Cloning and study of the pectate lyase gene of Erwinia carotovora

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

Bukanov, N.O.; Fonshtein, M.Yu.; Evtushenkov, A.N.

1986-04-01

The cloning of the gene of a secretable protein of Erwinia carotovora, pectate lyase, in Escherichia coli was described. Primary cloning was conducted using the phage vector lambda 47.1. In the gene library of E. carotovora obtained, eight phages carrying the gene sought were identified according to the appearance of enzymatic activity of the gene product, pectate lyase, in situ. The BamHI fragment of DNA, common to all these phages, was recloned on the plasmid pUC19. It was shown that the cloned pectate lyase gene is represented on the E. carotovora chromosome in one copy. Methods of production of representativemore » gene libraries on phage vectors from no less than 1 ..mu..g of cloned DNA even for the genomes of eukaryotes have now been developed. Vectors have been created, for example, lambda 47.1, permitting the selection only of hybrid molecules. A number of methods have been developed for the search for a required gene in the library, depending on whether the cloned gene can be expressed or not, and if it can, what properties it will impart to the hybrid clone containing it.« less

Comparative study on the in vitro effects of Pseudomonas aeruginosa and seaweed alginates on human gut microbiota

PubMed Central

Bai, Shaofeng; Chen, Huahai; Zhu, Liying; Liu, Wei; Yu, Hongwei D.; Wang, Xin; Yin, Yeshi

2017-01-01

Alginates pertain to organic polysaccharides that have been extensively used in food- and medicine-related industries. The present study obtained alginates from an alginate overproducing Pseudomonas aeruginosa PAO1 mutant by screening transposon mutagenesis libraries. The interaction between bacterial and seaweed alginates and gut microbiota were further studied by using an in vitro batch fermentation system. Thin-layer chromatography (TLC) analysis indicated that both bacterial and seaweed alginates can be completely degraded by fecal bacteria isolated from study volunteers, indicating that a minor structural difference between bacterial and seaweed alginates (O-acetylation and lack of G-G blocks) didn’t affect the digestion of alginates by human microbiota. Although, the digestion of bacterial and seaweed alginates was attributed to different Bacteroides xylanisolvens strains, they harbored similar alginate lyase genes. Genus Bacteroides with alginate-degrading capability were enriched in growth medium containing bacterial or seaweed alginates after in vitro fermentation. Short-chain fatty acid (SCFA) production in both bacterial and seaweed alginates was also comparable, but was significantly higher than the same medium using starch. In summary, the present study has isolated an alginate-overproducing P. aeruginosa mutant strain. Both seaweed and bacterial alginates were degraded by human gut microbiota, and their regulatory function on gut microbiota was similar. PMID:28170428

Comparative study on the in vitro effects of Pseudomonas aeruginosa and seaweed alginates on human gut microbiota.

PubMed

Bai, Shaofeng; Chen, Huahai; Zhu, Liying; Liu, Wei; Yu, Hongwei D; Wang, Xin; Yin, Yeshi

2017-01-01

Alginates pertain to organic polysaccharides that have been extensively used in food- and medicine-related industries. The present study obtained alginates from an alginate overproducing Pseudomonas aeruginosa PAO1 mutant by screening transposon mutagenesis libraries. The interaction between bacterial and seaweed alginates and gut microbiota were further studied by using an in vitro batch fermentation system. Thin-layer chromatography (TLC) analysis indicated that both bacterial and seaweed alginates can be completely degraded by fecal bacteria isolated from study volunteers, indicating that a minor structural difference between bacterial and seaweed alginates (O-acetylation and lack of G-G blocks) didn't affect the digestion of alginates by human microbiota. Although, the digestion of bacterial and seaweed alginates was attributed to different Bacteroides xylanisolvens strains, they harbored similar alginate lyase genes. Genus Bacteroides with alginate-degrading capability were enriched in growth medium containing bacterial or seaweed alginates after in vitro fermentation. Short-chain fatty acid (SCFA) production in both bacterial and seaweed alginates was also comparable, but was significantly higher than the same medium using starch. In summary, the present study has isolated an alginate-overproducing P. aeruginosa mutant strain. Both seaweed and bacterial alginates were degraded by human gut microbiota, and their regulatory function on gut microbiota was similar.

Identification of 2-keto-3-deoxy-d-Gluconate Kinase and 2-keto-3-deoxy-d-Phosphogluconate Aldolase in an Alginate-Assimilating Bacterium, Flavobacterium sp. Strain UMI-01

PubMed Central

Nishiyama, Ryuji; Inoue, Akira; Ojima, Takao

2017-01-01

Recently, we identified an alginate-assimilating gene cluster in the genome of Flavobacterium sp. strain UMI-01, a member of Bacteroidetes. Alginate lyase genes and a 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH) reductase gene in the cluster have already been characterized; however, 2-keto-3-deoxy-d-gluconate (KDG) kinase and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase genes, i.e., flkin and flald, still remained uncharacterized. The amino acid sequences deduced from flkin and flald showed low identities with those of corresponding enzymes of Saccharophagus degradans 2-40T, a member of Proteobacteria (Kim et al., Process Biochem., 2016). This led us to consider that the DEH-assimilating enzymes of Bacteroidetes species are somewhat deviated from those of Proteobacteria species. Thus, in the present study, we first assessed the characteristics in the primary structures of KDG kinase and KDG aldolase of the strain UMI-01, and then investigated the enzymatic properties of recombinant enzymes, recFlKin and recFlAld, expressed by an Escherichia coli expression system. Multiple-sequence alignment among KDG kinases and KDG aldolases from several Proteobacteria and Bacteroidetes species indicated that the strain UMI-01 enzymes showed considerably low sequence identities (15%–25%) with the Proteobacteria enzymes, while they showed relatively high identities (47%–68%) with the Bacteroidetes enzymes. Phylogenetic analyses for these enzymes indicated the distant relationship between the Proteobacteria enzymes and the Bacteroidetes enzymes, i.e., they formed distinct clusters in the phylogenetic tree. recFlKin and recFlAld produced with the genes flkin and flald, respectively, were confirmed to show KDG kinase and KDPG aldolase activities. Namely, recFlKin produced 1.7 mM KDPG in a reaction mixture containing 2.5 mM KDG and 2.5 mM ATP in a 90-min reaction, while recFlAld produced 1.2 mM pyruvate in the reaction mixture containing 5 mM KDPG at the equilibrium

Genome-wide characterization of the Pectate Lyase-like (PLL) genes in Brassica rapa.

PubMed

Jiang, Jingjing; Yao, Lina; Miao, Ying; Cao, Jiashu

2013-11-01

Pectate lyases (PL) depolymerize demethylated pectin (pectate, EC 4.2.2.2) by catalyzing the eliminative cleavage of α-1,4-glycosidic linked galacturonan. Pectate Lyase-like (PLL) genes are one of the largest and most complex families in plants. However, studies on the phylogeny, gene structure, and expression of PLL genes are limited. To understand the potential functions of PLL genes in plants, we characterized their intron-exon structure, phylogenetic relationships, and protein structures, and measured their expression patterns in various tissues, specifically the reproductive tissues in Brassica rapa. Sequence alignments revealed two characteristic motifs in PLL genes. The chromosome location analysis indicated that 18 of the 46 PLL genes were located in the least fractionated sub-genome (LF) of B. rapa, while 16 were located in the medium fractionated sub-genome (MF1) and 12 in the more fractionated sub-genome (MF2). Quantitative RT-PCR analysis showed that BrPLL genes were expressed in various tissues, with most of them being expressed in flowers. Detailed qRT-PCR analysis identified 11 pollen specific PLL genes and several other genes with unique spatial expression patterns. In addition, some duplicated genes showed similar expression patterns. The phylogenetic analysis identified three PLL gene subfamilies in plants, among which subfamily II might have evolved from gene neofunctionalization or subfunctionalization. Therefore, this study opens the possibility for exploring the roles of PLL genes during plant development.

Manipulation of Strawberry Fruit Softening by Antisense Expression of a Pectate Lyase Gene1

PubMed Central

Jiménez-Bermúdez, Silvia; Redondo-Nevado, José; Muñoz-Blanco, Juan; Caballero, José L.; López-Aranda, José M.; Valpuesta, Victoriano; Pliego-Alfaro, Fernando; Quesada, Miguel A.; Mercado, José A.

2002-01-01

Strawberry (Fragaria × ananassa, Duch., cv Chandler) is a soft fruit with a short postharvest life, mainly due to a rapid lost of firm texture. To control the strawberry fruit softening, we obtained transgenic plants that incorporate an antisense sequence of a strawberry pectate lyase gene under the control of the 35S promoter. Forty-one independent transgenic lines (Apel lines) were obtained, propagated in the greenhouse for agronomical analysis, and compared with control plants, non-transformed plants, and transgenic lines transformed with the pGUSINT plasmid. Total yield was significantly reduced in 33 of the 41 Apel lines. At the stage of full ripen, no differences in color, size, shape, and weight were observed between Apel and control fruit. However, in most of the Apel lines, ripened fruits were significantly firmer than controls. Six Apel lines were selected for further analysis. In all these lines, the pectate lyase gene expression in ripened fruit was 30% lower than in control, being totally suppressed in three of them. Cell wall material isolated from ripened Apel fruit showed a lower degree of in vitro swelling and a lower amount of ionically bound pectins than control fruit. An analysis of firmness at three different stages of fruit development (green, white, and red) showed that the highest reduction of softening in Apel fruit occurred during the transition from the white to the red stage. The postharvest softening of Apel fruit was also diminished. Our results indicate that pectate lyase gene is an excellent candidate for biotechnological improvement of fruit softening in strawberry. PMID:11842178

Expression and Bioinformatics Analysis of Pectate Lyase Gene from Bacillus subtilis521

NASA Astrophysics Data System (ADS)

Xiao, Jing; Lu, Fu-Ping; Li, Yu; Li, Jin-Ting

In order to exploit new genetic resources, Pectate lyase(PEL) gene was amplified by PCR using the genome DNA from an alkaline Bacillus subtilis521. The PCR product was inserted into pET22b(+) vector. The recombinant plasmids were cloned in E.coli DH5α and then expressed in E.coli BL21. When cultured in the optimized medium, the positive clones E.coli BL21(pET22b(+)pel)showed intracellular pectate lyase activity of 90.0 U/mL. It was indicated that we had obtained the correct PEL gene. The pel has an open reading frame of 1263 nucleotides and codes for a product of 420 amino acids with a calculated molecular mass of 45.5 kD. Based on computer assisted analysis, a signal peptides and two conserved domains were revealed. The sequence analysis for PEL showed that it shares 26-82% homology with other strains in GenBank. In addition, the advanced structure of PEL were also predicted and analysed. This study will help to the experimental design of PEL fermentation and production purification and enzyme evolution.

Characterization of the first alginolytic operons in a marine bacterium: from their emergence in marine Flavobacteriia to their independent transfers to marine Proteobacteria and human gut Bacteroides.

