducing photosynthesis in cyanobacteria requires respiration. A mutant deficient in glycogen phosphorylase (∆GlgP) was prepared in Synechocystis sp. PCC 6803 to suppress respiration. The accumulated glycogen in ΔGlgP was 250-450% of that accumulated in wild type (WT). The rate of dark respiration in ΔGlgP was 25% of that in WT. In the dark, P700(+) reduction was suppressed in ΔGlgP, and the rate corresponded to that in (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone)-treated WT, supporting a lower respiration rate in ∆GlgP. Photosynthetic O2-evolution rate reached a steady-state value much slower in ∆GlgP than in WT. This retardation was solved by addition of d-glucose. Furthermore, we found that the contents of Calvin cycle intermediates in ∆GlgP were lower than those in WT under dark conditions. These observations indicated that respiration provided the carbon source for regeneration of ribulose 1,5-bisphosphate in order to drive the rapid start of photosynthesis.

Molecular Evolution of a Type 1 Wild-Vaccine Poliovirus Recombinant during Widespread Circulation in China

PubMed Central

Liu, Hong-Mei; Zheng, Du-Ping; Zhang, Li-Bi; Oberste, M. Steven; Pallansch, Mark A.; Kew, Olen M.

2000-01-01

Type 1 wild-vaccine recombinant polioviruses were isolated from poliomyelitis patients in China from 1991 to 1993. We compared the sequences of 34 recombinant isolates over the 1,353-nucleotide (nt) genomic interval (nt 2480 to 3832) encoding the major capsid protein, VP1, and the protease, 2A. All recombinants had a 367-nt block of sequence (nt 3271 to 3637) derived from the Sabin 1 oral poliovirus vaccine strain spanning the 3′-terminal sequences of VP1 (115 nt) and the 5′ half of 2A (252 nt). The remaining VP1 sequences were closely (up to 99.5%) related to those of a major genotype of wild type 1 poliovirus endemic to China up to 1994. In contrast, the non-vaccine-derived sequences at the 3′ half of 2A were more distantly related (<90% nucleotide sequence match) to those of other contemporary wild polioviruses from China. The vaccine-derived sequences of the earliest (April 1991) isolates completely matched those of Sabin 1. Later isolates diverged from the early isolates primarily by accumulation of synonymous base substitutions (at a rate of ∼3.7 × 10−2 substitutions per synonymous site per year) over the entire VP1-2A interval. Distinct evolutionary lineages were found in different Chinese provinces. From the combined epidemiologic and evolutionary analyses, we propose that the recombinant virus arose during mixed infection of a single individual in northern China in early 1991 and that its progeny spread by multiple independent chains of transmission into some of the most populous areas of China within a year of the initiating infection. PMID:11070012

Molecular Characterization of a Functional Type VI Secretion System from a Clinical Isolate of Aeromonas hydrophila

PubMed Central

Suarez, Giovanni; Sierra, Johanna C.; Sha, Jian; Wang, Shaofei; Erova, Tatiana E.; Fadl, Amin A.; Foltz, Sheri M.; Horneman, Amy J.; Chopra, Ashok K.

2008-01-01

Our laboratory recently molecularly characterized the type II secretion system (T2SS)- associated cytotoxic enterotoxin (Act) and the T3SS-secreted AexU effector from a diarrheal isolate SSU of Aeromonas hydrophila. The role of these toxin proteins in the pathogenesis of A. hydrophila infections was subsequently delineated in in vitro and in vivo models. In this study, we characterized the new type 6 secretion system (T6SS) from isolate SSU of A. hydrophila and demonstrated its role in bacterial virulence. Study of the role of T6SS in bacterial virulence is in its infancy, and there are, accordingly, only limited, recent reports directed toward a better understanding its role in bacterial pathogenesis. We have provided evidence that the virulence-associated secretion (vas) genes vasH (Sigma 54-dependent transcriptional regulator) and vasK (encoding protein of unknown function) are essential for expression of the genes encoding the T6SS and/or they constituted important components of the T6SS. Deletion of the vasH gene prevented expression of the potential translocon hemolysin coregulated protein (Hcp) encoding gene from bacteria, while the vasK gene deletion prevented secretion but not translocation of Hcp into host cells. The secretion of Hcp was independent of the T3SS and the flagellar system. We demonstrated that secreted Hcp could bind to the murine RAW 264.7 macrophages from outside, in addition to its ability to be translocated into host cells. Further, the vasH and vasK mutants were less toxic to murine macrophages and human epithelial HeLa cells, and these mutants were more efficiently phagocytosed by macrophages. We also provided evidence that the expression of the hcp gene in the HeLa cell resulted in apoptosis of the host cells. Finally, the vasH and vasK mutants of A. hydrophila were less virulent in a septicemic mouse model of infection, and animals immunized with recombinant Hcp were protected from subsequent challenge with the wild-type (WT) bacterium. In addition, mice infected with the WT A. hydrophila had circulating antibodies to Hcp, indicating an important role of T6SS in the pathogenesis of A. hydrophila infections. Taken together, we have characterized the T6SS from Aeromonas for the first time and provided new features of this secretion system not yet known for other pathogens. PMID:18037263

CaV3.1 isoform of T-type calcium channels supports excitability of rat and mouse ventral tegmental area neurons.

PubMed

Tracy, Matthew E; Tesic, Vesna; Stamenic, Tamara Timic; Joksimovic, Srdjan M; Busquet, Nicolas; Jevtovic-Todorovic, Vesna; Todorovic, Slobodan M

2018-03-23

Recent data have implicated voltage-gated calcium channels in the regulation of the excitability of neurons within the mesolimbic reward system. While the attention of most research has centered on high voltage L-type calcium channel activity, the presence and role of the low voltage-gated T-type calcium channel (T-channels) has not been well explored. Hence, we investigated T-channel properties in the neurons of the ventral tegmental area (VTA) utilizing wild-type (WT) rats and mice, Ca V 3.1 knock-out (KO) mice, and TH-eGFP knock-in (KI) rats in acute horizontal brain slices of adolescent animals. In voltage-clamp experiments, we first assessed T-channel activity in WT rats with characteristic properties of voltage-dependent activation and inactivation, as well as characteristic crisscrossing patterns of macroscopic current kinetics. T-current kinetics were similar in WT mice and WT rats but T-currents were abolished in Ca V 3.1 KO mice. In ensuing current-clamp experiments, we observed the presence of hyperpolarization-induced rebound burst firing in a subset of neurons in WT rats, as well as dopaminergic and non-dopaminergic neurons in TH-eGFP KI rats. Following the application of a pan-selective T-channel blocker TTA-P2, rebound bursting was significantly inhibited in all tested cells. In a behavioral assessment, the acute locomotor increase induced by a MK-801 (Dizocilpine) injection in WT mice was abolished in Ca V 3.1 KO mice, suggesting a tangible role for 3.1 T-type channels in drug response. We conclude that pharmacological targeting of Ca V 3.1 isoform of T-channels may be a novel approach for the treatment of disorders of mesolimbic reward system. Copyright © 2018. Published by Elsevier Ltd.

IL-4 Knock out Mice Display Anxiety-like Behavior

PubMed Central

Moon, Morgan L.; Joesting, Jennifer J.; Blevins, Neil A.; Lawson, Marcus A.; Gainey, Stephen J.; Towers, Albert E.; McNeil, Leslie K.; Freund, Gregory G.

2015-01-01

Inflammation is a recognized antecedent and coincident factor when examining the biology of anxiety. Little is known, however, about how reductions in endogenous anti-inflammatory mediators impact anxiety. Therefore, mood- cognition- and anxiety-associated/like behaviors were examined in IL-4 knock out (KO) mice and wild-type (WT) mice. In comparison to WT mice, IL-4 KO mice demonstrated decreased burrowing and increased social exploration. No differences were seen in forced swim or saccharine preference testing. IL-4 KO mice had similar performance to WT mice in the Morris water maze and during object location and novel object recognition. In the elevated zero-maze, IL-4 KO mice, in comparison to WT mice, demonstrated anxiety-like behavior. Anxiety-like behavior in IL-4 KO mice was not observed, however, during open-field testing. Taken together, these data indicate that IL-4 KO mice display state, but not trait, anxiety suggesting that reductions in endogenous anti-inflammatory bioactives can engender subtypes of anxiety. PMID:25772794

IL-4 Knock Out Mice Display Anxiety-Like Behavior.

PubMed

Moon, Morgan L; Joesting, Jennifer J; Blevins, Neil A; Lawson, Marcus A; Gainey, Stephen J; Towers, Albert E; McNeil, Leslie K; Freund, Gregory G

2015-07-01

Inflammation is a recognized antecedent and coincident factor when examining the biology of anxiety. Little is known, however, about how reductions in endogenous anti-inflammatory mediators impact anxiety. Therefore, mood- cognition- and anxiety-associated/like behaviors were examined in IL-4 knock out (KO) mice and wild-type (WT) mice. In comparison to WT mice, IL-4 KO mice demonstrated decreased burrowing and increased social exploration. No differences were seen in forced swim or saccharine preference testing. IL-4 KO mice had similar performance to WT mice in the Morris water maze and during object location and novel object recognition. In the elevated zero-maze, IL-4 KO mice, in comparison to WT mice, demonstrated anxiety-like behavior. Anxiety-like behavior in IL-4 KO mice was not observed, however, during open-field testing. Taken together, these data indicate that IL-4 KO mice display state, but not trait, anxiety suggesting that reductions in endogenous anti-inflammatory bioactives can engender subtypes of anxiety.

Evaluation of the behavioral characteristics of the mdx mouse model of duchenne muscular dystrophy through operant conditioning procedures.

PubMed

Lewon, Matthew; Peters, Christina M; Van Ry, Pam M; Burkin, Dean J; Hunter, Kenneth W; Hayes, Linda J

2017-09-01

The mdx mouse is an important nonhuman model for Duchenne muscular dystrophy (DMD) research. Characterizing the behavioral traits of the strain relative to congenic wild-type (WT) mice may enhance our understanding of the cognitive deficits observed in some humans with DMD and contribute to treatment development and evaluation. In this paper we report the results of a number of experiments comparing the behavior of mdx to WT mice in operant conditioning procedures designed to assess learning and memory. We found that mdx outperformed WT in all learning and memory tasks involving food reinforcement, and this appeared to be related to the differential effects of the food deprivation motivating operation on mdx mice. Conversely, WT outperformed mdx in an escape/avoidance learning task. These results suggest motivational differences between the strains and demonstrate the potential utility of operant conditioning procedures in the assessment of the behavioral characteristics of the mdx mouse. Copyright © 2017 Elsevier B.V. All rights reserved.

Discovery of wt RET and V804M RET Inhibitors: From Hit to Lead.

PubMed

Mologni, Luca; Dalla Via, Martina; Chilin, Adriana; Palumbo, Manlio; Marzaro, Giovanni

2017-08-22

Oncogenic activation of RET kinase has been found in several neoplastic diseases, like medullary thyroid carcinoma, multiple endocrine neoplasia, papillary thyroid carcinoma, and non-small-cell lung cancer. Currently approved RET inhibitors were not originally designed to be RET inhibitors, and their potency against RET kinase has not been optimized. Hence, novel compounds able to inhibit both wild-type RET ( wt RET) and its mutants (e.g., V804M RET) are needed. Herein we present the development and the preliminary evaluation of a new sub-micromolar wt RET/ V804M RET inhibitor, N-(2-fluoro-5-trifluoromethylphenyl)-N'-{4'-[(2''-benzamido)pyridin-4''-ylamino]phenyl}urea (69), endowed with a 4-anilinopyridine structure, starting from our previously identified 4-anilinopyrimidine hit compound. Profiling against a panel of kinases indicated 69 as a multi cKIT/ wt RET/ V804M RET inhibitor. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determination of Isavuconazole Susceptibility of Aspergillus and Candida Species by the EUCAST Method

PubMed Central

Howard, Susan J.; Lass-Flörl, Cornelia; Cuenca-Estrella, Manuel; Gomez-Lopez, Alicia

2013-01-01

Isavuconazole is a novel expanded-spectrum triazole, which has recently been approved by the FDA as an orphan drug to treat invasive aspergillosis and is currently being studied in phase III clinical trials for invasive candidiasis. The susceptibility of relatively few clinical isolates has been reported. In this study, the isavuconazole susceptibilities of 1,237 Aspergillus and 2,010 Candida geographically diverse clinical isolates were determined by EUCAST methodology at four European mycology laboratories, producing the largest multicenter data set thus far for this compound. In addition, a blinded collection of 30 cyp51A mutant Aspergillus fumigatus clinical isolates and 10 wild-type isolates was tested. From these two data sets, the following preliminary epidemiological cutoff (ECOFF) values were suggested: 2 mg/liter for Aspergillus fumigatus, Aspergillus terreus, and Aspergillus flavus; 4 mg/liter for Aspergillus niger; 0.25 mg/liter for Aspergillus nidulans; and 0.03 mg/liter for Candida albicans, Candida parapsilosis, and Candida tropicalis. Unfortunately, ECOFFs could not be determined for Candida glabrata or Candida krusei due to an unexplained interlaboratory MIC variation. For the blinded collection of A. fumigatus isolates, all MICs were ≤2 mg/liter for wild-type isolates. Differential isavuconazole MICs were observed for triazole-resistant A. fumigatus isolates with different cyp51A alterations: TR34/L98H mutants had elevated isavuconazole MICs, whereas isolates with G54 and M220 alterations had MICs in the wild-type range, suggesting that the efficacy of isavuconazole may not be affected by these alterations. This study will be an aid in interpreting isavuconazole MICs for clinical care and an important step in the future process of setting official clinical breakpoints. PMID:23959309

Renoprotective impact of estrogen receptor α and its splice variants in female mice with type 1 diabetes.

PubMed

Irsik, Debra L; Romero-Aleshire, Melissa Jill; Chavez, Erin M; Fallet, Rachel W; Brooks, Heddwen L; Carmines, Pamela K; Lane, Pascale H

2018-04-18

Estrogen has been implicated in the regulation of growth and immune function in the kidney, which expresses the full-length estrogen receptor α (ERα66), its ERα splice variants, and estrogen receptor β (ERβ). Thus, we hypothesized that these splice variants may inhibit glomerular enlargement that occurs early in type 1 diabetes (T1D). T1D was induced by streptozotocin (STZ) injection in 8-12 wk-old female mice lacking ERα66 (ERα66KO) or all ERα variants (αERKO), and their wild-type (WT) littermates. Basal renal ERα36 protein expression was reduced in the ERα66KO model and was downregulated by T1D in WT mice. T1D did not alter ERα46 or ERβ in WT-STZ; however, ERα46 was decreased modestly in ERα66KO. Renal hypertrophy was evident in all diabetic mice. F4/80-positive immunostaining was reduced in ERα66KO, compared with WT and αERKO mice, but was higher in STZ than in WT mice across all genotypes. Glomerular area was greater in WT and αERKO than in ERα66KO mice, with T1D-induced glomerular enlargement apparent in WT-STZ and αERKO-STZ, but not in ERα66KO-STZ. Proteinuria and hyperfiltration were evident in ERα66KO-STZ and αERKO-STZ, but not in WT-STZ mice. These data indicate that ERα splice variants may exert an inhibitory influence on glomerular enlargement and macrophage infiltration during T1D; however, effects of splice variants are masked in the presence of the full-length ERα66, suggesting that ERα66 acts in opposition to its splice variants to influence these parameters. In contrast, hyperfiltration and proteinuria in T1D are attenuated via an ERα66-dependent mechanism that is unaffected by splice variant status.