PubMed

Thomas, François; Barbeyron, Tristan; Tonon, Thierry; Génicot, Sabine; Czjzek, Mirjam; Michel, Gurvan

2012-09-01

Alginate constitutes a significant part of seaweed biomass and thus a crucial nutrient for numerous marine heterotrophic bacteria. However, the mechanisms for alginate assimilation remain largely unknown in marine microorganisms. We show here that the genome of the marine flavobacterium Zobellia galactanivorans contains seven putative alginate lyase genes, five of them localized within two clusters comprising additional carbohydrate-related genes. The transcription of these genes and the alginolytic activity were strongly induced when Z. galactanivorans used alginate as sole carbon source. These clusters were shown to be transcribed as polycistronic mRNAs and thus to constitute operons. Several candidate enzymes were successfully overexpressed in Escherichia coli, purified and their activity tested. Particularly, AlyA1, AlyA4, AlyA5 and AlyA7 are confirmed as active alginate lyases. Zg2622 and Zg2614 are a dehydrogenase and a kinase, respectively, further converting the terminal unsaturated monosaccharides released by alginate lyases into 2-keto-3-deoxy-6-phosphogluconate. In-depth phylogenomic analyses reveal that such alginolytic operons originated from an ancestral marine flavobacterium and were independently transferred to marine proteobacteria and Japanese gut Bacteroides. These bacteria thus gained the capacity to assimilate the main polysaccharide of brown algae, an adaptive advantage in coastal environments but also in the gut microbiota of specific human population. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

The Syndrome of 17,20 Lyase Deficiency

PubMed Central

2012-01-01

Context: Disorders of steroidogenesis have been instrumental in delineating human steroidogenic pathways. Each genetic disorder seemed to correspond to a different steroidogenic activity, helping to identify several enzymes. Beginning in 1972, several patients have been reported as having “17,20 lyase deficiency,” but there have been inconsistent genetic findings. Objective: This manuscript reviews the biochemistry, genetics, and clinical disorders of 17,20 lyase activity, which converts 21-carbon precursors of glucocorticoids to 19-carbon precursors of sex steroids. Findings: A single enzyme, cytochrome P450c17, catalyzes both 17α-hydroxylase activity and 17,20 lyase activity. The 17,20 lyase activity is especially sensitive to the activities of the accessory proteins P450 oxidoreductase and cytochrome b5. The first cases of genetically and biochemically proven 17,20 lyase deficiency were reported in 1997, in which specific P450c17 mutations were identified that lost 17,20 lyase activity but not 17α-hydroxylase activity when assayed in vitro. Subsequent work identified other P450c17 mutations and mutations in the genes encoding P450 oxidoreductase and cytochrome b5. Recently, the initially reported cases from 1972 were found to carry mutations in two aldo-keto reductases, AKR1C2 and AKR1C4. These AKR1C isozymes catalyze 3α-hydroxysteroid dehydrogenase activity in the so-called “backdoor pathway” by which the fetal testis produces dihydrotestosterone without the intermediacy of testosterone. Conclusions: 17,20 Lyase deficiency should be considered a syndrome with multiple causes, and not a single disease. Study of this very rare disorder has substantially advanced our understanding of the pathways, mechanisms, and control of androgen synthesis. Mutations in other, as-yet unidentified genes may also cause this phenotype. PMID:22072737

Molecular characterization of a Penicillium chrysogenum exo-rhamnogalacturonan lyase that is structurally distinct from other polysaccharide lyase family proteins.

PubMed

Iwai, Marin; Kawakami, Takuya; Ikemoto, Takeshi; Fujiwara, Daisuke; Takenaka, Shigeo; Nakazawa, Masami; Ueda, Mitsuhiro; Sakamoto, Tatsuji

2015-10-01

We previously described an endo-acting rhamnogalacturonan (RG) lyase, termed PcRGL4A, of Penicillium chrysogenum 31B. Here, we describe a second RG lyase, called PcRGLX. We determined the cDNA sequence of the Pcrglx gene, which encodes PcRGLX. Based on analyses using a BLAST search and a conserved domain search, PcRGLX was found to be structurally distinct from known RG lyases and might belong to a new polysaccharide lyase family together with uncharacterized fungal proteins of Nectria haematococca, Aspergillus oryzae, and Fusarium oxysporum. The Pcrglx cDNA gene product (rPcRGLX) expressed in Escherichia coli demonstrated specific activity against RG but not against homogalacturonan. Divalent cations were not essential for the enzymatic activity of rPcRGLX. rPcRGLX mainly released unsaturated galacturonosyl rhamnose (ΔGR) from RG backbones used as the substrate from the initial stage of the reaction, indicating that the enzyme can be classified as an exo-acting RG lyase (EC 4.2.2.24). This is the first report of an RG lyase with this mode of action in Eukaryota. rPcRGLX acted synergistically with PcRGL4A to degrade soybean RG and released ΔGR. This ΔGR was partially decorated with galactose (Gal) residues, indicating that rPcRGLX preferred oligomeric RGs to polymeric RGs, that the enzyme did not require Gal decoration of RG backbones for degradation, and that the enzyme bypassed the Gal side chains of RG backbones. These characteristics of rPcRGLX might be useful in the determination of complex structures of pectins.

Cationic carbon quantum dots derived from alginate for gene delivery: One-step synthesis and cellular uptake.

PubMed

Zhou, Jie; Deng, Wenwen; Wang, Yan; Cao, Xia; Chen, Jingjing; Wang, Qiang; Xu, Wenqian; Du, Pan; Yu, Qingtong; Chen, Jiaxin; Spector, Myron; Yu, Jiangnan; Xu, Ximing

2016-09-15

Carbon quantum dots (CQDs), unlike semiconductor quantum dots, possess fine biocompatibility, excellent upconversion properties, high photostability and low toxicity. Here, we report multifunctional CQDs which were developed using alginate, 3% hydrogen peroxide and double distilled water through a facile, eco-friendly and inexpensive one-step hydrothermal carbonization route. In this reaction, the alginate served as both the carbon source and the cationization agent. The resulting CQDs exhibited strong and stable fluorescence with water-dispersible and positively-charged properties which could serve as an excellent DNA condensation. As non-viral gene vector being used for the first time, the CQDs showed considerably high transfection efficiency (comparable to Lipofectamine2000 and significantly higher than PEI, p<0.05) and negligible toxicity. The photoluminescence properties of CQDs also permitted easy tracking of the cellular-uptake. The findings showed that both caveolae- and clathrin-mediated endocytosis pathways were involved in the internalization process of CQDs/pDNA complexes. Taken together, the alginate-derived photoluminescent CQDs hold great potential in biomedical applications due to their dual role as efficient non-viral gene vectors and bioimaging probes. This manuscript describes a facile and simple one-step hydrothermal carbonization route for preparing optically tunable photoluminescent carbon quantum dots (CQDs) from a novel raw material, alginate. These CQDs enjoy low cytotoxicity, positive zeta potential, excellent ability to condense macromolecular DNA, and most importantly, notably high transfection efficiency. The interesting finding is that the negatively-charged alginate can convert into positively charged CQDs without adding any cationic reagents. The significance of this study is that the cationic carbon quantum dots play dual roles as both non-viral gene vectors and bioimaging probes at the same time, which are most desirable in many

Molecular variability and evolution of the pectate lyase (pel-2) parasitism gene in cyst nematodes parasitizing different solanaceous plants.

PubMed

Geric Stare, Barbara; Fouville, Didier; Širca, Saša; Gallot, Aurore; Urek, Gregor; Grenier, Eric

2011-02-01

While pectate lyases are major parasitism factors in plant-parasitic nematodes, there is little information on the variability of these genes within species and their utility as pathotype or host range molecular markers. We have analysed polymorphisms of pectate lyase 2 (pel-2) gene, which degrades the unesterified polygalacturonate (pectate) of the host cell-wall, in the genus Globodera. Molecular variability of the pel-2 gene and the predicted protein was evaluated in populations of G. rostochiensis, G. pallida, G. "mexicana" and G. tabacum. Seventy eight pel-2 sequences were obtained and aligned. Point mutations were observed at 373 positions, 57% of these affect the coding part of the gene and produce 129 aa replacements. The observed polymorphism does not correlate either to the pathotypes proposed in potato cyst nematodes (PCN) or the subspecies described in tobacco cyst nematodes. The trees reveal a topology different from the admitted species topology as G. rostochiensis and G. pallida sequences are more similar to each other than to G. tabacum. Species-specific sites, potentially applicable for identification, and sites distinguishing PCN from tobacco cyst nematodes, were identified. As both G. rostochiensis and G. pallida display the same host range, but distinct from G. tabacum, which cannot parasitize potato plants, it is tempting to speculate that pel-2 genes polymorphism may be implicated in this adaptation, a view supported by the fact that no active pectate lyase 2 was found in G. "mexicana", a close relative of G. pallida that is unable to develop on cultivated potato varieties.

A Rice PECTATE LYASE-LIKE Gene Is Required for Plant Growth and Leaf Senescence.

PubMed

Leng, Yujia; Yang, Yaolong; Ren, Deyong; Huang, Lichao; Dai, Liping; Wang, Yuqiong; Chen, Long; Tu, Zhengjun; Gao, Yihong; Li, Xueyong; Zhu, Li; Hu, Jiang; Zhang, Guangheng; Gao, Zhenyu; Guo, Longbiao; Kong, Zhaosheng; Lin, Yongjun; Qian, Qian; Zeng, Dali

2017-06-01

To better understand the molecular mechanisms behind plant growth and leaf senescence in monocot plants, we identified a mutant exhibiting dwarfism and an early-senescence leaf phenotype, termed dwarf and early-senescence leaf1 ( del1 ). Histological analysis showed that the abnormal growth was caused by a reduction in cell number. Further investigation revealed that the decline in cell number in del1 was affected by the cell cycle. Physiological analysis, transmission electron microscopy, and TUNEL assays showed that leaf senescence was triggered by the accumulation of reactive oxygen species. The DEL1 gene was cloned using a map-based approach. It was shown to encode a pectate lyase (PEL) precursor that contains a PelC domain. DEL1 contains all the conserved residues of PEL and has strong similarity with plant PelC. DEL1 is expressed in all tissues but predominantly in elongating tissues. Functional analysis revealed that mutation of DEL1 decreased the total PEL enzymatic activity, increased the degree of methylesterified homogalacturonan, and altered the cell wall composition and structure. In addition, transcriptome assay revealed that a set of cell wall function- and senescence-related gene expression was altered in del1 plants. Our research indicates that DEL1 is involved in both the maintenance of normal cell division and the induction of leaf senescence. These findings reveal a new molecular mechanism for plant growth and leaf senescence mediated by PECTATE LYASE-LIKE genes. © 2017 American Society of Plant Biologists. All Rights Reserved.

Silencing of grapevine pectate lyase-like genes VvPLL2 and VvPLL3 confers resistance against Erysiphe necator and differentially modulates gene expression

USDA-ARS?s Scientific Manuscript database

Broad-spectrum resistance against powdery mildew (PM) has been reported by silencing susceptibility genes in the model plant Arabidopsis. Here we used artificial microRNA constructs in PM-susceptible Vitis vinifera cv. Chardonnay to stably silence two pectate lyase-like orthologs (VvPLL2 and VvPLL3)...

Evolution of Sulfobacillus thermosulfidooxidans secreting alginate during bioleaching of chalcopyrite concentrate.

PubMed

Yu, R-L; Liu, A; Liu, Y; Yu, Z; Peng, T; Wu, X; Shen, L; Liu, Y; Li, J; Liu, X; Qiu, G; Chen, M; Zeng, W

2017-06-01

To explore the distribution disciplinarian of alginate on the chalcopyrite concentrate surface during bioleaching. The evolution of Sulfobacillus thermosulfidooxidans secreting alginate during bioleaching of chalcopyrite concentrate was investigated through gas chromatography coupled with mass spectrometry (GC-MS) and confocal laser scanning microscope (CLSM), and the critical synthetic genes (algA, algC, algD) of alginate were analysed by real-time polymerase chain reaction (RT-PCR). The GC-MS analysis results indicated that there was a little amount of alginate formed on the mineral surface at the early stage, while increasing largely to the maximum value at the intermediate stage, and then kept a stable value at the end stage. The CLSM analysis of chalcopyrite slice showed the same variation trend of alginate content on the mineral surface. Furthermore, the RT-PCR results showed that during the early stage of bioleaching, the expressions of the algA, algC and the algD genes were all overexpressed. However, at the final stage, the algD gene expression decreased in a large scale, and the algA and algC decreased slightly. This expression pattern was attributed to the fact that algA and algC genes were involved in several biosynthesis reactions, but the algD gene only participated in the alginate biosynthesis and this was considered as the key gene to control alginate synthesis. The content of alginate on the mineral surface increased largely at the beginning of bioleaching, and remained stable at the end of bioleaching due to the restriction of algD gene expression. Our findings provide valuable information to explore the relationship between alginate formation and bioleaching of chalcopyrite. © 2017 The Society for Applied Microbiology.

Homozygous Mutation G539R in the Gene for P450 Oxidoreductase in a Family Previously Diagnosed as Having 17,20-Lyase Deficiency

PubMed Central

Hershkovitz, Eli; Parvari, Ruthi; Wudy, Stefan A.; Hartmann, Michaela F.; Gomes, Larissa G.; Loewental, Neta; Miller, Walter L.

2008-01-01

Context: Very few patients have been described with isolated 17,20-lyase deficiency who have had their mutations in P450c17 (17α-hydroxylase/17,20-lyase) proven by DNA sequencing and in vitro characterization of the mutations. Most patients with 17,20-lyase deficiency have mutations in the domain of P450c17 that interact with the electron-donating redox partner, P450 oxidoreductase (POR). Objective: Our objective was to clarify the genetic and functional basis of isolated 17,20-lyase deficiency in familial cases who were previously reported as having 17,20-lyase deficiency. Patients: Four undervirilized males of an extended Bedouin family were investigated. One of these has previously been reported to carry mutations in the CYP17A1 gene encoding P450c17 causing isolated 17,20-lyase deficiency. Methods: Serum hormones were evaluated before and after stimulation with ACTH. Urinary steroid metabolites were profiled by gas chromatography-mass spectrometry. Exons 1 and 8 of CYP17A1 previously reported to harbor mutations in one of these patients and all 15 coding exons of POR were sequenced. Results: Gas chromatography-mass spectrometry (GC-MS) urinary steroid profiling and serum steroid measurements showed combined deficiencies of 17,20-lyase and 21-hydroxylase. Sequencing of exons 1 and 8 of CYP17A1 in two different laboratories showed no mutations. Sequencing of POR showed that all four patients were homozygous for G539R, a previously studied mutation that retains 46% of normal capacity to support the 17α-hydroxylase activity but only 8% of the 17,20-lyase activity of P450c17. Conclusion: POR deficiency can masquerade clinically as isolated 17,20-lyase deficiency. PMID:18559916

Homozygous mutation G539R in the gene for P450 oxidoreductase in a family previously diagnosed as having 17,20-lyase deficiency.

PubMed

Hershkovitz, Eli; Parvari, Ruthi; Wudy, Stefan A; Hartmann, Michaela F; Gomes, Larissa G; Loewental, Neta; Miller, Walter L

2008-09-01

Very few patients have been described with isolated 17,20-lyase deficiency who have had their mutations in P450c17 (17alpha-hydroxylase/17,20-lyase) proven by DNA sequencing and in vitro characterization of the mutations. Most patients with 17,20-lyase deficiency have mutations in the domain of P450c17 that interact with the electron-donating redox partner, P450 oxidoreductase (POR). Our objective was to clarify the genetic and functional basis of isolated 17,20-lyase deficiency in familial cases who were previously reported as having 17,20-lyase deficiency. Four undervirilized males of an extended Bedouin family were investigated. One of these has previously been reported to carry mutations in the CYP17A1 gene encoding P450c17 causing isolated 17,20-lyase deficiency. Serum hormones were evaluated before and after stimulation with ACTH. Urinary steroid metabolites were profiled by gas chromatography-mass spectrometry. Exons 1 and 8 of CYP17A1 previously reported to harbor mutations in one of these patients and all 15 coding exons of POR were sequenced. Gas chromatography-mass spectrometry (GC-MS) urinary steroid profiling and serum steroid measurements showed combined deficiencies of 17,20-lyase and 21-hydroxylase. Sequencing of exons 1 and 8 of CYP17A1 in two different laboratories showed no mutations. Sequencing of POR showed that all four patients were homozygous for G539R, a previously studied mutation that retains 46% of normal capacity to support the 17alpha-hydroxylase activity but only 8% of the 17,20-lyase activity of P450c17. POR deficiency can masquerade clinically as isolated 17,20-lyase deficiency.

Genomic Characterization of Phenylalanine Ammonia Lyase Gene in Buckwheat

PubMed Central

Thiyagarajan, Karthikeyan; Vitali, Fabio; Tolaini, Valentina; Galeffi, Patrizia; Cantale, Cristina; Vikram, Prashant; Singh, Sukhwinder; De Rossi, Patrizia; Nobili, Chiara; Procacci, Silvia; Del Fiore, Antonella; Antonini, Alessandro; Presenti, Ombretta; Brunori, Andrea

2016-01-01

Phenylalanine Ammonia Lyase (PAL) gene which plays a key role in bio-synthesis of medicinally important compounds, Rutin/quercetin was sequence characterized for its efficient genomics application. These compounds possessing anti-diabetic and anti-cancer properties and are predominantly produced by Fagopyrum spp. In the present study, PAL gene was sequenced from three Fagopyrum spp. (F. tataricum, F. esculentum and F. dibotrys) and showed the presence of three SNPs and four insertion/deletions at intra and inter specific level. Among them, the potential SNP (position 949th bp G>C) with Parsimony Informative Site was selected and successfully utilised to individuate the zygosity/allelic variation of 16 F. tataricum varieties. Insertion mutations were identified in coding region, which resulted the change of a stretch of 39 amino acids on the putative protein. Our Study revealed that autogamous species (F. tataricum) has lower frequency of observed SNPs as compared to allogamous species (F. dibotrys and F. esculentum). The identified SNPs in F. tataricum didn’t result to amino acid change, while in other two species it caused both conservative and non-conservative variations. Consistent pattern of SNPs across the species revealed their phylogenetic importance. We found two groups of F. tataricum and one of them was closely related with F. dibotrys. Sequence characterization information of PAL gene reported in present investigation can be utilized in genetic improvement of buckwheat in reference to its medicinal value. PMID:26990297

A Rice PECTATE LYASE-LIKE Gene Is Required for Plant Growth and Leaf Senescence1[OPEN

PubMed Central

Leng, Yujia; Yang, Yaolong; Ren, Deyong; Dai, Liping; Wang, Yuqiong; Chen, Long; Tu, Zhengjun; Gao, Yihong; Zhu, Li; Hu, Jiang; Gao, Zhenyu; Guo, Longbiao; Lin, Yongjun

2017-01-01

To better understand the molecular mechanisms behind plant growth and leaf senescence in monocot plants, we identified a mutant exhibiting dwarfism and an early-senescence leaf phenotype, termed dwarf and early-senescence leaf1 (del1). Histological analysis showed that the abnormal growth was caused by a reduction in cell number. Further investigation revealed that the decline in cell number in del1 was affected by the cell cycle. Physiological analysis, transmission electron microscopy, and TUNEL assays showed that leaf senescence was triggered by the accumulation of reactive oxygen species. The DEL1 gene was cloned using a map-based approach. It was shown to encode a pectate lyase (PEL) precursor that contains a PelC domain. DEL1 contains all the conserved residues of PEL and has strong similarity with plant PelC. DEL1 is expressed in all tissues but predominantly in elongating tissues. Functional analysis revealed that mutation of DEL1 decreased the total PEL enzymatic activity, increased the degree of methylesterified homogalacturonan, and altered the cell wall composition and structure. In addition, transcriptome assay revealed that a set of cell wall function- and senescence-related gene expression was altered in del1 plants. Our research indicates that DEL1 is involved in both the maintenance of normal cell division and the induction of leaf senescence. These findings reveal a new molecular mechanism for plant growth and leaf senescence mediated by PECTATE LYASE-LIKE genes. PMID:28455404

Regulation of L-phenylalanine ammonia-lyase by L-phenylalanine and nitrogen in Neurospora crassa.

PubMed Central

Sikora, L A; Marzluf, G A

1982-01-01

Neurospora crassa possesses an inducible L-phenylalanine ammonia-lyase that is expressed only when cells are derepressed for nitrogen in the presence of L-phenylalanine. Enzyme synthesis requires both induction by L-phenylalanine and simultaneous nitrogen catabolite derepression. Carbon limitation in the presence of phenylalanine does not elicit induction of L-phenylalanine ammonia-lyase. Specific induction by L-phenylalanine is required, and other amino acids completely failed to induce any lyase activity. The nit-2 gene is a major regulatory locus which is believed to mediate nitrogen catabolite repression in Neurospora. Mutants of nit-2 fail to express any phenylalanine ammonia-lyase activity under conditions of derepression and induction which lead to good enzyme induction in the wild type and in nit-2 revertants. The loss of lyase activity in nit-2 mutants does not result from inducer exclusion, which suggests that the nit-2 gene product has a direct role in controlling the expression of this enzyme. Substantial amounts of the enzyme were detected in the growth medium as well as in cell extracts. Inhibitors of protein synthesis or RNA synthesis block the induction of L-phenylalanine ammonia-lyase, suggesting that expression of this enzyme is controlled at the level of transcription. PMID:6210688

Isocitrate Lyase Is Essential for Pathogenicity of the Fungus Leptosphaeria maculans to Canola (Brassica napus)

PubMed Central

Idnurm, Alexander; Howlett, Barbara J.

2002-01-01

A pathogenicity gene has been identified in Leptosphaeria maculans, the ascomycetous fungus that causes blackleg disease of canola (Brassica napus). This gene encodes isocitrate lyase, a component of the glyoxylate cycle, and is essential for the successful colonization of B. napus. It was identified by a reverse genetics approach whereby a plasmid conferring hygromycin resistance was inserted randomly into the L. maculans genome. Twelve of 516 transformants tested had reduced pathogenicity on cotyledons of B. juncea and B. napus, and 1 of these 12 had a deletion of the isocitrate lyase gene, as well as an insertion of the hygromycin resistance gene. This mutant was unable to grow on fatty acids, including monolaurate, and the isocitrate lyase transcript was not detected. When the wild-type gene was reintroduced into the mutant, growth on monolaurate was restored and pathogenicity was partially restored. L. maculans isocitrate lyase is produced during infection of B. napus cotyledons, while the plant homologue is not. When 2.5% glucose was added to the inoculum of the isocitrate lyase mutant, lesions of sizes similar to those caused by wild-type isolate M1 developed on B. napus cotyledons. These findings suggest that the glyoxylate pathway is essential for disease development by this plant-pathogenic fungus, as has been shown recently for a fungal and bacterial pathogen of animals and a bacterial pathogen of plants. Involvement of the glyoxylate pathway in pathogenesis in animals and plants presents potential drug targets for control of diseases. PMID:12455691

The genetic and functional basis of isolated 17,20-lyase deficiency.

PubMed

Geller, D H; Auchus, R J; Mendonça, B B; Miller, W L

1997-10-01

Human male sexual differentiation requires production of fetal testicular testosterone, whose biosynthesis requires steroid 17,20-lyase activity. Patients with putative isolated 17,20-lyase deficiency have been reported. The existence of true isolated 17,20-lyase deficiency, however, has been questioned because 17 alpha-hydroxylase and 17,20-lyase activities are catalyzed by a single enzyme, microsomal cytochrome P450c17, and because the index case of apparent isolated 17,20-lyase deficiency had combined deficiencies of both activities. We studied two patients with clinical and hormonal findings suggestive of isolated 17,20-lyase deficiency. We found two patients homozygous for substitution mutations in CYP17, the gene encoding P450c17. When expressed in COS-1 cells, the mutants retained 17 alpha-hydroxylase activity but had minimal 17,20-lyase activity. Substrate competition experiments suggested that the mutations did not alter the enzyme's substrate-binding capacity, but co-transfection of cells with P450 oxidoreductase, the electron donor used by P450c17, indicated that the mutants had a diminished ability to interact with redox partners. Computer-graphic modelling of P450c17 suggests that both mutations lie in or near the redox-partner binding site, on the opposite side of the haem from the substrate-binding pocket. These mutations alter electrostatic charge distribution in the redox-partner binding site, so that electron transfer for the 17,20-lyase reaction is selectively lost or diverted to uncoupling reactions. These are the first proven cases of isolated 17,20-lyase deficiency, and they demonstrate a novel mechanism for loss of enzymatic activity.

Molecular Cloning and Sequence Analysis of a Phenylalanine Ammonia-Lyase Gene from Dendrobium

PubMed Central

Cai, Yongping; Lin, Yi

2013-01-01

In this study, a phenylalanine ammonia-lyase (PAL) gene was cloned from Dendrobium candidum using homology cloning and RACE. The full-length sequence and catalytic active sites that appear in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum are also found: PAL cDNA of D. candidum (designated Dc-PAL1, GenBank No. JQ765748) has 2,458 bps and contains a complete open reading frame (ORF) of 2,142 bps, which encodes 713 amino acid residues. The amino acid sequence of DcPAL1 has more than 80% sequence identity with the PAL genes of other plants, as indicated by multiple alignments. The dominant sites and catalytic active sites, which are similar to that showing in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum, are also found in DcPAL1. Phylogenetic tree analysis revealed that DcPAL is more closely related to PALs from orchidaceae plants than to those of other plants. The differential expression patterns of PAL in protocorm-like body, leaf, stem, and root, suggest that the PAL gene performs multiple physiological functions in Dendrobium candidum. PMID:23638048

The Phenylalanine Ammonia-Lyase Gene Family in Raspberry. Structure, Expression, and Evolution1

PubMed Central

Kumar, Amrita; Ellis, Brian E.