Maximizing PTH Anabolic Osteoporosis Therapy. Revision

DTIC Science & Technology

2016-09-01

Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 DISTRIBUTION STATEMENT: Approved for Public Release; Distribution Unlimited...SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S) U.S. Army Medical Research and Materiel Command Fort Detrick...mono-therapy and PTH+ anti-catabolic combination therapies on Nmp4-/- and wild type (WT) mice. The scope of the research comprises the following

Compatibility of a wild type and its genetically modified Sinorhizobium strain with two mycorrhizal fungi on Medicago species as affected by drought stress.

PubMed

Vázquez, M M.; Azcón, R; Barea, J M.

2001-07-01

The effect of double inoculation with two strains of Sinorhizobium meliloti [the wild type (WT) strain GR4 and its genetically modified (GM) derivative GR4(pCK3)], and two species of arbuscular mycorrhizal (AM) fungi (Glomus deserticola and Glomus intraradices) was examined in a microcosm system on three species of Medicago (M. nolana, M. rigidula, M. rotata). Two water regimes (80 and 100% water holding capacity, WHC) were assayed. The efficiency of each AM fungus increasing plant growth, nutrient content, nodulation and water-stress tolerance was related to the Sinorhizobium strains and Medicago species. This indicates selective and specific compatibilities between microsymbionts and the common host plant. Differential effects of the mycorrhizal isolates were not associated with their colonizing ability. Nodulation and mycorrhizal dependency (MD) changed in each plant genotype in accordance with the Sinorhizobium strain and AM fungi involved. Generally, Medicago sp. MD decreased under water-stress conditions even when these conditions did not affect AM colonization (%). Proline accumulation in non-mycorrhizal plant leaves was increased by water stress, except in M. rotata plants. Differences in proline accumulation in AM-colonized plants suggest that both the AM fungus and the Sinorhizobium strain were able to induce different degrees of osmotic adjustment. Mycorrhizal plants nodulated by the WT strain accumulated more proline in M. rigidula and M. rotata under water stress than non-mycorrhizal plants. Conversely, mycorrhizal plants nodulated by the GM strain accumulated less proline in response to both AM colonization and drought. These results indicated changes in the synthesis of this nitrogenous osmoregulator product associated with microbial inoculation and drought tolerance. Mycorrhizal plants nodulated by the GM Sinorhizobium strain seem to suffer less from the detrimental effect of water stress, since under water limitation relative plant growth, percentage of AM colonization, root dry weight and the highest R/S ratio remained the same. The fact that GM nodulated plants are better adapted to drought stress could be of practical interest and the management of GM microorganism inoculation may be crucial for biotechnological approaches to improving crop yield in dry environments.

Type II Kinase Inhibitors Show an Unexpected Inhibition Mode against Parkinson’s Disease-Linked LRRK2 Mutant G2019S

PubMed Central

Liu, Min; Bender, Samantha A.; Cuny, Gregory D; Sherman, Woody; Glicksman, Marcie; Ray, Soumya S.

2014-01-01

A number of well-known type II inhibitors (ATP non-competitive) that bind kinases in their DFG-out conformation were tested against wild-type LRRK2 and the most common Parkinson’s disease-linked mutation G2019S. We found that traditional type II inhibitors exhibit surprising variability in their inhibition mechanism between wild type (WT) and the G2019S mutant of LRRK2. The type II kinase inhibitors were found to work by an ATP-competitive fashion against the G2019S mutant, whereas they appear to follow the expected non-competitive mechanism against WT. Since the G2019S mutation lies in the DXG-motif (DYG in LRRK2 but DFG in most other kinases) of the activation loop, we explored the structural consequence of the mutation on loop dynamics using an enhanced sampling method called metadynamics. The simulations suggest that the G2019S mutation stabilizes the DYG-in state of LRRK2 through a series of hydrogen bonds, leading to an increase in the conformational barrier between the active and inactive forms of the enzyme and a relative stabilization of the active form. The conformational bias toward the active form of LRRK2 mutants has two primary consequences: 1) the mutant enzyme becomes hyperactive, a known contributor to the Parkinsonian phenotype, as a consequence of being “locked” into the activated state and 2) the mutation creates an unusual allosteric pocket that can bind type II inhibitors but in an ATP competitive fashion. Our results suggest that developing type II inhibitors, which are generally considered superior to type I inhibitors due to desirable selectivity profiles, might be especially challenging for the G2019S LRRK2 mutant. PMID:23379419

Fine Mapping of a Gene (ER4.1) that Causes Epidermal Reticulation of Tomato Fruit and Characterization of the Associated Transcriptome

PubMed Central

Cui, Lipeng; Qiu, Zhengkun; Wang, Zhirong; Gao, Jianchang; Guo, Yanmei; Huang, Zejun; Du, Yongchen; Wang, Xiaoxuan

2017-01-01

The hydrophobic cuticle that covers the surface of tomato (Solanum lycopersicum) fruit plays key roles in development and protection against biotic and abiotic stresses, including water loss, mechanical damage, UV radiation, pathogens, and pests. However, many details of the genes and regulatory mechanisms involved in cuticle biosynthesis in fleshy fruits are not well understood. In this study, we describe a novel tomato fruit phenotype, characterized by epidermal reticulation (ER) of green fruit and a higher water loss rate than wild type (WT) fruit. The ER phenotype is controlled by a single gene, ER4.1, derived from an introgressed chromosomal segment from the wild tomato species S. pennellii (LA0716). We performed fine mapping of the single dominant gene to an ~300 kb region and identified Solyc04g082540, Solyc04g082950, Solyc04g082630, and Solyc04g082910as potential candidate genes for the ER4.1 locus, based on comparative RNA-seq analysis of ER and WT fruit peels. In addition, the transcriptome analysis revealed that the expression levels of genes involved in cutin, wax and flavonoid biosynthesis were altered in the ER fruit compared with WT. This study provides new insights into the regulatory mechanisms and metabolism of the fruit cuticle. PMID:28798753

Isolation of Mycobacterium bovis from Free-Ranging Wildlife in South Korea.

PubMed

Jang, Yunho; Ryoo, Soyoon; Lee, Hyunkyoung; Kim, Narae; Lee, Hang; Park, So-Young; Song, Woong-Seog; Kim, Jong-Taek; Lee, Hee Soo; Myung Kim, Jae

2017-01-01

We demonstrate Mycobacterium bovis infection in wild boar ( Sus scrofa ) in South Korea. During 2012-15, we attempted to isolate M. bovis from 847 wild animals, mainly Korean water deer ( Hydropotes inermis argyropus), raccoon dogs ( Nyctereutes procyonoides ), and wild boar, from 11 regions in South Korea. We isolated M. bovis from three of 118 wild boar (2.5%) captured in Gyeonggi Province, where bovine tuberculosis (bTB) outbreaks have also occurred in livestock. Spoligotypes and mycobacterial interspersed repetitive units-variable number tandem repeats types of these M. bovis isolates (SB0140 and SB1040, 4-2-3-3-7-5-5-4-4-3-4-3 and 5-2-3-3-7-5-5-4-3-10-5-2; MIRU4, MIRU16, MIRU27, MIRU31, ETR-A, ETR-B, ETR-C, QUB11b, QUB26, QUB3336, VNTR2401, and VNTR3171) have also been identified from farmed livestock such as cattle ( Bos taurus coreanae), Formosan sika deer ( Cervus nippon taiouanus), and American elk ( Cervus canadensis ) in the country. In South Korea, bTB appears to be endemic in livestock, and there are numerous opportunities for contact between wild boar and livestock due to high population densities and broad activity ranges. Our results support the hypothesis that M. bovis is transmitted between domestic and wild animals.

Molecular characterization of wild-type polioviruses isolated in Greece during the 1996 outbreak in Albania.

PubMed

Kyriakopoulou, Zaharoula; Kottaridi, Christine; Dedepsidis, Evaggelos; Bolanaki, Eugenia; Levidiotou-Stefanou, Stamatina; Markoulatos, Panayotis

2006-03-01

During the present study three type 1 poliovirus strains isolated in Greece during the 1996 poliomyelitis outbreak in Albania were retrospectively investigated and determination of their relationship with other epidemic strains isolated in Albania or elsewhere during previous epidemics was attempted. SimPlot analysis revealed that the three Greek strains are the result of a recombination event in the VP2 coding region.

Macrophage migration inhibitory factor (MIF) knockout preserves cardiac homeostasis through alleviating Akt-mediated myocardial autophagy suppression in high-fat diet-induced obesity.

PubMed

Xu, X; Ren, J

2015-03-01

Macrophage migration inhibitory factor (MIF) has a role in the development of obesity and diabetes. However, whether MIF has a role in fat diet-induced obesity and associated cardiac anomalies still remains unknown. The aim of this study was to examine the impact of MIF knockout on high-fat diet-induced obesity, obesity-associated cardiac anomalies and the underlying mechanisms involved with a focus on Akt-mediated autophagy. Adult male wild-type (WT) and MIF knockout (MIF(-/-)) mice were placed on 45% high-fat diet for 5 months. Oxygen consumption, CO2 production, respiratory exchange ratio, locomotor activity and heat generation were measured using energy calorimeter. Echocardiographic, cardiomyocyte mechanical and intracellular Ca2+ properties were assessed. Apoptosis was examined using terminal dUTP nick end labeling staining and western blot analysis. Akt signaling pathway and autophagy markers were evaluated. Cardiomyocytes isolated from WT and MIF(-/-) mice were treated with recombinant mouse MIF (rmMIF). High-fat diet feeding elicited increased body weight gain, insulin resistance and caloric disturbance in WT and MIF(-/-) mice. High-fat diet induced unfavorable geometric, contractile and histological changes in the heart, the effects of which were alleviated by MIF knockout. In addition, fat diet-induced cardiac anomalies were associated with Akt activation and autophagy suppression, which were nullified by MIF deficiency. In cardiomyocytes from WT mice, autophagy was inhibited by exogenous rmMIF through Akt activation. In addition, MIF knockout rescued palmitic acid-induced suppression of cardiomyocyte autophagy, the effect of which was nullified by rmMIF. These results indicate that MIF knockout preserved obesity-associated cardiac anomalies without affecting fat diet-induced obesity, probably through restoring myocardial autophagy in an Akt-dependent manner. Our findings provide new insights for the role of MIF in obesity and associated cardiac anomalies.

Depressed Frank-Starling mechanism in the left ventricular muscle of the knock-in mouse model of dilated cardiomyopathy with troponin T deletion mutation ΔK210.

PubMed

Inoue, Takahiro; Kobirumaki-Shimozawa, Fuyu; Kagemoto, Tatsuya; Fujii, Teruyuki; Terui, Takako; Kusakari, Yoichiro; Hongo, Kenichi; Morimoto, Sachio; Ohtsuki, Iwao; Hashimoto, Kazuhiro; Fukuda, Norio

2013-10-01

It has been reported that the Frank-Starling mechanism is coordinately regulated in cardiac muscle via thin filament "on-off" equilibrium and titin-based lattice spacing changes. In the present study, we tested the hypothesis that the deletion mutation ΔK210 in the cardiac troponin T gene shifts the equilibrium toward the "off" state and accordingly attenuate the sarcomere length (SL) dependence of active force production, via reduced cross-bridge formation. Confocal imaging in isolated hearts revealed that the cardiomyocytes were enlarged, especially in the longitudinal direction, in ΔK210 hearts, with striation patterns similar to those in wild type (WT) hearts, suggesting that the number of sarcomeres is increased in cardiomyocytes but the sarcomere length remains unaltered. For analysis of the SL dependence of active force, skinned muscle preparations were obtained from the left ventricle of WT and knock-in (ΔK210) mice. An increase in SL from 1.90 to 2.20μm shifted the mid-point (pCa50) of the force-pCa curve leftward by ~0.21pCa units in WT preparations. In ΔK210 muscles, Ca(2+) sensitivity was lower by ~0.37pCa units, and the SL-dependent shift of pCa50, i.e., ΔpCa50, was less pronounced (~0.11pCa units), with and without protein kinase A treatment. The rate of active force redevelopment was lower in ΔK210 preparations than in WT preparations, showing blunted thin filament cooperative activation. An increase in thin filament cooperative activation upon an increase in the fraction of strongly bound cross-bridges by MgADP increased ΔpCa50 to ~0.21pCa units. The depressed Frank-Starling mechanism in ΔK210 hearts is the result of a reduction in thin filament cooperative activation. © 2013.

Sterol O-acyltransferase 1 deficiency improves defective insulin signaling in the brains of mice fed a high-fat diet.

PubMed

Xu, Ning; Meng, Hao; Liu, Tian-Yi; Feng, Ying-Li; Qi, Yuan; Zhang, Dong-Huan; Wang, Hong-Lei

2018-05-05

Insulin resistance induced by a high-fat diet (HFD) is related to metabolic diseases, and sterol O-acyltransferase 1 (SOAT1) is a key enzyme for the biosynthesis of cholesteryl ester. In the present study, wild-type (WT) mice and SOAT1-knockout (KO) mice with a C57BL6 background fed a HFD were used to explore the role of SOAT1 in the hypothalamus. The results show that the WT mice exhibited a significant increase in body weight as well as hepatic histologic changes; they also had a lower glucose and insulin tolerance than the WT mice fed a normal diet. However, the metabolic syndrome was attenuated in the SOAT1-KO HFD-fed mice. With regard to brain function, the SOAT1-KO HFD-fed mice showed improved cognitive function; they also manifested reduced levels of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6, which would otherwise be raised by a HFD. In addition, the HFD led to the overexpression of GFAP and phosphorylated NF-κB in the hypothalamus, changes that were reversed in the SOAT1-KO HFD-fed mice. Moreover, SOAT1-KO mice improved HFD-caused defective hypothalamic insulin resistance, as evidenced by the upregulation of p-insulin receptor (INSR), p-AKT and p-glycogen synthase kinase (GSK)-3β, while the downregulation of p-AMP-activated protein kinase (AMPK)-α and p-acetyl-CoA carboxylase (ACC)-α. In addition, similar results were observed in high fructose (HFR)-stimulated astrocytes (ASTs) isolated from WT or KO mice. These results suggest that SOAT1 plays an important role in hypothalamic insulin sensitivity, linked to cognitive impairment, in HFD-fed mice. Copyright © 2018. Published by Elsevier Inc.

A Non-specific Setaria italica Lipid Transfer Protein Gene Plays a Critical Role under Abiotic Stress.