2001-01-01

In raspberry (Rubus idaeus), development of fruit color and flavor are critically dependent on products of the phenylpropanoid pathway. To determine how these metabolic functions are integrated with the fruit ripening program, we are examining the properties and expression of key genes in the pathway. Here, we report that l- phenylalanine ammonia-lyase (PAL) is encoded in raspberry by a family of two genes (RiPAL1 and RiPAL2). RiPAL1 shares 88% amino acid sequence similarity to RiPAL2, but phylogenetic analysis places RiPAL1 and RiPAL2 in different clusters within the plant PAL gene family. The spatial and temporal expression patterns of the two genes were investigated in various vegetative and floral tissues using the reverse transcriptase competitor polymerase chain reaction assay. Although expression of both genes was detected in all tissues examined, RiPAL1 was associated with early fruit ripening events, whereas expression of RiPAL2 correlated more with later stages of flower and fruit development. Determination of the absolute levels of the two transcripts in various tissues showed that RiPAL1 transcripts were 3- to 10-fold more abundant than those of RiPAL2 in leaves, shoots, roots, young fruits, and ripe fruits. The two RiPAL genes therefore appear to be controlled by different regulatory mechanisms. PMID:11553751

Pectate lyase PelI of Erwinia chrysanthemi 3937 belongs to a new family.

PubMed Central

Shevchik, V E; Robert-Baudouy, J; Hugouvieux-Cotte-Pattat, N

1997-01-01

Erwinia chrysanthemi 3937 secretes five major isoenzymes of pectate lyases encoded by the pel4, pelB, pelC, pelD, and pelE genes and a set of secondary pectate lyases, two of which, pelL and pelZ, have been already identified. We cloned the pelI gene, encoding a ninth pectate lyase of E. chrysanthemi 3937. The pelI reading frame is 1,035 bases long, corresponding to a protein of 344 amino acids including a typical amino-terminal signal sequence of 19 amino acids. The purified mature PelI protein has an isoelectric point of about 9 and an apparent molecular mass of 34 kDa. PelI has a preference for partially methyl esterified pectin and presents an endo-cleaving activity with an alkaline pH optimum and an absolute requirement for Ca2+ ions. PelI is an extracellular protein secreted by the Out secretory pathway of E. chrysanthemi. The PelI protein is very active in the maceration of plant tissues. A pelI mutant displayed reduced pathogenicity on chicory leaves, but its virulence did not appear to be affected on potato tubers or Saintpaulia ionantha plants. The pelI gene constitutes an independent transcriptional unit. As shown for the other pel genes, the transcription of pelI is dependent on various environmental conditions. It is induced by pectic catabolic products and affected by growth phase, oxygen limitation, temperature, nitrogen starvation, and catabolite repression. Regulation of pelI expression appeared to be dependent on the three repressors of pectinase synthesis, KdgR, PecS, and PecT, and on the global activator of sugar catabolism, cyclic AMP receptor protein. A functional KdgR binding site was identified close to the putative pelI promoter. Analysis of the amino acid sequence of PelI revealed high homology with a pectate lyase from Erwinia carotovora subsp. carotovora (65% identity) and low homology with pectate lyases of the phytopathogenic fungus Nectria haematococca (Fusarium solani). This finding indicates that PelI belongs to pectate lyase class

Genome-wide characterization of phenylalanine ammonia-lyase gene family in watermelon (Citrullus lanatus).

PubMed

Dong, Chun-Juan; Shang, Qing-Mao

2013-07-01

Phenylalanine ammonia-lyase (PAL), the first enzyme in the phenylpropanoid pathway, plays a critical role in plant growth, development, and adaptation. PAL enzymes are encoded by a gene family in plants. Here, we report a genome-wide search for PAL genes in watermelon. A total of 12 PAL genes, designated ClPAL1-12, are identified . Nine are arranged in tandem in two duplication blocks located on chromosomes 4 and 7, and the other three ClPAL genes are distributed as single copies on chromosomes 2, 3, and 8. Both the cDNA and protein sequences of ClPALs share an overall high identity with each other. A phylogenetic analysis places 11 of the ClPALs into a separate cucurbit subclade, whereas ClPAL2, which belongs to neither monocots nor dicots, may serve as an ancestral PAL in plants. In the cucurbit subclade, seven ClPALs form homologous pairs with their counterparts from cucumber. Expression profiling reveals that 11 of the ClPAL genes are expressed and show preferential expression in the stems and male and female flowers. Six of the 12 ClPALs are moderately or strongly expressed in the fruits, particularly in the pulp, suggesting the potential roles of PAL in the development of fruit color and flavor. A promoter motif analysis of the ClPAL genes implies redundant but distinctive cis-regulatory structures for stress responsiveness. Finally, duplication events during the evolution and expansion of the ClPAL gene family are discussed, and the relationships between the ClPAL genes and their cucumber orthologs are estimated.

Metagenomics unveils the attributes of the alginolytic guilds of sediments from four distant cold coastal environments: Alginolytic guilds from cold sediments

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

Matos, Marina N.; Lozada, Mariana; Anselmino, Luciano E.

Alginates are abundant polysaccharides in brown algae that constitute an important energy source for marine heterotrophic bacteria. Despite the key role of alginate assimilation processes in the marine carbon cycle, little information is available on the bacterial populations involved in these processes. The goal of this work was to gain insight into the structure and functional traits of the alginolytic communities from sediments of cold coastal environments. Sediment metagenomes from high-latitude regions of both Hemispheres were interrogated for alginate lyase gene homolog sequences and their genomic context. Sediments contained highly abundant and diverse bacterial assemblages with alginolytic potential, including membersmore » of Bacteroidetes and Proteobacteria, as well as several poorly characterized taxa. Temperature and salinity were correlated to the variation in community structure. The microbial communities in Arctic and Antarctic sediments exhibited the most similar alginolytic profiles, whereas brackish sediments had a higher proportion of novel members. Examination of the gene context of the alginate lyase homologs revealed distinct patterns according to the phylogenetic origin of the scaffolds, with evidence of evolutionary relationships among lineages. This information is relevant for understanding carbon fluxes in cold coastal environments and provides valuable information for the development of biotechnological applications from brown algae biomass.« less

Exploiting fine-scale genetic and physiological variation of closely related microbes to reveal unknown enzyme functions.

PubMed

Badur, Ahmet H; Plutz, Matthew J; Yalamanchili, Geethika; Jagtap, Sujit Sadashiv; Schweder, Thomas; Unfried, Frank; Markert, Stephanie; Polz, Martin F; Hehemann, Jan-Hendrik; Rao, Christopher V

2017-08-04

Polysaccharide degradation by marine microbes represents one of the largest and most rapid heterotrophic transformations of organic matter in the environment. Microbes employ systems of complementary carbohydrate-specific enzymes to deconstruct algal or plant polysaccharides (glycans) into monosaccharides. Because of the high diversity of glycan substrates, the functions of these enzymes are often difficult to establish. One solution to this problem may lie within naturally occurring microdiversity; varying numbers of enzymes, due to gene loss, duplication, or transfer, among closely related environmental microbes create metabolic differences akin to those generated by knock-out strains engineered in the laboratory used to establish the functions of unknown genes. Inspired by this natural fine-scale microbial diversity, we show here that it can be used to develop hypotheses guiding biochemical experiments for establishing the role of these enzymes in nature. In this work, we investigated alginate degradation among closely related strains of the marine bacterium Vibrio splendidus One strain, V. splendidus 13B01, exhibited high extracellular alginate lyase activity compared with other V. splendidus strains. To identify the enzymes responsible for this high extracellular activity, we compared V. splendidus 13B01 with the previously characterized V. splendidus 12B01, which has low extracellular activity and lacks two alginate lyase genes present in V. splendidus 13B01. Using a combination of genomics, proteomics, biochemical, and functional screening, we identified a polysaccharide lyase family 7 enzyme that is unique to V. splendidus 13B01, secreted, and responsible for the rapid digestion of extracellular alginate. These results demonstrate the value of querying the enzymatic repertoires of closely related microbes to rapidly pinpoint key proteins with beneficial functions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Gene Delivery of TGF-β3 and BMP2 in an MSC-Laden Alginate Hydrogel for Articular Cartilage and Endochondral Bone Tissue Engineering.

PubMed

Gonzalez-Fernandez, Tomas; Tierney, Erica G; Cunniffe, Grainne M; O'Brien, Fergal J; Kelly, Daniel J

2016-05-01

Incorporating therapeutic genes into three-dimensional biomaterials is a promising strategy for enhancing tissue regeneration. Alginate hydrogels have been extensively investigated for cartilage and bone tissue engineering, including as carriers of transfected cells to sites of injury, making them an ideal gene delivery platform for cartilage and osteochondral tissue engineering. The objective of this study was to develop gene-activated alginate hydrogels capable of supporting nanohydroxyapatite (nHA)-mediated nonviral gene transfer to control the phenotype of mesenchymal stem cells (MSCs) for either cartilage or endochondral bone tissue engineering. To produce these gene-activated constructs, MSCs and nHA complexed with plasmid DNA (pDNA) encoding for transforming growth factor-beta 3 (pTGF-β3), bone morphogenetic protein 2 (pBMP2), or a combination of both (pTGF-β3-pBMP2) were encapsulated into alginate hydrogels. Initial analysis using reporter genes showed effective gene delivery and sustained overexpression of the transgenes were achieved. Confocal microscopy demonstrated that complexing the plasmid with nHA before hydrogel encapsulation led to transport of the plasmid into the nucleus of MSCs, which did not happen with naked pDNA. Gene delivery of TGF-β3 and BMP2 and subsequent cell-mediated expression of these therapeutic genes resulted in a significant increase in sulfated glycosaminoglycan and collagen production, particularly in the pTGF-β3-pBMP2 codelivery group in comparison to the delivery of either pTGF-β3 or pBMP2 in isolation. In addition, stronger staining for collagen type II deposition was observed in the pTGF-β3-pBMP2 codelivery group. In contrast, greater levels of calcium deposition were observed in the pTGF-β3- and pBMP2-only groups compared to codelivery, with a strong staining for collagen type X deposition, suggesting these constructs were supporting MSC hypertrophy and progression along an endochondral pathway. Together, these

Stemness of spermatogonial stem cells encapsulated in alginate hydrogel during cryopreservation.

PubMed

Pirnia, A; Parivar, K; Hemadi, M; Yaghmaei, P; Gholami, M

2017-06-01

This study investigated the effect of spermatogonial stem cell encapsulated in alginate hydrogel during cryopreservation, as cells were protected against damage during cryopreservation within the hydrogel. Spermatogonial stem cells were isolated from the testes of Balb/c mice pups (6 days old), purified in laminin-coated dishes and CD90.1 microbeads, encapsulated in alginate hydrogel and then cryopreserved. After thawing, cell viability and Spermatogonial stem cell (SSC) colony diameter were evaluated. After RNA was isolated and cDNA was synthesised, the expression of stemness genes was considered using RT real-time PCR. Finally, spermatogonial stem cells labelled with BrdU were transplanted to busulfan azoospermic mouse models. Lin28a and Sall4 genes were significantly upregulated after cryopreservation in alginate hydrogel. However, cell viability was significantly decreased. The diameter of colonies consisting of spermatogonial stem cells freeze-thawed in alginate microbeads showed no significant difference with fresh spermatogonial stem cells and the control group. The injection of freeze-thawed spermatogonial stem cells encapsulated in alginate hydrogel resulted in spermatogenesis recovery. Alginate mimics the extracellular matrices (ECM) for spermatogonial stem cells; therefore, it can support stemness potential during the cell cryopreservation process and restart spermatogenesis after transplantation. © 2016 Blackwell Verlag GmbH.

Iron-Regulated Expression of Alginate Production, Mucoid Phenotype, and Biofilm Formation by Pseudomonas aeruginosa

PubMed Central

Wiens, Jacinta R.; Vasil, Adriana I.; Schurr, Michael J.; Vasil, Michael L.

2014-01-01

ABSTRACT Pseudomonas aeruginosa strains of non-cystic fibrosis (non-CF) origin do not produce significant amounts of extracellular alginate and are nonmucoid. In CF, such isolates can become mucoid through mutation of one of the genes (mucA, mucB, mucC, or mucD) that produce regulatory factors that sequester AlgU, required for increased expression of alginate genes. Mutation of the muc genes in the nonmucoid PAO1, PA14, PAKS-1, and Ps388 strains led to increased levels of extracellular alginate and an obvious mucoid phenotype, but only under iron-limiting growth conditions (≤5 µM), not under iron-replete conditions (≥10 µM). In contrast, >50% of P. aeruginosa isolates from chronic CF pulmonary infections expressed increased levels of alginate and mucoidy both under iron-limiting and iron-replete conditions (i.e., iron-constitutive phenotype). No single iron regulatory factor (e.g., Fur, PvdS) was associated with this loss of iron-regulated alginate expression and mucoidy in these CF isolates. However, the loss of only pyoverdine production, or its uptake, abrogated the ability of P. aeruginosa to produce a robust biofilm that represents the Psl-type of biofilm. In contrast, we show that mutation of the pyoverdine and pyochelin biosynthesis genes and the pyoverdine receptor (FpvA) lead to iron-constitutive expression of the key alginate biosynthesis gene, algD, and an explicitly mucoid phenotype in both iron-limiting and iron-replete conditions. These data indicate that alginate production and mucoidy, in contrast to other types of biofilms produced by P. aeruginosa, are substantially enhanced under iron limitation. These results also have compelling implications in relation to the use of iron chelators in the treatment of P. aeruginosa CF infections. PMID:24496793

Jellyfish collagen and alginate: Combined marine materials for superior chondrogenesis of hMSC.

PubMed

Pustlauk, W; Paul, B; Gelinsky, M; Bernhardt, A

2016-07-01

Marine, hybrid constructs of porous scaffolds from fibrillized jellyfish collagen and alginate hydrogel are mimicking both of the main tissue components of cartilage, thus being a promising approach for chondrogenic differentiation of human mesenchymal stem cells (hMSC). Investigating their potential for articular cartilage repair, the present study examined scaffolds being either infiltrated with an alginate-cell-suspension (ACS) or seeded with hMSC and embedded in alginate after cell adhesion (EAS). Hybrid constructs with 2×10(5) and 4.5×10(5)hMSC/scaffold were compared to hMSC encapsulated in pure alginate discs, both chondrogenically stimulated for 21days. Typical round, chondrocyte-like morphology was observed in pure alginate gels and ACS scaffolds, while cells in EAS were elongated and tightly attached to the collagen pores. Col 2 gene expression was comparable in all scaffold types examined. However, the Col 2/Col 1 ratio was higher for pure alginate discs and ACS scaffolds compared to EAS. In contrast, cells in EAS scaffolds displayed higher gene expression of Sox 9, Col 11 and ACAN compared to ACS and pure alginate. Secretion of sulfated glycosaminoglycans (sGAG) was comparable for ACS and EAS scaffolds. In conclusion hybrid constructs of jellyfish collagen and alginate support hMSC chondrogenic differentiation and provide more stable and constructs compared to pure hydrogels. Copyright © 2016 Elsevier B.V. All rights reserved.

Adenoviral transduction supports matrix expression of alginate cultured articular chondrocytes.

PubMed

Pohle, D; Kasch, R; Herlyn, P; Bader, R; Mittlmeier, T; Pützer, B M; Müller-Hilke, B

2012-09-01

The present study examines the effects of adenoviral (Ad) transduction of human primary chondrocyte on transgene expression and matrix production. Primary chondrocytes were isolated from healthy articular cartilage and from cartilage with mild osteoarthritis (OA), transduced with an Ad vector and either immediately cultured in alginate or expanded in monolayer before alginate culture. Proteoglycan production was measured using dimethylmethylene blue (DMMB) assay and matrix gene expression was quantified by real-time PCR. Viral infection of primary chondrocytes results in a stable long time transgene expression for up to 13 weeks. Ad transduction does not significantly alter gene expression and matrix production if chondrocytes are immediately embedded in alginate. However, if expanded prior to three dimension (3D) culture in alginate, chondrocytes produce not only more proteoglycans compared to non-transduced controls, but also display an increased anabolic and decreased catabolic activity compared to non-transduced controls. We therefore suggest that successful autologous chondrocyte transplantation (ACT) should combine adenoviral transduction of primary chondrocytes with expansion in monolayer followed by 3D culture. Future studies will be needed to investigate whether the subsequent matrix production can be further improved by using Ad vectors bearing genes encoding matrix proteins. Copyright © 2012 Wiley Periodicals, Inc.

Xylella fastidiosa esterase rather than hydroxynitrile lyase.

PubMed

Torrelo, Guzman; Ribeiro de Souza, Fayene Zeferino; Carrilho, Emanuel; Hanefeld, Ulf

2015-03-02

In 2009, we reported that the product of the gene SCJ21.16 (XFa0032) from Xylella fastidiosa, a xylem-restricted plant pathogen that causes a range of diseases in several important crops, encodes a protein (XfHNL) with putative hydroxynitrile lyase activity. Sequence analysis and activity tests indicated that XfHNL exhibits an α/β-hydrolase fold and could be classified as a member of the family of FAD-independent HNLs. Here we provide a more detailed sequence analysis and new experimental data. Using pure heterologously expressed XfHNL we show that this enzyme cannot catalyse the cleavage/synthesis of mandelonitrile and that this protein is in fact a non-enantioselective esterase. Homology modelling and ligand docking simulations were used to study the active site and support these results. This finding could help elucidate the common ancestor of esterases and hydroxynitrile lyases with an α/β -hydrolase fold. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Silencing of SlPL, which encodes a pectate lyase in tomato, confers enhanced fruit firmness, prolonged shelf-life and reduced susceptibility to grey mould.

PubMed

Yang, Lu; Huang, Wei; Xiong, Fangjie; Xian, Zhiqiang; Su, Deding; Ren, Maozhi; Li, Zhengguo

2017-12-01

Pectate lyase genes have been documented as excellent candidates for improvement of fruit firmness. However, implementation of pectate lyase in regulating fruit postharvest deterioration has not been fully explored. In this report, 22 individual pectate lyase genes in tomato were identified, and one pectate lyase gene SlPL (Solyc03g111690) showed dominant expression during fruit maturation. RNA interference of SlPL resulted in enhanced fruit firmness and changes in pericarp cells. More importantly, the SlPL-RNAi fruit demonstrated greater antirotting and pathogen-resisting ability. Compared to wild-type, SlPL-RNAi fruit had higher levels of cellulose and hemicellulose, whereas the level of water-soluble pectin was lower. Consistent with this, the activities of peroxidase, superoxide dismutase and catalase were higher in SlPL-RNAi fruit, and the malondialdehyde concentration was lower. RNA-Seq results showed large amounts of differentially expressed genes involved in hormone signalling, cell wall modification, oxidative stress and pathogen resistance. Collectively, these data demonstrate that pectate lyase plays an important role in both fruit softening and pathogen resistance. This may advance knowledge of postharvest fruit preservation in tomato and other fleshy fruit. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Characterisation of the willow phenylalanine ammonia-lyase (PAL) gene family reveals expression differences compared with poplar

PubMed Central

de Jong, Femke; Hanley, Steven J.; Beale, Michael H.; Karp, Angela

2015-01-01

Willow is an important biomass crop for the bioenergy industry, and therefore optimal growth with minimal effects of biotic and abiotic stress is essential. The phenylpropanoid pathway is responsible for the biosynthesis of not only lignin but also of flavonoids, condensed tannins, benzenoids and phenolic glycosides which all have a role in protecting the plant against biotic and abiotic stress. All products of the phenylpropanoid pathway are important for the healthy growth of short rotation cropping species such as willow. However, the phenylpropanoid pathway in willow remains largely uncharacterised. In the current study we identified and characterised five willow phenylalanine ammonia-lyase (PAL) genes, which encode enzymes that catalyse the deamination of l-phenylalanine to form trans-cinnamic acid, the entry point into the phenylpropanoid pathway. Willow PAL1, PAL2, PAL3 and PAL4 genes were orthologous to the poplar genes. However no orthologue of PAL5 appears to be present in willow. Moreover, two tandemly repeated PAL2 orthologues were identified in a single contig. Willow PALs show similar sub-cellular localisation to the poplar genes. However, the enzyme kinetics and gene expression of the willow PAL genes differed slightly, with willow PAL2 being more widely expressed than its poplar orthologues implying a wider role for PALs in the production of flavonoids, condensed tannins, benzenoids, and phenolic glycosides, in willow. PMID:26070140

Suppressed phenylalanine ammonia-lyase activity after heat shock in transgenic Nicotiana plumbaginifolia containing an Arabidopsis HSP18.2-parsley PAL2 chimera gene.

PubMed

Moriwaki, M; Yamakawa, T; Washino, T; Kodama, T; Igarashi, Y

1999-01-01

The activity of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) after heat shock (HS) in leaves and buds of transgenic Nicotiana plumbaginifolia containing an Arabidopsis HSP18.2 promoter-parsley phenylalanine ammonia-lyase 2 (HSP18.2-PAL2) chimera gene was examined. Immediately after HS treatment at 44 degrees C for 5 h, the PAL activity in both transgenic and normal (untransformed) plants was 35-38% lower than that before HS. At normal temperature (25-26 degrees C), the PAL activity recovered within 5 h of ending the HS treatment in normal plants, but not until 12-24 h in transgenic plants containing the HSP18.2-PAL2 gene. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed the presence of parsley PAL2 mRNA in transgenic plants, which remained for 8-12 h following 5-h HS at 44 degrees C; the mRNA was not observed before HS. The content of chlorogenic acid (CGA; 3-caffeoylquinic acid) decreased drastically 8-12 h after HS in transgenic plants, but only slightly in normal plants. Thus, the decrease in PAL activity accompanied by expression of the parsley PAL2 gene after HS treatment corresponded to the decrease in CGA synthesis. These results might be attributed to post-transcriptional degradation of endogenous PAL mRNA triggered by transcription of the transgene.

Gene inactivation in the plant pathogen Glomerella cingulata: three strategies for the disruption of the pectin lyase gene pnlA.

PubMed

Bowen, J K; Templeton, M D; Sharrock, K R; Crowhurst, R N; Rikkerink, E H

1995-01-20

The feasibility of performing routine transformation-mediated mutagenesis in Glomerella cingulata was analysed by adopting three one-step gene disruption strategies targeted at the pectin lyase gene pnlA. The efficiencies of disruption following transformation with gene replacement- or gene truncation-disruption vectors were compared. To effect replacement-disruption, G. cingulata was transformed with a vector carrying DNA from the pnlA locus in which the majority of the coding sequence had been replaced by the gene for hygromycin B resistance. Two of the five transformants investigated contained an inactivated pnlA gene (pnlA-); both also contained ectopically integrated vector sequences. The efficacy of gene disruption by transformation with two gene truncation-disruption vectors was also assessed. Both vectors carried at 5' and 3' truncated copy of the pnlA coding sequence, adjacent to the gene for hygromycin B resistance. The promoter sequences controlling the selectable marker differed in the two vectors. In one vector the homologous G. cingulata gpdA promoter controlled hygromycin B phosphotransferase expression (homologous truncation vector), whereas in the second vector promoter elements were from the Aspergillus nidulans gpdA gene (heterologous truncation vector). Following transformation with the homologous truncation vector, nine transformants were analysed by Southern hybridisation; no transformants contained a disrupted pnlA gene. Of nineteen heterologous truncation vector transformants, three contained a disrupted pnlA gene; Southern analysis revealed single integrations of vector sequence at pnlA in two of these transformants. pnlA mRNA was not detected by Northern hybridisation in pnlA- transformants. pnlA- transformants failed to produce a PNLA protein with a pI identical to one normally detected in wild-type isolates by silver and activity staining of isoelectric focussing gels. Pathogenesis on Capsicum and apple was unaffected by disruption of

Control of Alginate Core Size in Alginate-Poly (Lactic-Co-Glycolic) Acid Microparticles

NASA Astrophysics Data System (ADS)

Lio, Daniel; Yeo, David; Xu, Chenjie

2016-01-01

Core-shell alginate-poly (lactic-co-glycolic) acid (PLGA) microparticles are potential candidates to improve hydrophilic drug loading while facilitating controlled release. This report studies the influence of the alginate core size on the drug release profile of alginate-PLGA microparticles and its size. Microparticles are synthesized through double-emulsion fabrication via a concurrent ionotropic gelation and solvent extraction. The size of alginate core ranges from approximately 10, 50, to 100 μm when the emulsification method at the first step is homogenization, vortexing, or magnetic stirring, respectively. The second step emulsification for all three conditions is performed with magnetic stirring. Interestingly, although the alginate core has different sizes, alginate-PLGA microparticle diameter does not change. However, drug release profiles are dramatically different for microparticles comprising different-sized alginate cores. Specifically, taking calcein as a model drug, microparticles containing the smallest alginate core (10 μm) show the slowest release over a period of 26 days with burst release less than 1 %.