PubMed

Pan, Yanlin; Li, Jianrui; Jiao, Licong; Li, Cong; Zhu, Dengyun; Yu, Jingjuan

2016-01-01

Lipid transfer proteins (LTPs) are a class of cysteine-rich soluble proteins having small molecular weights. LTPs participate in flower and seed development, cuticular wax deposition, also play important roles in pathogen and abiotic stress responses. A non-specific LTP gene ( SiLTP ) was isolated from a foxtail millet ( Setaria italica ) suppression subtractive hybridization library enriched for differentially expressed genes after abiotic stress treatments. A semi-quantitative reverse transcriptase PCR analysis showed that SiLTP was expressed in all foxtail millet tissues. Additionally, the SiLTP promoter drove GUS expression in root tips, stems, leaves, flowers, and siliques of transgenic Arabidopsis . Quantitative real-time PCR indicated that the SiLTP expression was induced by NaCl, polyethylene glycol, and abscisic acid (ABA). SiLTP was localized in the cytoplasm of tobacco leaf epidermal cells and maize protoplasts. The ectopic expression of SiLTP in tobacco resulted in higher levels of salt and drought tolerance than in the wild type (WT). To further assess the function of SiLTP, SiLTP overexpression (OE) and RNA interference (RNAi)-based transgenic foxtail millet were obtained. SiLTP -OE lines performed better under salt and drought stresses compared with WT plants. In contrast, the RNAi lines were much more sensitive to salt and drought compared than WT. Electrophoretic mobility shift assays and yeast one-hybrids indicated that the transcription factor ABA-responsive DRE-binding protein (SiARDP) could bind to the dehydration-responsive element of SiLTP promoter in vitro and in vivo , respectively. Moreover, the SiLTP expression levels were higher in SiARDP -OE plants compared than the WT. These results confirmed that SiLTP plays important roles in improving salt and drought stress tolerance of foxtail millet, and may partly be upregulated by SiARDP. SiLTP may provide an effective genetic resource for molecular breeding in crops to enhance salt and drought tolerance levels.

Paraspinal Muscle Passive Stiffness Remodels in Direct Response to Spine Stiffness: A Study Using the ENT1-Deficient Mouse.

PubMed

Gsell, Kelsey Y; Zwambag, Derek P; Fournier, Dale E; Séguin, Cheryle A; Brown, Stephen H M

2017-10-01

Basic science study of the relationship between the structural properties of the spine and its surrounding musculature. To determine whether an increase in spine stiffness causes an inverse compensatory change in the passive stiffness of the adjacent paraspinal muscles. Intervertebral disc degeneration causes an increase in multifidus passive stiffness; this was hypothesized to compensate for a decrease in spine stiffness associated with disc degeneration. Mice lacking equilibrative nucleoside transporter 1 (ENT1) develop progressive ectopic calcification of the fibrous connective tissues of the spine, which affects the lumbar spine by 6 months of age and likely creates a mechanically stiffer spine. Experiments were conducted on four groups of mice (n = 8 mice/group): wild-type (WT) and ENT1 knockout (KO) at 2 or 8 months of age. Lumbar spines were removed and tested in cyclic axial compression to determine neutral zone length and stiffness. Single muscle fibers and bundles of fibers were isolated from lumbar multifidus and erector spinae, as well as tibialis anterior (a non-spine-related control) and tested to determine elastic modulus (passive stiffness). At 2 months of age, neither spine nor muscle stiffness was different between KO and WT. At 8 months of age, compared with WT the lumbar spines of ENT1 KO mice had a stiffer and shorter neutral zone, and the paraspinal muscle fibers were less stiff; however, fiber bundles were not different. In addition, tibialis anterior was not different between KO and WT. This work has confirmed that calcification of spinal connective tissues in the ENT1 KO mouse results in a stiffened spine, whereas the concurrent decrease in muscle fiber elastic modulus in the adjacent paraspinal muscles suggests a direct compensatory relationship between the stiffness of the spine and the muscles that are attached to it. N/A.

Annexin A6 interacts with p65 and stimulates NF-κB activity and catabolic events in articular chondrocytes.

PubMed

Campbell, Kirk A; Minashima, Takeshi; Zhang, Ying; Hadley, Scott; Lee, You Jin; Giovinazzo, Joseph; Quirno, Martin; Kirsch, Thorsten

2013-12-01

ANXA6, the gene for annexin A6, is highly expressed in osteoarthritic (OA) articular chondrocytes but not in healthy articular chondrocytes. This study was undertaken to determine whether annexin A6 affects catabolic events in these cells. Articular chondrocytes were isolated from Anxa6-knockout mice, wild-type (WT) mice, and human articular cartilage in which ANXA6 was overexpressed. Cells were treated with interleukin-1β (IL-1β) or tumor necrosis factor α (TNFα), and expression of catabolic genes and activation of NF-κB were determined by real-time polymerase chain reaction and luciferase reporter assay. Anxa6(-/-) and WT mouse knee joints were injected with IL-1β or the medial collateral ligament was transected and partial resection of the medial meniscus was performed to determine the role of Anxa6 in IL-1β-mediated cartilage destruction and OA progression. The mechanism by which Anxa6 stimulates NF-κB activity was determined by coimmunoprecipitation and immunoblot analysis of nuclear and cytoplasmic fractions of IL-1β-treated Anxa6(-/-) and WT mouse chondrocytes for p65 and Anxa6. Loss of Anxa6 resulted in decreased NF-κB activation and catabolic marker messenger RNA (mRNA) levels in IL-1β- or TNFα-treated articular chondrocytes, whereas overexpression of ANXA6 resulted in increased NF-κB activity and catabolic marker mRNA levels. Annexin A6 interacted with p65, and loss of Anxa6 caused decreased nuclear translocation and retention of the active p50/p65 NF-κB complex. Cartilage destruction in Anxa6(-/-) mouse knee joints after IL-1β injection or partial medial meniscectomy was reduced as compared to that in WT mouse joints. Our data define a role of annexin A6 in the modulation of NF-κB activity and in the stimulation of catabolic events in articular chondrocytes. Copyright © 2013 by the American College of Rheumatology.

ABC transporters P-gp and Bcrp do not limit the brain uptake of the novel antipsychotic and anticonvulsant drug cannabidiol in mice

PubMed Central

Brzozowska, Natalia; Li, Kong M.; Wang, Xiao Suo; Booth, Jessica; Stuart, Jordyn; McGregor, Iain S.

2016-01-01

Cannabidiol (CBD) is currently being investigated as a novel therapeutic for the treatment of CNS disorders like schizophrenia and epilepsy. ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) mediate pharmacoresistance in these disorders. P-gp and Bcrp are expressed at the blood brain barrier (BBB) and reduce the brain uptake of substrate drugs including various antipsychotics and anticonvulsants. It is therefore important to assess whether CBD is prone to treatment resistance mediated by P-gp and Bcrp. Moreover, it has become common practice in the drug development of CNS agents to screen against ABC transporters to help isolate lead compounds with optimal pharmacokinetic properties. The current study aimed to assess whether P-gp and Bcrp impacts the brain transport of CBD by comparing CBD tissue concentrations in wild-type (WT) mice versus mice devoid of ABC transporter genes. P-gp knockout (Abcb1a/b−∕−), Bcrp knockout (Abcg2−∕−), combined P-gp/Bcrp knockout (Abcb1a/b−∕−Abcg2−∕−) and WT mice were injected with CBD, before brain and plasma samples were collected at various time-points. CBD results were compared with the positive control risperidone and 9-hydroxy risperidone, antipsychotic drugs that are established ABC transporter substrates. Brain and plasma concentrations of CBD were not greater in P-gp, Bcrp or P-gp/Bcrp knockout mice than WT mice. In comparison, the brain/plasma concentration ratios of risperidone and 9-hydroxy risperidone were profoundly higher in P-gp knockout mice than WT mice. These results suggest that CBD is not a substrate of P-gp or Bcrp and may be free from the complication of reduced brain uptake by these transporters. Such findings provide favorable evidence for the therapeutic development of CBD in the treatment of various CNS disorders. PMID:27257556

ABC transporters P-gp and Bcrp do not limit the brain uptake of the novel antipsychotic and anticonvulsant drug cannabidiol in mice.

PubMed

Brzozowska, Natalia; Li, Kong M; Wang, Xiao Suo; Booth, Jessica; Stuart, Jordyn; McGregor, Iain S; Arnold, Jonathon C

2016-01-01

Cannabidiol (CBD) is currently being investigated as a novel therapeutic for the treatment of CNS disorders like schizophrenia and epilepsy. ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) mediate pharmacoresistance in these disorders. P-gp and Bcrp are expressed at the blood brain barrier (BBB) and reduce the brain uptake of substrate drugs including various antipsychotics and anticonvulsants. It is therefore important to assess whether CBD is prone to treatment resistance mediated by P-gp and Bcrp. Moreover, it has become common practice in the drug development of CNS agents to screen against ABC transporters to help isolate lead compounds with optimal pharmacokinetic properties. The current study aimed to assess whether P-gp and Bcrp impacts the brain transport of CBD by comparing CBD tissue concentrations in wild-type (WT) mice versus mice devoid of ABC transporter genes. P-gp knockout (Abcb1a/b (-∕-)), Bcrp knockout (Abcg2 (-∕-)), combined P-gp/Bcrp knockout (Abcb1a/b (-∕-) Abcg2 (-∕-)) and WT mice were injected with CBD, before brain and plasma samples were collected at various time-points. CBD results were compared with the positive control risperidone and 9-hydroxy risperidone, antipsychotic drugs that are established ABC transporter substrates. Brain and plasma concentrations of CBD were not greater in P-gp, Bcrp or P-gp/Bcrp knockout mice than WT mice. In comparison, the brain/plasma concentration ratios of risperidone and 9-hydroxy risperidone were profoundly higher in P-gp knockout mice than WT mice. These results suggest that CBD is not a substrate of P-gp or Bcrp and may be free from the complication of reduced brain uptake by these transporters. Such findings provide favorable evidence for the therapeutic development of CBD in the treatment of various CNS disorders.

A Human Strain of Oxalobacter (HC-1) Promotes Enteric Oxalate Secretion in the Small Intestine of Mice and Reduces Urinary Oxalate Excretion

PubMed Central

Hatch, Marguerite; Freel, Robert W.

2013-01-01

Enteric oxalate secretion that correlated with reductions in urinary oxalate excretion was previously reported in a mouse model of Primary Hyperoxaluria, and in wild type (WT) mice colonized with a wild rat strain (OXWR) of Oxalobacter (Am J Physiol 300: G461-G469, 2011). Since a human strain of the bacterium is more likely to be clinically used as a probiotic therapeutic, we tested the effects of HC-1 in WT. Following artificial colonization of WT mice with HC-1, the bacteria were confirmed to be present in the large intestine and, unexpectedly, detected in the small intestine for varying periods of time. The main objective of the present study was to determine whether the presence of HC-1 promoted intestinal secretion in the more proximal segments of the gastrointestinal tract. In addition, we determined whether HC-1 colonization led to reductions in urinary oxalate excretion in these mice. The results show that the human Oxalobacter strain promotes a robust net secretion of oxalate in the distal ileum as well as in the caecum and distal colon and these changes in transport correlate with the beneficial effect of reducing renal excretion of oxalate. We conclude that OXWR effects on intestinal oxalate transport and oxalate homeostasis are not unique to the wild rat strain and that, mechanistically, HC-1 has significant potential for use as a probiotic treatment for hyperoxaluria especially if it is also targeted to the upper and lower gastrointestinal tract. PMID:23959075

Advanced Glycated End-Products Affect HIF-Transcriptional Activity in Renal Cells

PubMed Central

Bondeva, Tzvetanka; Heinzig, Juliane; Ruhe, Carola

2013-01-01

Advanced glycated end-products (AGEs) are ligands of the receptor for AGEs and increase in diabetic disease. MAPK organizer 1 (Morg1) via its binding partner prolyl-hydroxylase domain (PHD)-3 presumably plays a role in the regulation of hypoxia-inducible factor (HIF)-1α and HIF-2α transcriptional activation. The purpose of this study was to analyze the influence of AGEs on Morg1 expression and its correlation to PHD3 activity and HIF-transcriptional activity in various renal cell types. The addition of glycated BSA (AGE-BSA) significantly up-regulated Morg1 mRNA levels in murine mesangial cells and down-regulated it in murine proximal tubular cells and differentiated podocytes. These effects were reversible when the cells were preincubated with a receptor for α-AGE antibody. AGE-BSA treatment induced a relocalization of the Morg1 cellular distribution compared with nonglycated control-BSA. Analysis of PHD3 activity demonstrated an elevated PHD3 enzymatic activity in murine mesangial cells but an inhibition in murine proximal tubular cells and podocytes after the addition of AGE-BSA. HIF-transcriptional activity was also affected by AGE-BSA treatment. Reporter gene assays and EMSAs showed that AGEs regulate HIF- transcriptional activity under nonhypoxic conditions in a cell type-specific manner. In proximal tubular cells, AGE-BSA stimulation elevated mainly HIF-1α transcriptional activity and to a lesser extent HIF-2α. We also detected an increased expression of the HIF-1α and the HIF-2α proteins in kidneys from Morg1 heterozygous (HZ) placebo mice compared with the Morg1 wild-type (WT) placebo-treated mice, and the HIF-1α protein expression in the Morg1 HZ streptozotocin-treated mice was significantly higher than the WT streptozotocin-treated mice. Analysis of isolated mesangial cells from Morg1 HZ (±) and WT mice showed an inhibited PHD3 activity and an increased HIF-transcriptional activity in cells with only one Morg1 allele. These findings are important for a better understanding of the molecular mechanisms of diabetic nephropathy. PMID:24030251

Cyclophilin B Deficiency Causes Abnormal Dentin Collagen Matrix.

PubMed

Terajima, Masahiko; Taga, Yuki; Cabral, Wayne A; Nagasawa, Masako; Sumida, Noriko; Hattori, Shunji; Marini, Joan C; Yamauchi, Mitsuo

2017-08-04

Cyclophilin B (CypB) is an endoplasmic reticulum-resident protein that regulates collagen folding, and also contributes to prolyl 3-hydroxylation (P3H) and lysine (Lys) hydroxylation of collagen. In this study, we characterized dentin type I collagen in CypB null (KO) mice, a model of recessive osteogenesis imperfecta type IX, and compared to those of wild-type (WT) and heterozygous (Het) mice. Mass spectrometric analysis demonstrated that the extent of P3H in KO collagen was significantly diminished compared to WT/Het. Lys hydroxylation in KO was significantly diminished at the helical cross-linking sites, α1/α2(I) Lys-87 and α1(I) Lys-930, leading to a significant increase in the under-hydroxylated cross-links and a decrease in fully hydroxylated cross-links. The extent of glycosylation of hydroxylysine residues was, except α1(I) Lys-87, generally higher in KO than WT/Het. Some of these molecular phenotypes were distinct from other KO tissues reported previously, indicating the dentin-specific control mechanism through CypB. Histological analysis revealed that the width of predentin was greater and irregular, and collagen fibrils were sparse and significantly smaller in KO than WT/Het. These results indicate a critical role of CypB in dentin matrix formation, suggesting a possible association between recessive osteogenesis imperfecta and dentin defects that have not been clinically detected.

Influence of the CCR-5/MIP-1 α Axis in the Pathogenesis of Rocio Virus Encephalitis in a Mouse Model

PubMed Central

Chávez, Juliana H.; França, Rafael F. O.; Oliveira, Carlo J. F.; de Aquino, Maria T. P.; Farias, Kleber J. S.; Machado, Paula R. L.; de Oliveira, Thelma F. M.; Yokosawa, Jonny; Soares, Edson G.; da Silva, João S.; da Fonseca, Benedito A. L.; Figueiredo, Luiz T. M.

2013-01-01

Rocio virus (ROCV) caused an outbreak of human encephalitis during the 1970s in Brazil and its immunopathogenesis remains poorly understood. CC-chemokine receptor 5 (CCR5) is a chemokine receptor that binds to macrophage inflammatory protein (MIP-1 α). Both molecules are associated with inflammatory cells migration during infections. In this study, we demonstrated the importance of the CCR5 and MIP-1 α, in the outcome of viral encephalitis of ROCV-infected mice. CCR5 and MIP-1 α knockout mice survived longer than wild-type (WT) ROCV-infected animals. In addition, knockout mice had reduced inflammation in the brain. Assessment of brain viral load showed mice virus detection five days post-infection in wild-type and CCR5−/− mice, while MIP-1 α−/− mice had lower viral loads seven days post-infection. Knockout mice required a higher lethal dose than wild-type mice as well. The CCR5/MIP-1 α axis may contribute to migration of infected cells to the brain and consequently affect the pathogenesis during ROCV infection. PMID:24080631

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

Bruening, W.; Nakagama, H.: Bardessy, N.

Wilms` tumor (WT), an embryonal malignancy of the kidney, occurs most frequently in children under the age of 5 years, affecting {approximately}1 in 10,000 individuals. The WT1 tumor suppressor gene, residing at 11p13, is structurally altered in {approximately}10-15% of WT cases. Individuals with germline mutations within the WT1 gene suffer from predisposition to WT and developmental defects of the urogenital system. Patients with heterozygous deletions of the WT1 gene, or mutations predicted to cause inactivation of one WT1 allele, suffer relatively mild genital system defects (notably hypospadias and cryptorchidism in males) and a predisposition to WT. These results suggest thatmore » developing genital system development is sensitive to the absolute concentrations of the WT1 gene products. Patients with missense mutations within the WT1 gene, however, can suffer from a much more severe disorder known as Denys-Drash syndrome (DDS). This syndrome is characterized by intersex disorders, renal nephropathy, and a predisposition to WTs. The increased severity of the developmental defects associated with DDS, compared to those individuals with mild genital system anomalies and WTs, suggests that mutations defined in patients with DDS behave in a dominant-negative fashion. We have identified a novel WT1 mutation in a patient with DDS. This mutation, predicted to produce a truncated WT1 polypeptide encompassing exons 1, 2, and 3, defines a domain capable of behaving as an antimorph. We have also demonstrated that WT1 can self-associate in vivo using yeast two-hybrid systems. Deletion analysis have mapped the interacting domains to the amino terminus of the WT1 polypeptide, within exons 1 and 2. These results provide a molecular mechanism to explain how WT1 mutations can function in a dominant-negative fashion to eliminate wild-type WT1 activity, leading to DDS.« less

Analysis of four connexin26 mutant gap junctions and hemichannels reveals variations in hexamer stability.

PubMed

Ambrosi, Cinzia; Boassa, Daniela; Pranskevich, Jennifer; Smock, Amy; Oshima, Atsunori; Xu, Ji; Nicholson, Bruce J; Sosinsky, Gina E

2010-05-19

Connexin26 is a ubiquitous gap junction protein that serves critical homeostatic functions. Four single-site mutations found in the transmembrane helices (M1-M4) cause different types of dysfunctional channels: 1), Cx26T135A in M3 produces a closed channel; 2), Cx26M34A in M1 severely decreases channel activity; 3), Cx26P87L in M2 has been implicated in defective channel gating; and 4), Cx26V84L in M2, a nonsyndromic deafness mutant, retains normal dye coupling and electrophysiological properties but is deficient in IP(3) transfer. These mutations do not affect Cx26 trafficking in mammalian cells, and make normal-appearing channels in baculovirus-infected Sf9 membranes when imaged by negative stain electron microscopy. Upon dodecylmaltoside solubilization of the membrane fraction, Cx26M34A and Cx26V84L are stable as hexamers or dodecamers, but Cx26T135A and Cx26P87L oligomers are not. This instability is also found in Cx26T135A and Cx26P87L hemichannels isolated from mammalian cells. In this work, coexpression of both wild-type Cx26 and Cx26P87L in Sf9 cells rescued P87L hexamer stability. Similarly, in paired Xenopus oocytes, coexpression with wild-type restored function. In contrast, the stability of Cx26T135A hemichannels could not be rescued by coexpression with WT. Thus, T135 and P87 residues are in positions that are important for oligomer stability and can affect gap junction gating. Copyright (c) 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Strain-specific reverse transcriptase PCR assay: means to distinguish candidate vaccine from wild-type strains of respiratory syncytial virus.

PubMed Central

Zheng, H; Peret, T C; Randolph, V B; Crowley, J C; Anderson, L J

1996-01-01

Candidate live-virus vaccines for respiratory syncytial virus are being developed and are beginning to be evaluated in clinical trials. To distinguish candidate vaccine strains from wild-type strains isolated during these trials, we developed PCR assays specific to two sets of candidate vaccine strains. The two sets were a group A strain (3A), its three attenuated, temperature-sensitive variant strains, a group B strain (2B), and its four attenuated, temperature-sensitive variant strains. The PCR assays were evaluated by testing 18 group A wild-type strains, the 3A strains, 9 group B wild-type strains, and the 2B strains. PCR specific to group A wild-type strains amplified only group A wild-type strains, and 3A-specific PCR amplified only 3A strains. PCR specific to group B wild-type strains amplified all group A and group B strains but gave a 688-bp product for group B wild-type strains, a 279-bp product for 2B strains, a 547-bp product for all group A strains, and an additional 688-bp product for some group A strains, including 3A strains. These types of PCR assays can, in conjunction with other methods, be used to efficiently distinguish candidate vaccine strains from other respiratory syncytial virus strains. PMID:8789010

Microbial short chain fatty acid metabolites lower blood pressure via endothelial G protein-coupled receptor 41.

PubMed

Natarajan, Niranjana; Hori, Daijiro; Flavahan, Sheila; Steppan, Jochen; Flavahan, Nicholas A; Berkowitz, Dan E; Pluznick, Jennifer L

2016-11-01

Short chain fatty acid (SCFA) metabolites are byproducts of gut microbial metabolism that are known to affect host physiology via host G protein-coupled receptor (GPCRs). We previously showed that an acute SCFA bolus decreases blood pressure (BP) in anesthetized mice, an effect mediated primarily via Gpr41. In this study, our aims were to identify the cellular localization of Gpr41 and to determine its role in BP regulation. We localized Gpr41 to the vascular endothelium using RT-PCR: Gpr41 is detected in intact vessels (with endothelium) but is absent from denuded vessels (without endothelium). Furthermore, using pressure myography we confirmed that SCFAs dilate resistance vessels in an endothelium-dependent manner. Since we previously found that Gpr41 mediates a hypotensive response to acute SCFA administration, we hypothesized that Gpr41 knockout (KO) mice would be hypertensive. Here, we report that Gpr41 KO mice have isolated systolic hypertension compared with wild-type (WT) mice; diastolic BP was not different between WT and KO. Older Gpr41 KO mice also exhibited elevated pulse wave velocity, consistent with a phenotype of systolic hypertension; however, there was no increase in ex vivo aorta stiffness (measured by mechanical tensile testing). Plasma renin concentrations were also similar in KO and WT mice. The systolic hypertension in Gpr41 KO is not salt sensitive, as it is not significantly altered on either a high- or low-salt diet. In sum, these studies suggest that endothelial Gpr41 lowers baseline BP, likely by decreasing active vascular tone without altering passive characteristics of the blood vessels, and that Gpr41 KO mice have hypertension of a vascular origin. Copyright © 2016 the American Physiological Society.

Galectin-3 drives oligodendrocyte differentiation to control myelin integrity and function

PubMed Central

Pasquini, L A; Millet, V; Hoyos, H C; Giannoni, J P; Croci, D O; Marder, M; Liu, F T; Rabinovich, G A; Pasquini, J M

2011-01-01

Galectins control critical pathophysiological processes, including the progression and resolution of central nervous system (CNS) inflammation. In spite of considerable progress in dissecting their role within lymphoid organs, their functions within the inflamed CNS remain elusive. Here, we investigated the role of galectin–glycan interactions in the control of oligodendrocyte (OLG) differentiation, myelin integrity and function. Both galectin-1 and -3 were abundant in astrocytes and microglia. Although galectin-1 was abundant in immature but not in differentiated OLGs, galectin-3 was upregulated during OLG differentiation. Biochemical analysis revealed increased activity of metalloproteinases responsible for cleaving galectin-3 during OLG differentiation and modulating its biological activity. Exposure to galectin-3 promoted OLG differentiation in a dose- and carbohydrate-dependent fashion consistent with the ‘glycosylation signature' of immature versus differentiated OLG. Accordingly, conditioned media from galectin-3-expressing, but not galectin-3-deficient (Lgals3−/−) microglia, successfully promoted OLG differentiation. Supporting these findings, morphometric analysis showed a significant decrease in the frequency of myelinated axons, myelin turns (lamellae) and g-ratio in the corpus callosum and striatum of Lgals3−/− compared with wild-type (WT) mice. Moreover, the myelin structure was loosely wrapped around the axons and less smooth in Lgals3−/− mice versus WT mice. Behavior analysis revealed decreased anxiety in Lgals3−/− mice similar to that observed during early demyelination induced by cuprizone intoxication. Finally, commitment toward the oligodendroglial fate was favored in neurospheres isolated from WT but not Lgals3−/− mice. Hence, glial-derived galectin-3, but not galectin-1, promotes OLG differentiation, thus contributing to myelin integrity and function with critical implications in the recovery of inflammatory demyelinating disorders. PMID:21566659

A Novel WRKY Transcription Factor, MuWRKY3 (Macrotyloma uniflorum Lam. Verdc.) Enhances Drought Stress Tolerance in Transgenic Groundnut (Arachis hypogaea L.) Plants.

PubMed

Kiranmai, Kurnool; Lokanadha Rao, Gunupuru; Pandurangaiah, Merum; Nareshkumar, Ambekar; Amaranatha Reddy, Vennapusa; Lokesh, Uppala; Venkatesh, Boya; Anthony Johnson, A M; Sudhakar, Chinta

2018-01-01

Drought stress has adverse effects on growth, water relations, photosynthesis and yield of groundnut. WRKY transcription factors (TFs) are the plant-specific TFs which regulate several down-stream stress-responsive genes and play an essential role in plant biotic and abiotic stress responses. We found that WRKY3 gene is highly up-regulated under drought stress conditions and therefore isolated a new WRKY3TF gene from a drought-adapted horsegram ( Macrotyloma uniflorum Lam. Verdc.). Conserved domain studies revealed that protein encoded by this gene contains highly conserved regions of two WRKY domains and two C2H2 zinc-finger motifs. The fusion protein localization studies of transient MuWRKY 3-YFP revealed its nuclear localization. Overexpression of MuWRKY3 TF gene in groundnut ( Arachis hypogaea L.) showed increased tolerance to drought stress compared to wild-type (WT) plants. MuWRKY3 groundnut transgenics displayed lesser and delayed wilting symptoms than WT plants after 10-days of drought stress imposition. The transgenic groundnut plants expressing MuWRKY3 showed less accumulation of malondialdehyde, hydrogen peroxide (H 2 O 2 ), and superoxide anion (O 2 ∙- ), accompanied by more free proline, total soluble sugar content, and activities of antioxidant enzymes than WT plants under drought stress. Moreover, a series of stress-related LEA, HSP, MIPS, APX, SOD , and CAT genes found up-regulated in the transgenic groundnut plants. The study demonstrates that nuclear-localized MuWRKY3 TF regulates the expression of stress-responsive genes and the activity of ROS scavenging enzymes which results in improved drought tolerance in groundnut. We conclude that MuWRKY3 may serve as a new putative candidate gene for the improvement of stress resistance in plants.

A Novel WRKY Transcription Factor, MuWRKY3 (Macrotyloma uniflorum Lam. Verdc.) Enhances Drought Stress Tolerance in Transgenic Groundnut (Arachis hypogaea L.) Plants

PubMed Central

Kiranmai, Kurnool; Lokanadha Rao, Gunupuru; Pandurangaiah, Merum; Nareshkumar, Ambekar; Amaranatha Reddy, Vennapusa; Lokesh, Uppala; Venkatesh, Boya; Anthony Johnson, A. M.; Sudhakar, Chinta

2018-01-01

Drought stress has adverse effects on growth, water relations, photosynthesis and yield of groundnut. WRKY transcription factors (TFs) are the plant-specific TFs which regulate several down-stream stress-responsive genes and play an essential role in plant biotic and abiotic stress responses. We found that WRKY3 gene is highly up-regulated under drought stress conditions and therefore isolated a new WRKY3TF gene from a drought-adapted horsegram (Macrotyloma uniflorum Lam. Verdc.). Conserved domain studies revealed that protein encoded by this gene contains highly conserved regions of two WRKY domains and two C2H2 zinc-finger motifs. The fusion protein localization studies of transient MuWRKY3-YFP revealed its nuclear localization. Overexpression of MuWRKY3 TF gene in groundnut (Arachis hypogaea L.) showed increased tolerance to drought stress compared to wild-type (WT) plants. MuWRKY3 groundnut transgenics displayed lesser and delayed wilting symptoms than WT plants after 10-days of drought stress imposition. The transgenic groundnut plants expressing MuWRKY3 showed less accumulation of malondialdehyde, hydrogen peroxide (H2O2), and superoxide anion (O2∙-), accompanied by more free proline, total soluble sugar content, and activities of antioxidant enzymes than WT plants under drought stress. Moreover, a series of stress-related LEA, HSP, MIPS, APX, SOD, and CAT genes found up-regulated in the transgenic groundnut plants. The study demonstrates that nuclear-localized MuWRKY3 TF regulates the expression of stress-responsive genes and the activity of ROS scavenging enzymes which results in improved drought tolerance in groundnut. We conclude that MuWRKY3 may serve as a new putative candidate gene for the improvement of stress resistance in plants. PMID:29616059

Lack of specificity of antibodies raised against CLN3, the lysosomal/endosomal transmembrane protein mutated in juvenile Batten disease

PubMed Central

Nelson, Tarah

2017-01-01

Juvenile CLN3 (Batten) disease, a fatal, childhood neurodegenerative disorder, results from mutations in the CLN3 gene encoding a lysosomal/endosomal transmembrane protein. The exact physiological function of CLN3 is still unknown and it is unclear how CLN3 mutations lead to selective neurodegeneration. To study the tissue expression and subcellular localization of the CLN3 protein, a number of anti-CLN3 antibodies have been generated using either the whole CLN3 protein or short peptides from CLN3 for immunization. The specificity of these antibodies, however, has never been tested properly. Using immunoblot experiments, we show that commercially available or researcher-generated anti-CLN3 antibodies lack specificity: they detect the same protein bands in wild-type (WT) and Cln3−/− mouse brain and kidney extracts prepared with different detergents, in membrane proteins isolated from the cerebellum, cerebral hemisphere and kidney of WT and Cln3−/− mice, in cell extracts of WT and Cln3−/− mouse embryonic fibroblast cultures, and in lysates of BHK cells lacking or overexpressing human CLN3. Protein BLAST searches with sequences from peptides used to generate anti-CLN3 antibodies identified short motifs present in a number of different mouse and human proteins, providing a plausible explanation for the lack of specificity of anti-CLN3 antibodies. Our data provide evidence that immunization against a transmembrane protein with low to medium expression level does not necessarily generate specific antibodies. Because of the possible cross-reactivity to other proteins, the specificity of an antibody should always be checked using tissue samples from an appropriate knock-out animal or using knock-out cells. PMID:29089465

Remodeling of repolarization and arrhythmia susceptibility in a myosin-binding protein C knockout mouse model.