A novel C-S lyase from the latex-producing plant Taraxacum brevicorniculatum displays alanine aminotransferase and l-cystine lyase activity.

PubMed

Munt, Oliver; Prüfer, Dirk; Schulze Gronover, Christian

2013-01-01

We isolated a novel pyridoxal-5-phosphate-dependent l-cystine lyase from the dandelion Taraxacum brevicorniculatum. Real time qPCR analysis showed that C-S lyase from Taraxacum brevicorniculatum (TbCSL) mRNA is expressed in all plant tissues, although at relatively low levels in the latex and pedicel. The 1251 bp TbCSL cDNA encodes a protein with a calculated molecular mass of 46,127 kDa. It is homologous to tyrosine and alanine aminotransferases (AlaATs) as well as to an Arabidopsis thaliana carbon-sulfur lyase (C-S lyase) (SUR1), which has a role in glucosinolate metabolism. TbCSL displayed in vitrol-cystine lyase and AlaAT activities of 4 and 19nkatmg(-1) protein, respectively. However, we detected no in vitro tyrosine aminotransferase (TyrAT) activity and RNAi knockdown of the enzyme had no effect on phenotype, showing that TbCSL substrates might be channeled into redundant pathways. TbCSL is in vivo localized in the cytosol and functions as a C-S lyase or an aminotransferase in planta, but the purified enzyme converts at least two substrates specifically, and can thus be utilized for further in vitro applications. Copyright © 2012 Elsevier GmbH. All rights reserved.

Insight into the role of substrate-binding residues in conferring substrate specificity for the multifunctional polysaccharide lyase Smlt1473.

PubMed

MacDonald, Logan C; Berger, Bryan W

2014-06-27

Anionic polysaccharides are of growing interest in the biotechnology industry due to their potential pharmaceutical applications in drug delivery and wound treatment. Chemical composition and polymer length strongly influence the physical and biological properties of the polysaccharide and thus its potential industrial and medical applications. One promising approach to determining monomer composition and controlling the degree of polymerization involves the use of polysaccharide lyases, which catalyze the depolymerization of anionic polysaccharides via a β-elimination mechanism. Utilization of these enzymes for the production of custom-made oligosaccharides requires a high degree of control over substrate specificity. Previously, we characterized a polysaccharide lyase (Smlt1473) from Stenotrophomonas maltophilia k279a, which exhibited significant activity against hyaluronan (HA), poly-β-d-glucuronic acid (poly-GlcUA), and poly-β-d-mannuronic acid (poly-ManA) in a pH-regulated manner. Here, we utilize a sequence structure guided approach based on a homology model of Smlt1473 to identify nine putative substrate-binding residues and examine their effect on substrate specificity via site-directed mutagenesis. Interestingly, single point mutations H221F and R312L resulted in increased activity and specificity toward poly-ManA and poly-GlcUA, respectively. Furthermore, a W171A mutant nearly eliminated HA activity, while increasing poly-ManA and poly-GlcUA activity by at least 35%. The effect of these mutations was analyzed by comparison with the high resolution structure of Sphingomonas sp. A1-III alginate lyase in complex with poly-ManA tetrasaccharide and by taking into account the structural differences between HA, poly-GlcUA, and poly-ManA. Overall, our results demonstrate that even minor changes in active site architecture have a significant effect on the substrate specificity of Smlt1473, whose structural plasticity could be applied to the design of highly

Culture of hESC-derived pancreatic progenitors in alginate-based scaffolds.

PubMed

Formo, Kjetil; Cho, Candy H-H; Vallier, Ludovic; Strand, Berit L

2015-12-01

The effect of alginate-based scaffolds with added basement membrane proteins on the in vitro development of hESC-derived pancreatic progenitors was investigated. Cell clusters were encapsulated in scaffolds containing the basement membrane proteins collagen IV, laminin, fibronectin, or extracellular matrix-derived peptides, and maintained in culture for up to 46 days. The cells remained viable throughout the experiment with no signs of central necrosis. Whereas nonencapsulated cells aggregated into larger clusters, some of which showed signs of morphological changes and tissue organization, the alginate matrix stabilized the cluster size and displayed more homogeneous cell morphologies, allowing culture for long periods of time. For all conditions tested, a stable or declining expression of insulin and PDX1 and an increase in glucagon and somatostatin over time indicated a progressive reduction in beta cell-related gene expression. Alginate scaffolds can provide a chemically defined, xeno-free and easily scalable alternative for culture of pancreatic progenitors. Although no increase in insulin and PDX1 gene expression after alginate-immobilized cell culture was seen in this study, further optimization of the matrix physicochemical and biological properties and of the medium composition may still be a relevant strategy to promote the stabilization or maturation of stem cell-derived beta cells. © 2015 Wiley Periodicals, Inc.

Metabolism of β-valine via a CoA-dependent ammonia lyase pathway.

PubMed

Otzen, Marleen; Crismaru, Ciprian G; Postema, Christiaan P; Wijma, Hein J; Heberling, Matthew M; Szymanski, Wiktor; de Wildeman, Stefaan; Janssen, Dick B

2015-11-01

Pseudomonas species strain SBV1 can rapidly grow on medium containing β-valine as a sole nitrogen source. The tertiary amine feature of β-valine prevents direct deamination reactions catalyzed by aminotransferases, amino acid dehydrogenases, and amino acid oxidases. However, lyase- or aminomutase-mediated conversions would be possible. To identify enzymes involved in the degradation of β-valine, a PsSBV1 gene library was prepared and used to complement the β-valine growth deficiency of a closely related Pseudomonas strain. This resulted in the identification of a gene encoding β-valinyl-coenzyme A ligase (BvaA) and two genes encoding β-valinyl-CoA ammonia lyases (BvaB1 and BvaB2). The BvaA protein demonstrated high sequence identity to several known phenylacetate CoA ligases. Purified BvaA enzyme did not convert phenyl acetic acid but was able to activate β-valine in an adenosine triphosphate (ATP)- and CoA-dependent manner. The substrate range of the enzyme appears to be narrow, converting only β-valine and to a lesser extent, 3-aminobutyrate and β-alanine. Characterization of BvaB1 and BvaB2 revealed that both enzymes were able to deaminate β-valinyl-CoA to produce 3-methylcrotonyl-CoA, a common intermediate in the leucine degradation pathway. Interestingly, BvaB1 and BvaB2 demonstrated no significant sequence identity to known CoA-dependent ammonia lyases, suggesting they belong to a new family of enzymes. BLAST searches revealed that BvaB1 and BvaB2 show high sequence identity to each other and to several enoyl-CoA hydratases, a class of enzymes that catalyze a similar reaction with water instead of amine as the leaving group.

Defluviitalea phaphyphila sp. nov., a Novel Thermophilic Bacterium That Degrades Brown Algae.

PubMed

Ji, Shi-Qi; Wang, Bing; Lu, Ming; Li, Fu-Li

2016-02-01

Brown algae are one of the largest groups of oceanic primary producers for CO2 removal and carbon sinks for coastal regions. However, the mechanism for brown alga assimilation remains largely unknown in thermophilic microorganisms. In this work, a thermophilic alginolytic community was enriched from coastal sediment, from which an obligate anaerobic and thermophilic bacterial strain, designated Alg1, was isolated. Alg1 shared a 16S rRNA gene identity of 94.6% with Defluviitalea saccharophila LIND6LT2(T). Phenotypic, chemotaxonomic, and phylogenetic studies suggested strain Alg1 represented a novel species of the genus Defluviitalea, for which the name Defluviitalea phaphyphila sp. nov. is proposed. Alg1 exhibited an intriguing ability to convert carbohydrates of brown algae, including alginate, laminarin, and mannitol, to ethanol and acetic acid. Three gene clusters participating in this process were predicted to be in the genome, and candidate enzymes were successfully expressed, purified, and characterized. Six alginate lyases were demonstrated to synergistically deconstruct alginate into unsaturated monosaccharide, followed by one uronic acid reductase and two 2-keto-3-deoxy-d-gluconate (KDG) kinases to produce pyruvate. A nonclassical mannitol 1-phosphate dehydrogenase, catalyzing D-mannitol 1-phosphate to fructose 1-phosphate in the presence of NAD(+), and one laminarase also were disclosed. This work revealed that a thermophilic brown alga-decomposing system containing numerous novel thermophilic alginate lyases and a unique mannitol 1-phosphate dehydrogenase was adopted by the natural ethanologenic strain Alg1 during the process of evolution in hostile habitats. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

21 CFR 184.1610 - Potassium alginate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Potassium alginate. 184.1610 Section 184.1610 Food... GRAS § 184.1610 Potassium alginate. (a) Potassium alginate (CAS Reg. No. 9005-36-1) is the potassium salt of alginic acid, a natural polyuronide constituent of certain brown algae. Potassium alginate is...

21 CFR 184.1187 - Calcium alginate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Calcium alginate. 184.1187 Section 184.1187 Food... GRAS § 184.1187 Calcium alginate. (a) Calcium alginate (CAS Reg. No. 9005-35-0) is the calcium salt of alginic acid, a natural polyuronide constituent of certain brown algae. Calcium alginate is prepared by...

21 CFR 184.1724 - Sodium alginate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sodium alginate. 184.1724 Section 184.1724 Food... GRAS § 184.1724 Sodium alginate. (a) Sodium alginate (CAS Reg. No. 9005-38-3) is the sodium salt of alginic acid, a natural polyuronide constituent of certain brown algae. Sodium alginate is prepared by the...

21 CFR 184.1724 - Sodium alginate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium alginate. 184.1724 Section 184.1724 Food and... Substances Affirmed as GRAS § 184.1724 Sodium alginate. (a) Sodium alginate (CAS Reg. No. 9005-38-3) is the sodium salt of alginic acid, a natural polyuronide constituent of certain brown algae. Sodium alginate is...

Isocitrate Lyase from Flax 1

PubMed Central

Khan, Fazal R.; McFadden, Bruce A.

1982-01-01

The cleavage of Ds-isocitrate catalyzed by isocitrate lyase from Linum usitatissimum results in the ordered release of succinate and glyoxylate. The glyoxylate analog 3-bromopyruvate irreversibly inactivates the flax enzyme in a process exhibiting saturation kinetics and protection by glyoxylate or isocitrate or the competitive inhibitor l-tartrate. Succinate provides considerably less protection. Results with 3-bromopyruvate suggest that this reagent modifies plant and prokaryotic isocitrate lyases differently. Treatment of the tetrameric 264,000-dalton flax enzyme with carboxypeptidase A results in a release of one histidine/subunit which is concordant with loss of activity. The only N-terminal residue is methionine. Treatment of flax enzyme with diethylpyrocarbonate at pH 6.5 selectively modifies two histidines per 67,000-dalton subunit. The reaction of one histidine residue is abolished by the binding of l-tartrate and the modification of one is coincident with inactivation. The carboxy-terminal and active-site modifications establish that one histidine residue/monomer is essential in the flax enzyme and considerably extend information heretofore available only for fungal and bacterial isocitrate lyase. PMID:16662648

Characterization of free and alginate-polylysine-alginate microencapsulated Erwinia herbicola for the conversion of ammonia, pyruvate, and phenol into L-tyrosine

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

Lloyd-George, I.; Chang, T.M.S.