PubMed

Toib, Amir; Zhang, Chen; Borghetti, Giulia; Zhang, Xiaoxiao; Wallner, Markus; Yang, Yijun; Troupes, Constantine D; Kubo, Hajime; Sharp, Thomas E; Feldsott, Eric; Berretta, Remus M; Zalavadia, Neil; Trappanese, Danielle M; Harper, Shavonn; Gross, Polina; Chen, Xiongwen; Mohsin, Sadia; Houser, Steven R

2017-09-01

Hypertrophic cardiomyopathy (HCM) is one of the most common genetic cardiac diseases and among the leading causes of sudden cardiac death (SCD) in the young. The cellular mechanisms leading to SCD in HCM are not well known. Prolongation of the action potential (AP) duration (APD) is a common feature predisposing hypertrophied hearts to SCD. Previous studies have explored the roles of inward Na + and Ca 2+ in the development of HCM, but the role of repolarizing K + currents has not been defined. The objective of this study was to characterize the arrhythmogenic phenotype and cellular electrophysiological properties of mice with HCM, induced by myosin-binding protein C (MyBPC) knockout (KO), and to test the hypothesis that remodeling of repolarizing K + currents causes APD prolongation in MyBPC KO myocytes. We demonstrated that MyBPC KO mice developed severe hypertrophy and cardiac dysfunction compared with wild-type (WT) control mice. Telemetric electrocardiographic recordings of awake mice revealed prolongation of the corrected QT interval in the KO compared with WT control mice, with overt ventricular arrhythmias. Whole cell current- and voltage-clamp experiments comparing KO with WT mice demonstrated ventricular myocyte hypertrophy, AP prolongation, and decreased repolarizing K + currents. Quantitative RT-PCR analysis revealed decreased mRNA levels of several key K + channel subunits. In conclusion, decrease in repolarizing K + currents in MyBPC KO ventricular myocytes contributes to AP and corrected QT interval prolongation and could account for the arrhythmia susceptibility. NEW & NOTEWORTHY Ventricular myocytes isolated from the myosin-binding protein C knockout hypertrophic cardiomyopathy mouse model demonstrate decreased repolarizing K + currents and action potential and QT interval prolongation, linking cellular repolarization abnormalities with arrhythmia susceptibility and the risk for sudden cardiac death in hypertrophic cardiomyopathy. Copyright © 2017 the American Physiological Society.

Mesenchymal change and drug resistance in neuroblastoma.

PubMed

Naiditch, Jessica A; Jie, Chunfa; Lautz, Timothy B; Yu, Songtao; Clark, Sandra; Voronov, Dimitry; Chu, Fei; Madonna, Mary Beth

2015-01-01

Metastatic initiation has many phenotypic similarities to epithelial-to-mesenchymal transition, including loss of cell-cell adhesion, increased invasiveness, and increased cell mobility. We have previously demonstrated that drug resistance is associated with a metastatic phenotype in neuroblastoma (NB). The purpose of this project was to determine if the development of doxorubicin resistance is associated with characteristics of mesenchymal change in human NB cells. Total RNA was isolated from wild type (WT) and doxorubicin-resistant (DoxR) human NB cell lines (SK-N-SH and SK-N-BE(2)C) and analyzed using the Illumina Human HT-12 version 4 Expression BeadChip. Differentially expressed genes (DEGs) were identified. Volcano plots and heat maps were generated. Genes of interest with a fold change in expression >1.5 and an adjusted P < 0.1 were analyzed. Immunofluorescence (IF) and Western blot analysis confirmed microarray results of interest. Matrigel invasion assay and migration wounding assays were performed. Volcano plots and heat maps visually demonstrated a similar pattern of DEGs in the SK-N-SH and SK-N-BE(2)C DoxR cell lines relative to their parental WT lines. Venn diagramming revealed 1594 DEGs common to both DoxR cell lines relative to their parental cell lines. Network analysis pointed to several significantly upregulated epithelial-to-mesenchymal transition pathways, through TGF-beta pathways via RhoA, PI3K, and ILK and via SMADs, as well as via notch signaling pathways. DoxR cell lines displayed a more invasive phenotype than respective WT cell lines. Human SK-N-SH and SK-N-BE(2)C NB cells display characteristics of mesenchymal change via multiple pathways in the transition to a drug-resistant state. Copyright © 2015 Elsevier Inc. All rights reserved.

CXCR6 Plays a Critical Role in Angiotensin II-induced Renal Injury and Fibrosis

PubMed Central

Xia, Yunfeng; Jin, Xiaogao; Yan, Jingyin; Entman, Mark L.; Wang, Yanlin

2014-01-01

Objective Recent studies have shown that angiotensin II (Ang II) plays a critical role in the pathogenesis and progression of hypertensive kidney disease. However, the signaling mechanisms are poorly understood. In this study, we investigated the role of CXCR6 in Ang II-induced renal injury and fibrosis. Approach and Results Wild-type and CXCR6-GFP knockin mice were treated with Ang II via subcutaneous osmotic minipumps at 1500 ng/kg/min after unilateral nephrectomy for up to 4 weeks. WT and CXCR6-GFP knockin mice had virtually identical blood pressure at baseline. Ang II treatment led to an increase in blood pressure that was similar between WT and CXCR6-GFP knockin mice. CXCR6-GFP knockin mice were protected from Ang II-induced renal dysfunction, proteinuria, and fibrosis. CXCR6-GFP knockin mice accumulated fewer bone marrow-derived fibroblasts and myofibroblasts and produced less extracellular matrix protein in the kidneys following Ang II treatment. Furthermore, CXCR6-GFP knockin mice exhibited fewer F4/80+ macrophages and CD3+ T cells and expressed less proinflammatory cytokines in the kidneys after Ang II treatment. Finally, wild-type mice engrafted with CXCR6−/− bone marrow cells displayed fewer bone marrow-derived fibroblasts, macrophages, and T cells in the kidney after Ang II treatment compared with wild-type mice engrafted with CXCR6+/+ bone marrow cells. Conclusions Our results indicate that CXCR6 plays a pivotal role in the development of Ang II-induced renal injury and fibrosis through regulation of macrophage and T cell infiltration and bone marrow-derived fibroblast accumulation. PMID:24855055

ERp29 Regulates ΔF508 and Wild-type Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Trafficking to the Plasma Membrane in Cystic Fibrosis (CF) and Non-CF Epithelial Cells*

PubMed Central

Suaud, Laurence; Miller, Katelyn; Alvey, Lora; Yan, Wusheng; Robay, Amal; Kebler, Catherine; Kreindler, James L.; Guttentag, Susan; Hubbard, Michael J.; Rubenstein, Ronald C.

2011-01-01

Sodium 4-phenylbutyrate (4PBA) improves the intracellular trafficking of ΔF508-CFTR in cystic fibrosis (CF) epithelial cells. The underlying mechanism is uncertain, but 4PBA modulates the expression of some cytosolic molecular chaperones. To identify other 4PBA-regulated proteins that might regulate ΔF508-CFTR trafficking, we performed a differential display RT-PCR screen on IB3-1 CF bronchiolar epithelial cells exposed to 4PBA. One transcript up-regulated by 4PBA encoded ERp29, a luminal resident of the endoplasmic reticulum (ER) thought to be a novel molecular chaperone. We tested the hypothesis that ERp29 is a 4PBA-regulated ER chaperone that influences ΔF508-CFTR trafficking. ERp29 mRNA and protein expression was significantly increased (∼1.5-fold) in 4PBA-treated IB3-1 cells. In Xenopus oocytes, ERp29 overexpression increased the functional expression of both wild-type and ΔF508-CFTR over 3-fold and increased wild-type cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane expression. In CFBE41o− WT-CFTR cells, expression of and short circuit currents mediated by CFTR decreased upon depletion of ERp29 as did maturation of newly synthesized CFTR. In IB3-1 cells, ΔF508-CFTR co-immunoprecipitated with endogenous ERp29, and overexpression of ERp29 led to increased ΔF508-CFTR expression at the plasma membrane. These data suggest that ERp29 is a 4PBA-regulated ER chaperone that regulates WT-CFTR biogenesis and can promote ΔF508-CFTR trafficking in CF epithelial cells. PMID:21525008

ERp29 regulates DeltaF508 and wild-type cystic fibrosis transmembrane conductance regulator (CFTR) trafficking to the plasma membrane in cystic fibrosis (CF) and non-CF epithelial cells.

PubMed

Suaud, Laurence; Miller, Katelyn; Alvey, Lora; Yan, Wusheng; Robay, Amal; Kebler, Catherine; Kreindler, James L; Guttentag, Susan; Hubbard, Michael J; Rubenstein, Ronald C

2011-06-17

Sodium 4-phenylbutyrate (4PBA) improves the intracellular trafficking of ΔF508-CFTR in cystic fibrosis (CF) epithelial cells. The underlying mechanism is uncertain, but 4PBA modulates the expression of some cytosolic molecular chaperones. To identify other 4PBA-regulated proteins that might regulate ΔF508-CFTR trafficking, we performed a differential display RT-PCR screen on IB3-1 CF bronchiolar epithelial cells exposed to 4PBA. One transcript up-regulated by 4PBA encoded ERp29, a luminal resident of the endoplasmic reticulum (ER) thought to be a novel molecular chaperone. We tested the hypothesis that ERp29 is a 4PBA-regulated ER chaperone that influences ΔF508-CFTR trafficking. ERp29 mRNA and protein expression was significantly increased (∼1.5-fold) in 4PBA-treated IB3-1 cells. In Xenopus oocytes, ERp29 overexpression increased the functional expression of both wild-type and ΔF508-CFTR over 3-fold and increased wild-type cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane expression. In CFBE41o- WT-CFTR cells, expression of and short circuit currents mediated by CFTR decreased upon depletion of ERp29 as did maturation of newly synthesized CFTR. In IB3-1 cells, ΔF508-CFTR co-immunoprecipitated with endogenous ERp29, and overexpression of ERp29 led to increased ΔF508-CFTR expression at the plasma membrane. These data suggest that ERp29 is a 4PBA-regulated ER chaperone that regulates WT-CFTR biogenesis and can promote ΔF508-CFTR trafficking in CF epithelial cells.

Characterization of H7 Influenza A Virus in Wild and Domestic Birds in Korea

PubMed Central

Kang, Hyun-Mi; Park, Ha-Young; Lee, Kyu-Jun; Choi, Jun-Gu; Lee, Eun-Kyoung; Song, Byung-Min; Lee, Hee-Soo; Lee, Youn-Jeong

2014-01-01

During surveillance programs in Korea between January 2006 and March 2011, 31 H7 avian influenza viruses were isolated from wild birds and domestic ducks and genetically characterized using large-scale sequence data. All Korean H7 viruses belonged to the Eurasian lineage, which showed substantial genetic diversity, in particular in the wild birds. The Korean H7 viruses from poultry were closely related to those of wild birds. Interestingly, two viruses originating in domestic ducks in our study had the same gene constellations in all segment genes as viruses originating in wild birds. The Korean H7 isolates contained avian-type receptors (Q226 and G228), no NA stalk deletion (positions 69–73), no C-terminal deletion (positions 218–230) in NS1, and no substitutions in PB2-627, PB1-368, and M2-31, compared with H7N9 viruses. In pathogenicity experiments, none of the Korean H7 isolates tested induced clinical signs in domestic ducks or mice. Furthermore, while they replicated poorly, with low titers (10 0.7–1.3EID50/50 µl) in domestic ducks, all five viruses replicated well (up to 7–10 dpi, 10 0.7–4.3EID50/50 µl) in the lungs of mice, without prior adaptation. Our results suggest that domestic Korean viruses were transferred directly from wild birds through at least two independent introductions. Our data did not indicate that wild birds carried poultry viruses between Korea and China, but rather, that wild-type H7 viruses were introduced several times into different poultry populations in eastern Asia. PMID:24776918

Using whole-genome sequencing to determine appropriate streptomycin epidemiological cutoffs for Salmonella and Escherichia coli.

PubMed

Tyson, Gregory H; Li, Cong; Ayers, Sherry; McDermott, Patrick F; Zhao, Shaohua

2016-02-01

For Enterobacteriaceae such as Salmonella spp. and Escherichia coli, no unified interpretive resistance criteria exist for streptomycin, an epidemiologically important antibiotic. As part of the National Antimicrobial Resistance Monitoring System, we had previously used a minimum inhibitory concentration of ≥ 64 μg mL(-1) as an epidemiological cutoff value (ECV) to define non-wild-type isolates. To identify whether this ECV correlated with genetic determinants of resistance, we performed whole-genome sequencing of 463 Salmonella and E. coli isolates to identify streptomycin resistance genotypes. From this analysis, we found that using a streptomycin resistance breakpoint of ≥ 64 μg mL(-1) classified over 20% of strains possessing aadA or strA/strB resistance genes as wild-type. Therefore, to improve the concordance between genotypic and phenotypic data, we propose reducing the phenotypic cutoff values to ≥ 32 μg mL(-1) for both Salmonella and E. coli, to be used widely as ECVs to categorize non-wild-type isolates. Published by Oxford University Press on behalf of FEMS 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Antigenic analysis of genetic variants of Canine distemper virus.

PubMed

Anis, Eman; Holford, Amy L; Galyon, Gina D; Wilkes, Rebecca P

2018-06-01

Canine distemper virus (CDV) is an RNA virus of the genus Morbillivirus within the family Paramyxoviridae. CDV produces multi-systemic disease in dogs and other terrestrial carnivores. With the development of modified live vaccines in the 1950s and 1960s, the disease, with a few exceptions, has been successfully controlled. However, recently the cases of CDV in vaccinated dogs have been increasing throughout the world, including the United States. There are many reasons that can lead to vaccine failure, including antigenic differences between the vaccine strains and the currently circulating wild-type strains. Currently, there are at least three genetically different CDV lineages circulating in the US. Therefore, in this study, we evaluated various wild-type CDV and vaccine isolates to determine if the genetic differences observed among various strains result in significant antigenic differences based on changes to the neutralizing epitopes. The results of a cross-neutralization assay revealed that there are antigenic differences among the tested CDV wild-type isolates as well as between the tested isolates and the vaccine strains currently used in the US. Therefore, these results suggest the need to develop an updated CDV vaccine. Copyright © 2018 Elsevier B.V. All rights reserved.

Sodium bicarbonate cotransporter NBCe2 gene variants increase sodium and bicarbonate transport in human renal proximal tubule cells.

PubMed

Gildea, John J; Xu, Peng; Kemp, Brandon A; Carlson, Julia M; Tran, Hanh T; Bigler Wang, Dora; Langouët-Astrié, Christophe J; McGrath, Helen E; Carey, Robert M; Jose, Pedro A; Felder, Robin A

2018-01-01

Salt sensitivity of blood pressure affects >30% of the hypertensive and >15% of the normotensive population. Variants of the electrogenic sodium bicarbonate cotransporter NBCe2 gene, SLC4A5, are associated with increased blood pressure in several ethnic groups. SLC4A5 variants are also highly associated with salt sensitivity, independent of hypertension. However, little is known about how NBCe2 contributes to salt sensitivity, although NBCe2 regulates renal tubular sodium bicarbonate transport. We hypothesized that SLC4A5 rs10177833 and rs7571842 increase NBCe2 expression and human renal proximal tubule cell (hRPTC) sodium transport and may be a cause of salt sensitivity of blood pressure. To characterize the hRPTC ion transport of wild-type (WT) and homozygous variants (HV) of SLC4A5. The expressions of NBCe2 mRNA and protein were not different between hRPTCs carrying WT or HV SLC4A5 before or after dopaminergic or angiotensin (II and III) stimulation. However, luminal to basolateral sodium transport, NHE3 protein, and Cl-/HCO3- exchanger activity in hRPTCs were higher in HV than WT SLC4A5. Increasing intracellular sodium enhanced the apical location of NBCe2 in HV hRPTCs (4.24±0.35% to 11.06±1.72% (P<0.05, N = 3, 2-way ANOVA, Holm-Sidak test)) as determined by Total Internal Reflection Fluorescence Microscopy (TIRFM). In hRPTCs isolated from kidney tissue, increasing intracellular sodium enhanced bicarbonate-dependent pH recovery rate and increased NBCe2 mRNA and protein expressions to a greater extent in HV than WT SLC4A5 (+38.00±6.23% vs HV normal salt (P<0.01, N = 4, 2-way ANOVA, Holm-Sidak test)). In hRPTCs isolated from freshly voided urine, bicarbonate-dependent pH recovery was also faster in those from salt-sensitive and carriers of HV SLC4A5 than from salt-resistant and carriers of WT SLC4A5. The faster NBCe2-specific bicarbonate-dependent pH recovery rate in HV SCL4A5 was normalized by SLC4A5- but not SLC4A4-shRNA. The binding of purified hepatocyte nuclear factor type 4A (HNF4A) to DNA was increased in hRPTCs carrying HV SLC4A5 rs7571842 but not rs10177833. The faster NBCe2-specific bicarbonate-dependent pH recovery rate in HV SCL4A5 was abolished by HNF4A antagonists. NBCe2 activity is stimulated by an increase in intracellular sodium and is hyper-responsive in hRPTCs carrying HV SLC4A5 rs7571842 through an aberrant HNF4A-mediated mechanism.

Sabin Vaccine Reversion in the Field: a Comprehensive Analysis of Sabin-Like Poliovirus Isolates in Nigeria

PubMed Central

Chang, Stewart; Iber, Jane; Zhao, Kun; Adeniji, Johnson A.; Bukbuk, David; Baba, Marycelin; Behrend, Matthew; Burns, Cara C.; Oberste, M. Steven

2015-01-01

ABSTRACT To assess the dynamics of genetic reversion of live poliovirus vaccine in humans, we studied molecular evolution in Sabin-like poliovirus isolates from Nigerian acute flaccid paralysis cases obtained from routine surveillance. We employed a novel modeling approach to infer substitution and recombination rates from whole-genome sequences and information about poliovirus infection dynamics and the individual vaccination history. We confirmed observations from a recent vaccine trial that VP1 substitution rates are increased for Sabin-like isolates relative to the rate for the wild type due to increased nonsynonymous substitution rates. We also inferred substitution rates for attenuating nucleotides and confirmed that reversion can occur in days to weeks after vaccination. We combine our observations for Sabin-like virus evolution with the molecular clock for VP1 of circulating wild-type strains to infer that the mean time from the initiating vaccine dose to the earliest detection of circulating vaccine-derived poliovirus (cVDPV) is 300 days for Sabin-like virus type 1, 210 days for Sabin-like virus type 2, and 390 days for Sabin-like virus type 3. Phylogenetic relationships indicated transient local transmission of Sabin-like virus type 3 and, possibly, Sabin-like virus type 1 during periods of low wild polio incidence. Comparison of Sabin-like virus recombinants with known Nigerian vaccine-derived poliovirus recombinants shows that while recombination with non-Sabin enteroviruses is associated with cVDPV, the recombination rates are similar for Sabin isolate-Sabin isolate and Sabin isolate–non-Sabin enterovirus recombination after accounting for the time from dosing to the time of detection. Our study provides a comprehensive picture of the evolutionary dynamics of the oral polio vaccine in the field. IMPORTANCE The global polio eradication effort has completed its 26th year. Despite success in eliminating wild poliovirus from most of the world, polio persists in populations where logistical, social, and political factors have not allowed vaccination programs of sustained high quality. One issue of critical importance is eliminating circulating vaccine-derived polioviruses (cVDPVs) that have properties indistinguishable from those of wild poliovirus and can cause paralytic disease. cVDPV emerges due to the genetic instability of the Sabin viruses used in the oral polio vaccine (OPV) in populations that have low levels of immunity to poliovirus. However, the dynamics responsible are incompletely understood because it has historically been difficult to gather and interpret data about evolution of the Sabin viruses used in OPV in regions where cVDPV has occurred. This study is the first to combine whole-genome sequencing of poliovirus isolates collected during routine surveillance with knowledge about the intrahost dynamics of poliovirus to provide quantitative insight into polio vaccine evolution in the field. PMID:26468545

Rapid identification of causal mutations in tomato EMS populations via mapping-by-sequencing.

PubMed

Garcia, Virginie; Bres, Cécile; Just, Daniel; Fernandez, Lucie; Tai, Fabienne Wong Jun; Mauxion, Jean-Philippe; Le Paslier, Marie-Christine; Bérard, Aurélie; Brunel, Dominique; Aoki, Koh; Alseekh, Saleh; Fernie, Alisdair R; Fraser, Paul D; Rothan, Christophe

2016-12-01

The tomato is the model species of choice for fleshy fruit development and for the Solanaceae family. Ethyl methanesulfonate (EMS) mutants of tomato have already proven their utility for analysis of gene function in plants, leading to improved breeding stocks and superior tomato varieties. However, until recently, the identification of causal mutations that underlie particular phenotypes has been a very lengthy task that many laboratories could not afford because of spatial and technical limitations. Here, we describe a simple protocol for identifying causal mutations in tomato using a mapping-by-sequencing strategy. Plants displaying phenotypes of interest are first isolated by screening an EMS mutant collection generated in the miniature cultivar Micro-Tom. A recombinant F 2 population is then produced by crossing the mutant with a wild-type (WT; non-mutagenized) genotype, and F 2 segregants displaying the same phenotype are subsequently pooled. Finally, whole-genome sequencing and analysis of allele distributions in the pools allow for the identification of the causal mutation. The whole process, from the isolation of the tomato mutant to the identification of the causal mutation, takes 6-12 months. This strategy overcomes many previous limitations, is simple to use and can be applied in most laboratories with limited facilities for plant culture and genotyping.

Capillary arterialization requires the bone-marrow-derived cell (BMC)-specific expression of chemokine (C-C motif) receptor-2, but BMCs do not transdifferentiate into microvascular smooth muscle.

PubMed

Nickerson, Meghan M; Burke, Caitlin W; Meisner, Joshua K; Shuptrine, Casey W; Song, Ji; Price, Richard J

2009-01-01

Chemokine (C-C motif) receptor-2 (CCR2) regulates arteriogenesis and angiogenesis, facilitating the MCP-1-dependent recruitment of growth factor-secreting bone marrow-derived cells (BMCs). Here, we tested the hypothesis that the BMC-specific expression of CCR2 is also required for new arteriole formation via capillary arterialization. Following non-ischemic saphenous artery occlusion, we measured the following in gracilis muscles: monocyte chemotactic protein-1 (MCP-1) in wild-type (WT) C57Bl/6J mice by ELISA, and capillary arterialization in WT-WT and CCR2(-/-)-WT (donor-host) bone marrow chimeric mice, as well as BMC transdifferentiation in EGFP(+)-WT mice, by smooth muscle (SM) alpha-actin immunochemistry. MCP-1 levels were significantly elevated 1 day after occlusion in WT mice. In WT-WT mice at day 7, compared to sham controls, arterial occlusion induced a 34% increase in arteriole length density, a 46% increase in SM alpha-actin(+) vessels, and a 45% increase in the fraction of vessels coated with SM alpha-actin, indicating significant capillary arterialization. However, in CCR2(-/-)-WT mice, no differences were observed between arterial occlusion and sham surgery. In EGFP(+)-WT mice, EGFP and SM alpha-actin never colocalized. We conclude that BMC-specific CCR2 expression is required for skeletal muscle capillary arterialization following arterial occlusion; however, BMCs do not transdifferentiate into smooth muscle.

Methicillin resistant Staphylococcus aureus (MRSA) carriage in different free-living wild animal species in Spain.

PubMed

Porrero, M Concepción; Mentaberre, Gregorio; Sánchez, Sergio; Fernández-Llario, Pedro; Gómez-Barrero, Susana; Navarro-Gonzalez, Nora; Serrano, Emmanuel; Casas-Díaz, Encarna; Marco, Ignasi; Fernández-Garayzabal, José-Francisco; Mateos, Ana; Vidal, Dolors; Lavín, Santiago; Domínguez, Lucas

2013-10-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a life-threatening pathogen in humans and its presence in animals is a public health concern. The aim of this study was to measure the prevalence of MRSA in free-living wild animals. Samples from red deer (n=273), Iberian ibex (n=212), Eurasian Griffon vulture (n=40) and wild boar (n=817) taken from different areas in Spain between June 2008 and November 2011 were analyzed. Characterization of the isolates was performed by spa typing, multi-locus sequence typing (MLST) and antimicrobial susceptibility testing. A low prevalence of MRSA was found with 13 isolates obtained from 12 animals (0.89%; 95% CI: 0.46-1.56). All MRSA sequence types belonged to ST398 (t011 and t1451) and ST1 (t127). Genotypes and antimicrobial susceptibility patterns (tetracycline resistance in ST398 and clindamycin-erythromycin-tetracycline resistance in ST1) suggest that the MRSA found probably originated in livestock (ST398) or humans (ST1). This is the first report of MRSA carriers in free-living wild animals in Europe. Although our data showed that MRSA prevalence is currently low, free-living wild animals might act as reservoir and represent a potential risk for human health. Copyright © 2013 Elsevier Ltd. All rights reserved.

Adverse effect of urease on salt stress during seed germination in Arabidopsis thaliana.

PubMed

Bu, Yuanyuan; Kou, Jing; Sun, Bo; Takano, Testuo; Liu, Shenkui

2015-05-22

Seed germination is a critical stage in the development of crops that grow in saline soils. We noticed that seeds of an Arabidopsis urease mutant have significantly increased salt stress tolerance. To understand why, we treated the wild type (WT) with a urease inhibitor and found that its salt stress tolerance was also improved. We hypothesized that urease acting on urea generates NH₄⁺, which probably exacerbates salt stress. As expected, the urease inhibitor significantly decreased the NH₄⁺ level in WT seeds. These findings suggest that blocking urease activity improves salt tolerance during seed germination by lowering the concentration of NH₄⁺. Copyright © 2015. Published by Elsevier B.V.

Role of medial prefrontal cortex Narp in the extinction of morphine conditioned place preference.

PubMed

Blouin, Ashley M; Han, Sungho; Pearce, Anne M; Cheng, Kailun; Lee, Jongah J; Johnson, Alexander W; Wang, Chuansong; During, Matthew J; Holland, Peter C; Shaham, Yavin; Baraban, Jay M; Reti, Irving M

2013-01-15

Narp knockout (KO) mice demonstrate an impaired extinction of morphine conditioned place preference (CPP). Because the medial prefrontal cortex (mPFC) has been implicated in extinction learning, we tested whether Narp cells in this region play a role in the extinction of morphine CPP. We found that intracranial injections of adenoassociated virus (AAV) expressing wild-type (WT) Narp into the mPFC of Narp KO mice rescued the extinction and the injection of AAV expressing a dominant negative form of Narp (NarpN) into the mPFC of WT mice impaired the extinction of morphine CPP. These findings suggest that Narp in the mPFC mediates the extinction of morphine CPP.

Gene expression patterns in the hippocampus during the development and aging of Glud1 (Glutamate Dehydrogenase 1) transgenic and wild type mice.

PubMed

Wang, Xinkun; Patel, Nilam D; Hui, Dongwei; Pal, Ranu; Hafez, Mohamed M; Sayed-Ahmed, Mohamed M; Al-Yahya, Abdulaziz A; Michaelis, Elias K

2014-03-04

Extraneuronal levels of the neurotransmitter glutamate in brain rise during aging. This is thought to lead to synaptic dysfunction and neuronal injury or death. To study the effects of glutamate hyperactivity in brain, we created transgenic (Tg) mice in which the gene for glutamate dehydrogenase (Glud1) is over-expressed in neurons and in which such overexpression leads to excess synaptic release of glutamate. In this study, we analyzed whole genome expression in the hippocampus, a region important for learning and memory, of 10 day to 20 month old Glud1 and wild type (wt) mice. During development, maturation and aging, both Tg and wt exhibited decreases in the expression of genes related to neurogenesis, neuronal migration, growth, and process elongation, and increases in genes related to neuro-inflammation, voltage-gated channel activity, and regulation of synaptic transmission. Categories of genes that were differentially expressed in Tg vs. wt during development were: synaptic function, cytoskeleton, protein ubiquitination, and mitochondria; and, those differentially expressed during aging were: synaptic function, vesicle transport, calcium signaling, protein kinase activity, cytoskeleton, neuron projection, mitochondria, and protein ubiquitination. Overall, the effects of Glud1 overexpression on the hippocampus transcriptome were greater in the mature and aged than the young. Glutamate hyperactivity caused gene expression changes in the hippocampus at all ages. Some of these changes may result in premature brain aging. The identification of these genomic expression differences is important in understanding the effects of glutamate dysregulation on neuronal function during aging or in neurodegenerative diseases.

Photo dynamics of BLUF domain mutant H44R of AppA from Rhodobacter sphaeroides

NASA Astrophysics Data System (ADS)

Zirak, P.; Penzkofer, A.; Hegemann, P.; Mathes, T.