1995-12-20

The whole cell tyrosine phenol-lyase activity of Erwinia herbicola was microencapsulated. The authors studied the use of this for the conversion of ammonia and pyruvate along with phenol or catechol, respectively, into L-tyrosine or dihydroxyphenyl-L-alanine (L-dopa). The reactions are relevant to the development of new methods for the production of L-tyrosine and L-dopa. The growth of E. herbicola at temperatures from 22 C to 32 C is stable, since at these temperatures the cells grow up to the stationary phase and remain there for at least 10 h. At 37 C the cells grow rapidly, but they also enter themore » death phase rapidly. There is only limited growth of E. herbicola at 42 C. Whole cells of E. herbicola were encapsulated within alginate-polylysine-alginate microcapsules (916 {+-} 100 {micro}m, mean {+-} std. dev.). The TPL activity of the cells catalyzed the production of L-tyrosine or dihydroxyphenol-L-alanine (L-dopa) from ammonia, pyruvate, and phenol or catechol, respectively. In the production of tyrosine, an integrated equation based on an ordered ter-uni rapid equilibrium mechanism can be used to find the kinetic parameters of TPL. In an adequately stirred system, the apparent values of the kinetic parameters of whole cell TPL are equal whether the cells are free or encapsulated. The apparent K{sub M} of tyrosine varies with the amount of whole cells in the system, ranging from 0.2 to 0.3 mM. The apparent K{sub M} for phenol is 0.5 mM. The apparent K{sub M} values for pyruvate and ammonia are an order of magnitude greater for whole cells than they are for the cell free enzyme.« less

Photo-crosslinked alginate hydrogels support enhanced matrix accumulation by nucleus pulposus cells in vivo.

PubMed

Chou, A I; Akintoye, S O; Nicoll, S B

2009-10-01

Intervertebral disc (IVD) degeneration is a major health concern in the United States. Replacement of the nucleus pulposus (NP) with injectable biomaterials represents a potential treatment strategy for IVD degeneration. The objective of this study was to characterize the extracellular matrix (ECM) assembly and functional properties of NP cell-encapsulated, photo-crosslinked alginate hydrogels in comparison to ionically crosslinked alginate constructs. Methacrylated alginate was synthesized by esterification of hydroxyl groups with methacrylic anhydride. Bovine NP cells were encapsulated in alginate hydrogels by ionic crosslinking using CaCl(2) or through photo-crosslinking upon exposure to long-wave UV light in the presence of a photoinitiator. The hydrogels were evaluated in vitro by gross and histological analysis and in vivo using a murine subcutaneous pouch model. In vivo samples were analyzed for gene expression, ECM localization and accumulation, and equilibrium mechanical properties. Ionically crosslinked hydrogels exhibited inferior proteoglycan accumulation in vitro and were unable to maintain structural integrity in vivo. In further studies, photo-crosslinked alginate hydrogels were implanted for up to 8 weeks to examine NP tissue formation. Photo-crosslinked hydrogels displayed temporal increases in gene expression and assembly of type II collagen and proteoglycans. Additionally, hydrogels remained intact over the duration of the study and the equilibrium Young's modulus increased from 1.24+/-0.09 kPa to 4.31+/-1.39 kPa, indicating the formation of functional matrix with properties comparable to those of the native NP. These findings support the use of photo-crosslinked alginate hydrogels as biomaterial scaffolds for NP replacement.

A Novel Aldo-Keto Reductase, HdRed, from the Pacific Abalone Haliotis discus hannai, Which Reduces Alginate-derived 4-Deoxy-l-erythro-5-hexoseulose Uronic Acid to 2-Keto-3-deoxy-d-gluconate*

PubMed Central

Mochizuki, Shogo; Nishiyama, Ryuji; Inoue, Akira; Ojima, Takao

2015-01-01

Abalone feeds on brown seaweeds and digests seaweeds' alginate with alginate lyases (EC 4.2.2.3). However, it has been unclear whether the end product of alginate lyases (i.e. unsaturated monouronate-derived 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH)) is assimilated by abalone itself, because DEH cannot be metabolized via the Embden-Meyerhof pathway of animals. Under these circumstances, we recently noticed the occurrence of an NADPH-dependent reductase, which reduced DEH to 2-keto-3-deoxy-d-gluconate, in hepatopancreas extract of the pacific abalone Haliotis discus hannai. In the present study, we characterized this enzyme to some extent. The DEH reductase, named HdRed in the present study, could be purified from the acetone-dried powder of hepatopancreas by ammonium sulfate fractionation followed by conventional column chromatographies. HdRed showed a single band of ∼40 kDa on SDS-PAGE and reduced DEH to 2-keto-3-deoxy-d-gluconate with an optimal temperature and pH at around 50 °C and 7.0, respectively. HdRed exhibited no appreciable activity toward 28 authentic compounds, including aldehyde, aldose, ketose, α-keto-acid, uronic acid, deoxy sugar, sugar alcohol, carboxylic acid, ketone, and ester. The amino acid sequence of 371 residues of HdRed deduced from the cDNA showed 18–60% identities to those of aldo-keto reductase (AKR) superfamily enzymes, such as human aldose reductase, halophilic bacterium reductase, and sea hare norsolorinic acid (a polyketide derivative) reductase-like protein. Catalytic residues and cofactor binding residues known in AKR superfamily enzymes were fairly well conserved in HdRed. Phylogenetic analysis for HdRed and AKR superfamily enzymes indicated that HdRed is an AKR belonging to a novel family. PMID:26555267

A Novel Aldo-Keto Reductase, HdRed, from the Pacific Abalone Haliotis discus hannai, Which Reduces Alginate-derived 4-Deoxy-L-erythro-5-hexoseulose Uronic Acid to 2-Keto-3-deoxy-D-gluconate.

PubMed

Mochizuki, Shogo; Nishiyama, Ryuji; Inoue, Akira; Ojima, Takao

2015-12-25

Abalone feeds on brown seaweeds and digests seaweeds' alginate with alginate lyases (EC 4.2.2.3). However, it has been unclear whether the end product of alginate lyases (i.e. unsaturated monouronate-derived 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH)) is assimilated by abalone itself, because DEH cannot be metabolized via the Embden-Meyerhof pathway of animals. Under these circumstances, we recently noticed the occurrence of an NADPH-dependent reductase, which reduced DEH to 2-keto-3-deoxy-D-gluconate, in hepatopancreas extract of the pacific abalone Haliotis discus hannai. In the present study, we characterized this enzyme to some extent. The DEH reductase, named HdRed in the present study, could be purified from the acetone-dried powder of hepatopancreas by ammonium sulfate fractionation followed by conventional column chromatographies. HdRed showed a single band of ∼ 40 kDa on SDS-PAGE and reduced DEH to 2-keto-3-deoxy-D-gluconate with an optimal temperature and pH at around 50 °C and 7.0, respectively. HdRed exhibited no appreciable activity toward 28 authentic compounds, including aldehyde, aldose, ketose, α-keto-acid, uronic acid, deoxy sugar, sugar alcohol, carboxylic acid, ketone, and ester. The amino acid sequence of 371 residues of HdRed deduced from the cDNA showed 18-60% identities to those of aldo-keto reductase (AKR) superfamily enzymes, such as human aldose reductase, halophilic bacterium reductase, and sea hare norsolorinic acid (a polyketide derivative) reductase-like protein. Catalytic residues and cofactor binding residues known in AKR superfamily enzymes were fairly well conserved in HdRed. Phylogenetic analysis for HdRed and AKR superfamily enzymes indicated that HdRed is an AKR belonging to a novel family. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Variations in chondrogenesis of human bone marrow-derived mesenchymal stem cells in fibrin/alginate blended hydrogels

PubMed Central

Ma, Kun; Titan, Ashley L.; Stafford, Melissa; Zheng, Chun hua; Levenston, Marc E.

2012-01-01

Fibrin and alginate hydrogels have been widely used to support chondrogenesis of bone marrow-derived mesenchymal stem cells (BM-MSCs) for articular cartilage and fibrocartilage tissue engineering, with distinct advantages and disadvantages to each material. Attempting to produce a gel scaffold exhibiting beneficial characteristics of both materials, we fabricated fibrin/alginate blended hydrogels at various blend ratios and evaluated the gel morphology, mechanical properties and their support for BM-MSC chondrogenesis. Results show that when the fibrin/alginate ratio decreased, the fibrin architecture transitioned from uniform to interconnected fibrous and finally to disconnected islands against an alginate background, with opposing trends in the alginate architecture. Fibrin maintained gel extensibility and promoted cell proliferation, while alginate improved the gel biostability and better supported glycosaminoglycan and collagen II production and chondrogenic gene expression. Blended gels had physical and biological characteristics intermediate between fibrin and alginate. Of the blends examined, FA 40:8 (40 mg/mL fibrinogen blended with 8 mg/mL alginate) was found to be the most appropriate group for future studies on tension-driven BM-MSC fibrochondrogenesis. As BM-MSC differentiation appeared to vary between fibrin and alginate regions of blended scaffolds, this study also highlighted the potential to develop spatially heterogeneous tissues through manipulating the heterogeneity of scaffold composition. PMID:22750738

21 CFR 184.1610 - Potassium alginate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Potassium alginate. 184.1610 Section 184.1610 Food... Specific Substances Affirmed as GRAS § 184.1610 Potassium alginate. (a) Potassium alginate (CAS Reg. No. 9005-36-1) is the potassium salt of alginic acid, a natural polyuronide constituent of certain brown...

21 CFR 184.1610 - Potassium alginate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Potassium alginate. 184.1610 Section 184.1610 Food... Specific Substances Affirmed as GRAS § 184.1610 Potassium alginate. (a) Potassium alginate (CAS Reg. No. 9005-36-1) is the potassium salt of alginic acid, a natural polyuronide constituent of certain brown...

A missense mutation in the human cytochrome b5 gene causes 46,XY disorder of sex development due to true isolated 17,20 lyase deficiency.

PubMed

Idkowiak, Jan; Randell, Tabitha; Dhir, Vivek; Patel, Pushpa; Shackleton, Cedric H L; Taylor, Norman F; Krone, Nils; Arlt, Wiebke

2012-03-01

Isolated 17,20 lyase deficiency is commonly defined by apparently normal 17α-hydroxylase activity but severely reduced 17,20 lyase activity of the bifunctional enzyme cytochrome P450 (CYP) enzyme 17A1 (CYP17A1), resulting in sex steroid deficiency but normal glucocorticoid and mineralocorticoid reserve. Cytochrome b5 (CYB5A) is thought to selectively enhance 17,20 lyase activity by facilitating the allosteric interaction of CYP17A1 with its electron donor P450 oxidoreductase (POR). We investigated a large consanguineous family including three siblings with 46,XY disorder of sex development (DSD) presenting with isolated 17,20 lyase deficiency. We investigated the clinical and biochemical phenotype, conducted genetic analyses, and functionally characterized the identified CYB5A mutation in cell-based CYP17A1 coexpression assays. All three siblings presented with 46,XY DSD, sex steroid deficiency, normal mineralocorticoids and glucocorticoids, and a urine steroid metabolome suggestive of isolated 17,20 lyase deficiency. CYP17A1 and POR sequences were normal, but we detected a homozygous CYB5A missense mutation (g.28,400A→T; p.H44L). Functional in vitro analysis revealed normal CYP17A1 17α-hydroxylase activity but severely impaired 17,20 lyase activity. In silico analysis suggested the disruption of CYB5A heme binding by p.H44L. We have identified the first human CYB5A missense mutation as the cause of isolated 17,20 lyase deficiency in three individuals with 46,XY DSD. Detailed review of previously reported cases with apparently isolated 17,20 lyase deficiency due to mutant CYP17A1 and POR reveals impaired 17α-hydroxylase activity as assessed by steroid metabolome analysis and short cosyntropin testing. This suggests that truly isolated 17,20 lyase deficiency is observed only in individuals with inactivating CYB5A mutations.