2007-05-01

The photo-cycle dynamics of the H44R mutant of the BLUF domain of the transcriptional anti-repressor protein AppA (AppA-H44R) from the non-sulfur anoxyphototropic purple bacterium Rhodobacter sphaeroides is studied in order to gain information on the involvement of His44 in the photo-cyclic mechanism of the AppA BLUF domain and to add information to the involved processes. The amino acid residue histidine at position 44 is replaced by arginine. A 12 nm red-shifted signalling state is formed upon blue-light excitation, while in wild-type AppA (AppA-wt) the red-shift is 16 nm. The recovery to the receptor dark state is approximately a factor of 2.5 faster ( τrec ≈ 6.5 min) than the recovery of the wild-type counterpart. Extended light exposure of the mutant causes photo-degradation of flavin (mainly free flavin conversion to lumichrome and re-equilibration between free and non-covalently bound flavin) and protein aggregation (showing up as light scattering). No photo-degradation was observed for AppA-wt. The quantum efficiency of signalling-state formation determined by intensity dependent absorption measurements is found to be ϕs ≈ 0.3 (for AppA-wt: ϕs ≈ 0.24). A two-component single-exponential fluorescence relaxation was observed, which is interpreted as fast fluorescence quenching to an equilibrium value by photo-induced electron transfer followed by slower fluorescence decay due to charge recombination. Based on the experimental findings, an extended photo-cycle model for BLUF domains is proposed.

Targeting glutamine metabolism in myeloproliferative neoplasms

PubMed Central

Zhan, Huichun; Ciano, Kristen; Dong, Katherine; Zucker, Stanley

2016-01-01

JAK2V617F mutation can be detected in the majority of myeloproliferative neoplasm (MPN) patients. The JAK2 inhibitor Ruxolitinib is the first FDA-approved treatment for MPNs. However, its use is limited by various dose related toxicities. Here, we studied the metabolic state and glutamine metabolism of BaF3-hEPOR-JAK2V617F and BaF3-hEPOR-JAK2WT cells. We found that the JAK2V617F-mutant cells were associated with increased oxygen consumption rate and extracellular acidification rate than the JAK2WT cells and there was an increased glutamine metabolism in JAK2V617F-mutant cells compared to wild-type cells. Glutaminase (GLS), the key enzyme in gluta-mine metabolism, was upregulated in the JAK2V617F-mutant BaF3 cells compared to the JAK2WT BaF3 cells. In MPN patient peripheral blood CD34+ cells, GLS expression was increased in JAK2V617F-mutant progenitor cells compared to JAK2 wild-type progenitor cells from the same patients and GLS levels were increased at the time of disease progression compared to at earlier time points. Moreover, GLS inhibitor increased the growth inhibitory effect of Ruxolitinib in both JAK2V617F-mutant cell lines and peripheral blood CD34+ cells from MPN patients. Therefore, GLS inhibitor should be further explored to enhance the therapeutic effectiveness of JAK2 inhibitor and allow the administration of lower doses of the drug to avoid its toxicity. PMID:26227854

Expression of interferon-induced antiviral genes is delayed in a STAT1 knockout mouse model of Crimean-Congo hemorrhagic fever.

PubMed

Bowick, Gavin C; Airo, Adriana M; Bente, Dennis A

2012-06-19

Crimean Congo hemorrhagic fever (CCHF) is a tick-borne hemorrhagic zoonosis associated with high mortality. Pathogenesis studies and the development of vaccines and antivirals against CCHF have been severely hampered by the lack of suitable animal model. We recently developed and characterized a mature mouse model for CCHF using mice carrying STAT1 knockout (KO). Given the importance of interferons in controlling viral infections, we investigated the expression of interferon pathway-associated genes in KO and wild-type (WT) mice challenged with CCHF virus. We expected that the absence of the STAT1 protein would result in minimal expression of IFN-related genes. Surprisingly, the KO mice showed high levels of IFN-stimulated gene expression, beginning on day 2 post-infection, while in WT mice challenged with virus the same genes were expressed at similar levels on day 1. We conclude that CCHF virus induces similar type I IFN responses in STAT1 KO and WT mice, but the delayed response in the KO mice permits rapid viral dissemination and fatal illness.

Stromal matrix metalloproteinase 2 regulates collagen expression and promotes the outgrowth of experimental metastases.

PubMed

Bates, Andreia L; Pickup, Michael W; Hallett, Miranda A; Dozier, E Ashley; Thomas, Stacy; Fingleton, Barbara

2015-04-01

Breast cancer survival rates decrease from 99% for patients with local disease to 25% for those with distant metastases. Matrix metalloproteinases (MMPs), including MMP2, are associated with metastatic progression. We found that loss of host MMP2 reduces the proliferation of experimental metastases in the lungs and identified fibroblasts in tumour-bearing lungs as the major source of MMP2. In vitro, spheroidal mammary tumour growth was increased by co-culture with control fibroblasts isolated from tumour-bearing lungs, but not when fibroblasts with stable Mmp2 knockdown were used. This result prompted us to assess whether MMP2 was responsible for a tumour-proliferative, activated fibroblast phenotype. To test this, we evaluated: (a) fibroblasts from wild-type tumour-bearing lungs, with or without shRNA-mediated MMP2 knockdown; and (b) normal, quiescent fibroblasts isolated from either WT or Mmp2(-/-) mice. Quantitative PCR revealed that Mmp2 knockdown attenuated expression of two markers of activation (α-smooth muscle actin and vimentin), but there was minimal expression in quiescent WT or Mmp2(-/-) fibroblasts, as expected. Placing quiescent fibroblasts under activating conditions led to increases in activation-associated transcripts in WT but not Mmp2(-/-) fibroblasts. Additionally, Mmp2 knockdown fibroblasts showed significantly decreased expression of the matrix transcripts collagen I, collagen IV and fibronectin. Addition of active TGFβ was sufficient to rescue the MMP2-dependent collagen I and IV expression, while MMP2-induced collagen expression was blocked by the addition of TGFβ1-neutralizing antibody. Gene expression data in stromal cells of human breast cancers reveal that MMP2 expression is also positively correlated with activation and matrix transcripts. Thus, we present a model whereby MMP2 production in tumour fibroblasts is important for TGFβ1 activity and subsequent activation of fibroblasts to a matrix-producing, proliferation-supportive phenotype. Overall, our results reveal a previously undefined role for MMP2 in metastatic outgrowth mediated by fibroblasts, and extend the mechanisms by which MMPs contribute to tumour progression. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The Active Form of the Saccharomyces cerevisiae Ribonucleotide Reductase Small Subunit Is a Heterodimer in Vitro and in Vivo†

PubMed Central

Perlstein, Deborah L.; Ge, Jie; Ortigosa, Allison D.; Robblee, John H.; Zhang, Zhen; Huang, Mingxia; Stubbe, JoAnne

2015-01-01

The class I ribonucleotide reductases (RNRs) are composed of two homodimeric subunits: R1 and R2. R2 houses a diferric-tyrosyl radical (Y•) cofactor. Saccharomyces cerevisiae has two R2s: Y2 (β2) and Y4 (β′2). Y4 is an unusual R2 because three residues required for iron binding have been mutated. While the heterodimer (ββ′) is thought to be the active form, several rnr4Δ strains are viable. To resolve this paradox, N-terminally epitope-tagged β and β′ were expressed in E. coli or integrated into the yeast genome. In vitro exchange studies reveal that when apo-(His6)-β2 (Hisβ2) is mixed with β′2, apo-Hisββ′ forms quantitatively within 2 min. In contrast, holo-ββ′ fails to exchange with apo-Hisβ2 to form holo-Hisββ and β′2. Isolation of genomically encoded tagged β or β′ from yeast extracts gave a 1:1 complex of β and β′, suggesting that ββ′ is the active form. The catalytic activity, protein concentrations, and Y• content of the rnr4Δ and wild type (wt) strains were compared to clarify the role of β′ in vivo. The Y• content of rnr4Δ is 15-fold less than that of wt, consistent with the observed low activity of rnr4Δ extracts (<0.01 nmol min−1 mg−1) versus wt (0.06 ± 0.01 nmol min−1 mg−1). FLAGβ2 isolated from the rnr4Δ strain has a specific activity of 2 nmol min−1 mg−1, similar to that of reconstituted apo-Hisβ2 (10 nmol min−1 mg−1), but significantly less than holo-Hisββ′ (~2000 nmol min−1 mg−1). These studies together demonstrate that β′ plays a crucial role in cluster assembly in vitro and in vivo and that the active form of the yeast R2 is ββ′. PMID:16285741

Expression of human cationic trypsinogen (PRSS1) in murine acinar cells promotes pancreatitis and apoptotic cell death

PubMed Central

Athwal, T; Huang, W; Mukherjee, R; Latawiec, D; Chvanov, M; Clarke, R; Smith, K; Campbell, F; Merriman, C; Criddle, D; Sutton, R; Neoptolemos, J; Vlatković, N

2014-01-01

Hereditary pancreatitis (HP) is an autosomal dominant disease that displays the features of both acute and chronic pancreatitis. Mutations in human cationic trypsinogen (PRSS1) are associated with HP and have provided some insight into the pathogenesis of pancreatitis, but mechanisms responsible for the initiation of pancreatitis have not been elucidated and the role of apoptosis and necrosis has been much debated. However, it has been generally accepted that trypsinogen, prematurely activated within the pancreatic acinar cell, has a major role in the initiation process. Functional studies of HP have been limited by the absence of an experimental system that authentically mimics disease development. We therefore developed a novel transgenic murine model system using wild-type (WT) human PRSS1 or two HP-associated mutants (R122H and N29I) to determine whether expression of human cationic trypsinogen in murine acinar cells promotes pancreatitis. The rat elastase promoter was used to target transgene expression to pancreatic acinar cells in three transgenic strains that were generated: Tg(Ela-PRSS1)NV, Tg(Ela-PRSS1*R122H)NV and Tg(Ela-PRSS1*N29I)NV. Mice were analysed histologically, immunohistochemically and biochemically. We found that transgene expression is restricted to pancreatic acinar cells and transgenic PRSS1 proteins are targeted to the pancreatic secretory pathway. Animals from all transgenic strains developed pancreatitis characterised by acinar cell vacuolisation, inflammatory infiltrates and fibrosis. Transgenic animals also developed more severe pancreatitis upon treatment with low-dose cerulein than controls, displaying significantly higher scores for oedema, inflammation and overall histopathology. Expression of PRSS1, WT or mutant, in acinar cells increased apoptosis in pancreatic tissues and isolated acinar cells. Moreover, studies of isolated acinar cells demonstrated that transgene expression promotes apoptosis rather than necrosis. We therefore conclude that expression of WT or mutant human PRSS1 in murine acinar cells induces apoptosis and is sufficient to promote spontaneous pancreatitis, which is enhanced in response to cellular insult. PMID:24722290

Proteomics of Aspergillus fumigatus Conidia-containing Phagolysosomes Identifies Processes Governing Immune Evasion.

PubMed

Schmidt, Hella; Vlaic, Sebastian; Krüger, Thomas; Schmidt, Franziska; Balkenhol, Johannes; Dandekar, Thomas; Guthke, Reinhard; Kniemeyer, Olaf; Heinekamp, Thorsten; Brakhage, Axel A

2018-06-01

Invasive infections by the human pathogenic fungus Aspergillus fumigatus start with the outgrowth of asexual, airborne spores (conidia) into the lung tissue of immunocompromised patients. The resident alveolar macrophages phagocytose conidia, which end up in phagolysosomes. However, A. fumigatus conidia resist phagocytic degradation to a certain degree. This is mainly attributable to the pigment 1,8-dihydroxynaphthalene (DHN) melanin located in the cell wall of conidia, which manipulates the phagolysosomal maturation and prevents their intracellular killing. To get insight in the underlying molecular mechanisms, we comparatively analyzed proteins of mouse macrophage phagolysosomes containing melanized wild-type (wt) or nonmelanized pksP mutant conidia. For this purpose, a protocol to isolate conidia-containing phagolysosomes was established and a reference protein map of phagolysosomes was generated. We identified 637 host and 22 A. fumigatus proteins that were differentially abundant in the phagolysosome. 472 of the host proteins were overrepresented in the pksP mutant and 165 in the wt conidia-containing phagolysosome. Eight of the fungal proteins were produced only in pksP mutant and 14 proteins in wt conidia-containing phagolysosomes. Bioinformatical analysis compiled a regulatory module, which indicates host processes affected by the fungus. These processes include vATPase-driven phagolysosomal acidification, Rab5 and Vamp8-dependent endocytic trafficking, signaling pathways, as well as recruitment of the Lamp1 phagolysosomal maturation marker and the lysosomal cysteine protease cathepsin Z. Western blotting and immunofluorescence analyses confirmed the proteome data and moreover showed differential abundance of the major metabolic regulator mTOR. Taken together, with the help of a protocol optimized to isolate A. fumigatus conidia-containing phagolysosomes and a potent bioinformatics algorithm, we were able to confirm A. fumigatus conidia-dependent modification of phagolysosomal processes that have been described before and beyond that, identify pathways that have not been implicated in A. fumigatus evasion strategy, yet.Mass spectrometry proteomics data are available via ProteomeXchange with identifiers PXD005724 and PXD006134. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Overexpression of 3-Ketoacyl-Acyl-Carrier Protein Synthase IIIs in Plants Reduces the Rate of Lipid Synthesis1

PubMed Central

Dehesh, Katayoon; Tai, Heeyoung; Edwards, Patricia; Byrne, James; Jaworski, Jan G.

2001-01-01

A cDNA coding for 3-ketoacyl-acyl-carrier protein (ACP) synthase III (KAS III) from spinach (Spinacia oleracea; So KAS III) was used to isolate two closely related KAS III clones (Ch KAS III-1 and Ch KAS III-2) from Cuphea hookeriana. Both Ch KAS IIIs are expressed constitutively in all tissues examined. An increase in the levels of 16:0 was observed in tobacco (Nicotiana tabacum, WT-SR) leaves overexpressing So KAS III when under the control of the cauliflower mosaic virus-35S promoter and in Arabidopsis and rapeseed (Brassica napus) seeds overexpressing either of the Ch KAS IIIs driven by napin. These data indicate that this enzyme has a universal role in fatty acid biosynthesis, irrespective of the plant species from which it is derived or the tissue in which it is expressed. The transgenic rapeseed seeds also contained lower levels of oil as compared with the wild-type levels. In addition, the rate of lipid synthesis in transgenic rapeseed seeds was notably slower than that of the wild-type seeds. The results of the measurements of the levels of the acyl-ACP intermediates as well as any changes in levels of other fatty acid synthase enzymes suggest that malonyl-ACP, the carbon donor utilized by all the 3- ketoacyl-ACP synthases, is limiting in the transgenic plants. This further suggests that malonyl-coenzyme A is a potential limiting factor impacting the final oil content as well as further extension of 16:0. PMID:11161065

Overexpression of 3-ketoacyl-acyl-carrier protein synthase IIIs in plants reduces the rate of lipid synthesis.

PubMed

Dehesh, K; Tai, H; Edwards, P; Byrne, J; Jaworski, J G

2001-02-01

A cDNA coding for 3-ketoacyl-acyl-carrier protein (ACP) synthase III (KAS III) from spinach (Spinacia oleracea; So KAS III) was used to isolate two closely related KAS III clones (Ch KAS III-1 and Ch KAS III-2) from Cuphea hookeriana. Both Ch KAS IIIs are expressed constitutively in all tissues examined. An increase in the levels of 16:0 was observed in tobacco (Nicotiana tabacum, WT-SR) leaves overexpressing So KAS III when under the control of the cauliflower mosaic virus-35S promoter and in Arabidopsis and rapeseed (Brassica napus) seeds overexpressing either of the Ch KAS IIIs driven by napin. These data indicate that this enzyme has a universal role in fatty acid biosynthesis, irrespective of the plant species from which it is derived or the tissue in which it is expressed. The transgenic rapeseed seeds also contained lower levels of oil as compared with the wild-type levels. In addition, the rate of lipid synthesis in transgenic rapeseed seeds was notably slower than that of the wild-type seeds. The results of the measurements of the levels of the acyl-ACP intermediates as well as any changes in levels of other fatty acid synthase enzymes suggest that malonyl-ACP, the carbon donor utilized by all the 3- ketoacyl-ACP synthases, is limiting in the transgenic plants. This further suggests that malonyl-coenzyme A is a potential limiting factor impacting the final oil content as well as further extension of 16:0.

Sabin Vaccine Reversion in the Field: a Comprehensive Analysis of Sabin-Like Poliovirus Isolates in Nigeria.

PubMed

Famulare, Michael; Chang, Stewart; Iber, Jane; Zhao, Kun; Adeniji, Johnson A; Bukbuk, David; Baba, Marycelin; Behrend, Matthew; Burns, Cara C; Oberste, M Steven

2016-01-01

To assess the dynamics of genetic reversion of live poliovirus vaccine in humans, we studied molecular evolution in Sabin-like poliovirus isolates from Nigerian acute flaccid paralysis cases obtained from routine surveillance. We employed a novel modeling approach to infer substitution and recombination rates from whole-genome sequences and information about poliovirus infection dynamics and the individual vaccination history. We confirmed observations from a recent vaccine trial that VP1 substitution rates are increased for Sabin-like isolates relative to the rate for the wild type due to increased nonsynonymous substitution rates. We also inferred substitution rates for attenuating nucleotides and confirmed that reversion can occur in days to weeks after vaccination. We combine our observations for Sabin-like virus evolution with the molecular clock for VP1 of circulating wild-type strains to infer that the mean time from the initiating vaccine dose to the earliest detection of circulating vaccine-derived poliovirus (cVDPV) is 300 days for Sabin-like virus type 1, 210 days for Sabin-like virus type 2, and 390 days for Sabin-like virus type 3. Phylogenetic relationships indicated transient local transmission of Sabin-like virus type 3 and, possibly, Sabin-like virus type 1 during periods of low wild polio incidence. Comparison of Sabin-like virus recombinants with known Nigerian vaccine-derived poliovirus recombinants shows that while recombination with non-Sabin enteroviruses is associated with cVDPV, the recombination rates are similar for Sabin isolate-Sabin isolate and Sabin isolate-non-Sabin enterovirus recombination after accounting for the time from dosing to the time of detection. Our study provides a comprehensive picture of the evolutionary dynamics of the oral polio vaccine in the field. The global polio eradication effort has completed its 26th year. Despite success in eliminating wild poliovirus from most of the world, polio persists in populations where logistical, social, and political factors have not allowed vaccination programs of sustained high quality. One issue of critical importance is eliminating circulating vaccine-derived polioviruses (cVDPVs) that have properties indistinguishable from those of wild poliovirus and can cause paralytic disease. cVDPV emerges due to the genetic instability of the Sabin viruses used in the oral polio vaccine (OPV) in populations that have low levels of immunity to poliovirus. However, the dynamics responsible are incompletely understood because it has historically been difficult to gather and interpret data about evolution of the Sabin viruses used in OPV in regions where cVDPV has occurred. This study is the first to combine whole-genome sequencing of poliovirus isolates collected during routine surveillance with knowledge about the intrahost dynamics of poliovirus to provide quantitative insight into polio vaccine evolution in the field. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Aldosterone-Induced Vascular Remodeling and Endothelial Dysfunction Require Functional Angiotensin Type 1a Receptors.

PubMed

Briet, Marie; Barhoumi, Tlili; Mian, Muhammad Oneeb Rehman; Coelho, Suellen C; Ouerd, Sofiane; Rautureau, Yohann; Coffman, Thomas M; Paradis, Pierre; Schiffrin, Ernesto L

2016-05-01

We investigated the role of angiotensin type 1a receptors (AGTR1a) in vascular injury induced by aldosterone activation of mineralocorticoid receptors in Agtr1a(-/-) and wild-type (WT) mice infused with aldosterone for 14 days while receiving 1% NaCl in drinking water. Aldosterone increased systolic blood pressure (BP) by ≈30 mm Hg in WT mice and ≈50 mm Hg in Agtr1a(-/-) mice. Aldosterone induced aortic and small artery remodeling, impaired endothelium-dependent relaxation in WT mice, and enhanced fibronectin and collagen deposition and vascular inflammation. None of these vascular effects were observed in Agtr1a(-/-) mice. Aldosterone effects were prevented by the AGTR1 antagonist losartan in WT mice. In contrast to aldosterone, norepinephrine caused similar BP increase and mesenteric artery remodeling in WT and Agtr1a(-/-) mice. Agtr1a(-/-) mice infused with aldosterone did not increase sodium excretion in response to a sodium chloride challenge, suggesting that sodium retention could contribute to the exaggerated BP rise induced by aldosterone. Agtr1a(-/-) mice had decreased mesenteric artery expression of the calcium-activated potassium channel Kcnmb1, which may enhance myogenic tone and together with sodium retention, exacerbate BP responses to aldosterone/salt in Agtr1a(-/-) mice. We conclude that although aldosterone activation of mineralocorticoid receptors raises BP more in Agtr1a(-/-) mice, AGTR1a is required for mineralocorticoid receptor stimulation to induce vascular remodeling and inflammation and endothelial dysfunction. © 2016 American Heart Association, Inc.

Renal Liver-Type Fatty Acid Binding Protein (L-FABP) Attenuates Acute Kidney Injury in Aristolochic Acid Nephrotoxicity

PubMed Central

Matsui, Katsuomi; Kamijo-Ikemorif, Atsuko; Sugaya, Takeshi; Yasuda, Takashi; Kimura, Kenjiro

2011-01-01

Injection of aristolochic acid (AA) in mice causes AA-induced nephrotoxicity, in which oxidative stress contributes to development of tubulointerstitial damage (TID). Liver-type fatty acid binding protein (L-FABP) is expressed in human proximal tubules and has an endogenous antioxidative function. The renoprotection of renal L-FABP was examined in a model of AA-induced nephrotoxicity. Established human L-FABP (hL-FABP) transgenic (Tg) mice and wild-type (WT) mice were treated with AA for up to 5 days. Mice were sacrificed on days 1, 3, and 5 after the start of AA injection. Although mouse L-FABP was not expressed in proximal tubules of WT mice, hL-FABP was expressed in proximal tubules of Tg mice. The expression of renal hL-FABP was significantly increased in Tg mice administered AA (Tg-AA), compared with the control (saline-treated Tg mice). In WT-AA mice, there was high urinary excretion of Nε-(hexanoyl)-lysine, the production of heme oxygenase-1 and receptor for advanced glycation end products increased, and TID was provoked. In contrast, renal hL-FABP in Tg-AA mice suppressed production of Nε-(hexanoyl)lysine, heme oxygenase-1, and receptor for advanced glycation end products. Renal dysfunction was significantly milder in Tg-AA mice than in WT-AA mice. The degree of TID was significantly attenuated in Tg-AA mice, compared with WT-AA. In conclusion, renal hL-FABP reduced the oxidative stress in AA-induced nephrotoxicity and attenuated TID. PMID:21356355

Pglyrp-Regulated Gut Microflora Prevotella falsenii, Parabacteroides distasonis and Bacteroides eggerthii Enhance and Alistipes finegoldii Attenuates Colitis in Mice

PubMed Central

Dziarski, Roman; Dowd, Scot E.; Gupta, Dipika

2016-01-01

Dysbiosis is a hallmark of inflammatory bowel disease (IBD), but it is unclear which specific intestinal bacteria predispose to and which protect from IBD and how they are regulated. Peptidoglycan recognition proteins (Pglyrps) are antibacterial, participate in maintaining intestinal microflora, and modulate inflammatory responses. Mice deficient in any one of the four Pglyrp genes are more sensitive to dextran sulfate sodium (DSS)-induced colitis, and stools from Pglyrp-deficient mice transferred to wild type (WT) germ-free mice predispose them to much more severe colitis than stools from WT mice. However, the identities of these Pglyrp-regulated bacteria that predispose Pglyrp-deficient mice to colitis or protect WT mice from colitis are not known. Here we identified significant changes in β-diversity of stool bacteria in Pglyrp-deficient mice compared with WT mice. The most consistent changes in microbiome in all Pglyrp-deficient mice were in Bacteroidales, from which we selected four species, two with increased abundance (Prevotella falsenii and Parabacteroides distasonis) and two with decreased abundance (Bacteroides eggerthii and Alistipes finegoldii). We then gavaged WT mice with stock type strains of these species to test the hypothesis that they predispose to or protect from DSS-induced colitis. P. falsenii, P. distasonis, and B. eggerthii all enhanced DSS-induced colitis in both WT mice with otherwise undisturbed intestinal microflora and in WT mice with antibiotic-depleted intestinal microflora. By contrast, A. finegoldii (which is the most abundant species in WT mice) attenuated DSS-induced colitis both in WT mice with otherwise undisturbed intestinal microflora and in WT mice with antibiotic-depleted intestinal microflora, similar to the colitis protective effect of the entire normal microflora. These results identify P. falsenii, P. distasonis, and B. eggerthii as colitis-promoting species and A. finegoldii as colitis-protective species. PMID:26727498

Retention during processing and bioaccessibility of β-carotene in high β-carotene transgenic cassava root.

PubMed

Failla, Mark L; Chitchumroonchokchai, Chureeporn; Siritunga, Dimuth; De Moura, Fabiana F; Fregene, Martin; Manary, Mark J; Sayre, Richard T

2012-04-18

Cassava is a root crop that serves as a primary caloric source for many African communities despite its low content of β-carotene (βC). Carotenoid content of roots from wild type (WT) and three transgenic lines with high βC were compared after cooking and preparation of nonfermented and fermented flours according to traditional African methods. The various methods of processing all decreased βC content per gram dry weight regardless of genotype. The greatest loss of βC occurred during preparation of gari (dry fermentation followed by roasting) from WT and transgenic lines. The quantities of βC in cooked transgenic cassava root that partitioned into mixed micelles during in vitro digestion and transported into Caco-2 cells were significantly greater than those for identically processed WT root. These results suggest that transgenic high βC cassava will provide individuals with greater quantities of bioaccessible βC.

A Field Trial of TCE Phytoremediation by Genetically Modified Poplars Expressing Cytochrome P450 2E1.

PubMed

Legault, Emily K; James, C Andrew; Stewart, Keith; Muiznieks, Indulis; Doty, Sharon L; Strand, Stuart E

2017-06-06

A controlled field study was performed to evaluate the effectiveness of transgenic poplars for phytoremediation. Three hydraulically contained test beds were planted with 12 transgenic poplars, 12 wild type (WT) poplars, or left unplanted, and dosed with equivalent concentrations of trichloroethylene (TCE). Removal of TCE was enhanced in the transgenic tree bed, but not to the extent of the enhanced removal observed in laboratory studies. Total chlorinated ethene removal was 87% in the CYP2E1 bed, 85% in the WT bed, and 34% in the unplanted bed in 2012. Evapotranspiration of TCE from transgenic leaves was reduced by 80% and diffusion of TCE from transgenic stems was reduced by 90% compared to WT. Cis-dichloroethene and vinyl chloride levels were reduced in the transgenic tree bed. Chloride ion accumulated in the planted beds corresponding to the TCE loss, suggesting that contaminant dehalogenation was the primary loss fate.

p27(kip1) Knockout enhances collateralization in response to hindlimb ischemia.

PubMed

Ankri-Eliahoo, Galit; Weitz, Kevin; Cox, Timothy C; Tang, Gale L

2016-05-01

The natural response to arterial occlusive disease is enlargement of collaterals; however, the molecular factors that control collateralization are not well understood. The gene p27(Kip1) (p27) affects human response to arterial injury. Previous studies have shown that overexpression of p27 inhibits vascular endothelial and vascular smooth muscle cell (VSMC) proliferation and angiogenesis. To test the hypothesis that knockout of p27 would improve collateralization in reaction to ischemia, we performed in vivo and in vitro experiments using p27 knockout (p27(-/-)) and wild-type (wt) mice. Hindlimb ischemia was induced by left femoral artery ligation in p27(-/-) and wt (C57BL/6) female mice. The mice underwent weekly laser Doppler perfusion imaging of the footpads until sacrifice on postoperative day 28 followed by microcomputed tomography scanning of both hindlimbs. VSMCs were isolated from p27(-/-) and wt mice and used in migration and gel contraction assays in the absence and presence of the nonspecific matrix metalloproteinase (MMP) inhibitor BB94. MMP-2 and MMP-9 messenger RNA (mRNA) expression was measured by quantitative reverse transcription-polymerase chain reaction in p27(-/-) and wt VSMCs. p27(-/-) mice reperfused more effectively than wt mice by laser Doppler starting from day 7 (ischemic/nonischemic ratio, 0.33 ± 0.02 vs 0.25 ± 0.02; P < .05) and continuing through day 28 (0.45 ± 0.04 vs 0.31 ± 0.04; P < .05). The gracilis collateral diameter was similar for the nonischemic hindlimbs of the p27(-/-) and wt mice, and this collateral pathway increased similarly after ischemia as assessed by microcomputed tomography. However, the p27(-/-) mice significantly enlarged a novel collateral pathway that bridged directly between the femoral artery proximal to the ligation site and the saphenous or popliteal artery distal to the ligation site more than wt mice (158 ± 18.3 vs 82 ± 22 μm; P < .001). p27(-/-) VSMCs migrated more (79% ± 5% vs 56% ± 6%; P < .05) and caused more gel contraction (18% ± 5% of the initial area vs 43% ± 4%; P < .05) than wt cells. Migration and collagen contraction were abolished in p27(-/-) and wt cells by MMP inhibition. p27(-/-) cells expressed significantly more MMP-2 mRNA than wt cells did. Knockout of p27 enhances arterial collateralization in response to hindlimb ischemia through enlargement of a new collateral pathway. In vitro, knockout of p27 increases collagen gel contraction in addition to stimulating VSMC migration. We speculate that p27 may affect collateralization through its role in regulating MMP-2 expression. Published by Elsevier Inc.

Genetics of Eosinophilic Esophagitis

DTIC Science & Technology

2012-03-01

cells, left panel, and differential cell counts , right panel) in bronchoalveolar lavage fluid (BALF) in IL- 21R-/- mice compared to wild-type (WT). A...positive skin tests. A third group (30%) had multiple sensitivities to foods and pollens (GM total IgE 285 IU/ ml). Tests for IgE to carbohydrate antigens...milk sensitized, and those with multiple pollen allergies. The frequent occurrence of multiple associated sensitivities to grains, legumes, molds, and