21 CFR 184.1724 - Sodium alginate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sodium alginate. 184.1724 Section 184.1724 Food... Specific Substances Affirmed as GRAS § 184.1724 Sodium alginate. (a) Sodium alginate (CAS Reg. No. 9005-38-3) is the sodium salt of alginic acid, a natural polyuronide constituent of certain brown algae...

21 CFR 184.1724 - Sodium alginate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sodium alginate. 184.1724 Section 184.1724 Food... Specific Substances Affirmed as GRAS § 184.1724 Sodium alginate. (a) Sodium alginate (CAS Reg. No. 9005-38-3) is the sodium salt of alginic acid, a natural polyuronide constituent of certain brown algae...

21 CFR 184.1724 - Sodium alginate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sodium alginate. 184.1724 Section 184.1724 Food... Specific Substances Affirmed as GRAS § 184.1724 Sodium alginate. (a) Sodium alginate (CAS Reg. No. 9005-38-3) is the sodium salt of alginic acid, a natural polyuronide constituent of certain brown algae...

21 CFR 184.1187 - Calcium alginate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Calcium alginate. 184.1187 Section 184.1187 Food... Specific Substances Affirmed as GRAS § 184.1187 Calcium alginate. (a) Calcium alginate (CAS Reg. No. 9005-35-0) is the calcium salt of alginic acid, a natural polyuronide constituent of certain brown algae...

21 CFR 184.1187 - Calcium alginate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Calcium alginate. 184.1187 Section 184.1187 Food... Specific Substances Affirmed as GRAS § 184.1187 Calcium alginate. (a) Calcium alginate (CAS Reg. No. 9005-35-0) is the calcium salt of alginic acid, a natural polyuronide constituent of certain brown algae...

Proteomic analysis reveals large amounts of decomposition enzymes and major metabolic pathways involved in algicidal process of Trametes versicolor F21a.

PubMed

Gao, Xueyan; Wang, Congyan; Dai, Wei; Ren, Shenrong; Tao, Fang; He, Xingbing; Han, Guomin; Wang, Wei

2017-06-20

A recent algicidal mode indicates that fungal mycelia can wrap and eliminate almost all co-cultivated algal cells within a short time span. However, the underlying molecular mechanism is rarely understood. We applied proteomic analysis to investigate the algicidal process of Trametes versicolor F21a and identified 3,754 fungal proteins. Of these, 30 fungal enzymes with endo- or exoglycosidase activities such as β-1,3-glucanase, α-galactosidase, α-glucosidase, alginate lyase and chondroitin lyase were significantly up-regulated. These proteins belong to Glycoside Hydrolases, Auxiliary Activities, Carbohydrate Esterases and Polysaccharide Lyases, suggesting that these enzymes may degrade lipopolysaccharides, peptidoglycans and alginic acid of algal cells. Additionally, peptidase, exonuclease, manganese peroxidase and cytochrome c peroxidase, which decompose proteins and DNA or convert other small molecules of algal cells, could be other major decomposition enzymes. Gene Ontology and KEGG pathway enrichment analysis demonstrated that pyruvate metabolism and tricarboxylic acid cycle pathways play a critical role in response to adverse environment via increasing energy production to synthesize lytic enzymes or uptake molecules. Carbon metabolism, selenocompound metabolism, sulfur assimilation and metabolism, as well as several amino acid biosynthesis pathways could play vital roles in the synthesis of nutrients required by fungal mycelia.

A cDNA clone highly expressed in ripe banana fruit shows homology to pectate lyases.

PubMed

Dominguez-Puigjaner, E; LLop, I; Vendrell, M; Prat, S

1997-07-01

A cDNA clone (Ban17), encoding a protein homologous to pectate lyase, has been isolated from a cDNA library from climacteric banana fruit by means of differential screening. Northern analysis showed that Ban17 mRNA is first detected in early climacteric fruit, reaches a steady-state maximum at the climacteric peak, and declines thereafter in overripe fruit. Accumulation of the Ban17 transcript can be induced in green banana fruit by exogenous application of ethylene. The demonstrates that expression of this gene is under hormonal control, its induction being regulated by the rapid increase in ethylene production at the onset of ripening. The deduced amino acid sequence derived from the Ban17 cDNA shares significant identity with pectate lyases from pollen and plant pathogenic bacteria of the genus Erwinia. Similarity to bacterial pectate lyases that were proven to break down the pectic substances of the plant cell wall suggest that Ban17 might play a role in the loss of mesocarp firmness during fruit ripening.

Osteogenic differentiation of human mesenchymal stem cells in mineralized alginate matrices.

PubMed

Westhrin, Marita; Xie, Minli; Olderøy, Magnus Ø; Sikorski, Pawel; Strand, Berit L; Standal, Therese

2015-01-01

Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC) in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP) for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and alginate beads mineralized with ALP to generate a polymer/hydroxyapatite scaffold mimicking the composition of bone. The initial scaffold mineralization induced further mineralization of the beads with nanosized particles, and scanning electron micrographs demonstrated presence of collagen in the mineralized and unmineralized alginate beads cultured in osteogenic medium. Cells in both types of beads sustained high viability and metabolic activity for the duration of the study (21 days) as evaluated by live/dead staining and alamar blue assay. MSC in beads induced to differentiate in osteogenic direction expressed higher mRNA levels of osteoblast-specific genes (RUNX2, COL1AI, SP7, BGLAP) than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), markers for late osteoblasts/osteocytes. In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture. Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering.

Osteogenic Differentiation of Human Mesenchymal Stem Cells in Mineralized Alginate Matrices

PubMed Central

Westhrin, Marita; Xie, Minli; Olderøy, Magnus Ø.; Sikorski, Pawel

2015-01-01

Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC) in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP) for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and alginate beads mineralized with ALP to generate a polymer/hydroxyapatite scaffold mimicking the composition of bone. The initial scaffold mineralization induced further mineralization of the beads with nanosized particles, and scanning electron micrographs demonstrated presence of collagen in the mineralized and unmineralized alginate beads cultured in osteogenic medium. Cells in both types of beads sustained high viability and metabolic activity for the duration of the study (21 days) as evaluated by live/dead staining and alamar blue assay. MSC in beads induced to differentiate in osteogenic direction expressed higher mRNA levels of osteoblast-specific genes (RUNX2, COL1AI, SP7, BGLAP) than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), markers for late osteoblasts/osteocytes. In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture. Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering. PMID:25769043

Structure, inheritance, and expression of hybrid poplar (Populus trichocarpa x Populus deltoides) phenylalanine ammonia-lyase genes.

PubMed Central

Subramaniam, R; Reinold, S; Molitor, E K; Douglas, C J

1993-01-01

A heterologous probe encoding phenylalanine ammonia-lyase (PAL) was used to identify PAL clones in cDNA libraries made with RNA from young leaf tissue of two Populus deltoides x P. trichocarpa F1 hybrid clones. Sequence analysis of a 2.4-kb cDNA confirmed its identity as a full-length PAl clone. The predicted amino acid sequence is conserved in comparison with that of PAL genes from several other plants. Southern blot analysis of popular genomic DNA from parental and hybrid individuals, restriction site polymorphism in PAL cDNA clones, and sequence heterogeneity in the 3' ends of several cDNA clones suggested that PAL is encoded by at least two genes that can be distinguished by HindIII restriction site polymorphisms. Clones containing each type of PAL gene were isolated from a poplar genomic library. Analysis of the segregation of PAL-specific HindIII restriction fragment-length polymorphisms demonstrated the existence of two independently segregating PAL loci, one of which was mapped to a linkage group of the poplar genetic map. Developmentally regulated PAL expression in poplar was analyzed using RNA blots. Highest expression was observed in young stems, apical buds, and young leaves. Expression was lower in older stems and undetectable in mature leaves. Cellular localization of PAL expression by in situ hybridization showed very high levels of expression in subepidermal cells of leaves early during leaf development. In stems and petioles, expression was associated with subepidermal cells and vascular tissues. PMID:8108506

Regulation of L-phenylalanine ammonia-lyase from Rhizoctonia solani.

PubMed Central

Kalghatgi, K K; Subba Rao, P V

1976-01-01

Maximal levels of L-henylalanine ammonia-lyase activity were observed when the mycelial felts of Rhizoctonia solani were grown for 4.5 days on Byrde synthetic medium containing 3.5% glucose and 0.3% L-phenylalanine, Differential centrifugation studies have indicated that the enzyme is localized in the soluble fraction. The time course of induction of L-phenylalanine ammonia-lyase activity by L-phenylalanine showed a lag period of 1 to 1.5 h and reached a maximum around 4 to 6 h after the addition of the inducer to the medium. L-Phenylalanine, L-tyrosine, and L-tryptophan were nearly equally efficient inducers of the enzyme. D-Phenylalanine was as efficient as the L-isomer, whereas D-tyrosine was a poor inducer. Light, gibberellic acid, indole 3-acetic acid, and kinetin had no effect on the induction of L-phenylalanine ammonia-lyase activity. Cycloheximide did not inhibit the uptake of amino acids by the mycelia but completely blocked the incorporation of radioactive amino acids into soluble proteins and the development of L-phenylalanine ammonia-lyase activity. Actinomycin D inhibited both the incorporation of 32P into ribonucleic acid and the enzyme activity. Conclusive evidence for de novo synthesis of L-phenylalanine ammonia-lyase was obtained by the incorporation of radioactive amino acids into the enzyme. Electrophoretic analysis of the purified preparation showed a single protein band that coincided with radioactivity and L-phenylalanine ammonia-lyase activity. Glucose and intermediates of the tricarboxylic acid cycle, like citric acid, alpha-ketoglutaric acid, and succinic acid, and the metabolites of L-phenylalanine, like o-coumaric acid, o-hydroxyphenylacetic acid, and protocatechuic acid, significantly repressed L-phenylalanine ammonia-lyase activity. The observed repression was not relieved by cyclic adenosine 5'-triphosphate. Images PMID:1262311

Physicochemical properties of marine collagen-alginate biomaterial

NASA Astrophysics Data System (ADS)

Soon, K. S.; Hii, S. L.; Wong, C. L.; Leong, L. K.; Woo, K. K.

2017-12-01

Collagen base biomaterials are widely applied in the field of tissue engineering. However, these fibrous proteins in animal connective tissues are insufficient to fulfill the mechanical properties for such applications. Therefore, alginate as a natural polysaccharide was incorporated. In this study, Smooth wolf herring skins was collected from the local fish ball processing industry for collagen extraction using acid solubilisation method. On the other hand, alginate from brown seaweed (Sargassum polycystum) was extracted with calcium carbonate at 50 °C. The composite films of different collagen and alginate ratio were prepared by lyophilisation with pure collagen film as control. The effects of alginate on swelling behaviour, porosity, collagenase degradation and tensile strength of the composite films were investigated. Swelling behaviour increased with alginate content, 50 % alginate film achieved 1254.75 % swelling after 24 h. All composite films achieved more than 80 % porosity except the film with 80 % collagen (65.41 %). Porosity was highest in 100 % alginate (94.30 %). Highest tensile strength (1585.87 kPa) and young modulus (27.05 MPa) was found in 50 % alginate film. In addition, resistance to collagenase degradation was improved with alginate content, lowest degradation rate was determined in 80 % alginate film. Results indicated alginate is efficient in improving some mechanical properties of the composite film.

3D Cell Culture in Alginate Hydrogels

PubMed Central

Andersen, Therese; Auk-Emblem, Pia; Dornish, Michael

2015-01-01

This review compiles information regarding the use of alginate, and in particular alginate hydrogels, in culturing cells in 3D. Knowledge of alginate chemical structure and functionality are shown to be important parameters in design of alginate-based matrices for cell culture. Gel elasticity as well as hydrogel stability can be impacted by the type of alginate used, its concentration, the choice of gelation technique (ionic or covalent), and divalent cation chosen as the gel inducing ion. The use of peptide-coupled alginate can control cell–matrix interactions. Gelation of alginate with concomitant immobilization of cells can take various forms. Droplets or beads have been utilized since the 1980s for immobilizing cells. Newer matrices such as macroporous scaffolds are now entering the 3D cell culture product market. Finally, delayed gelling, injectable, alginate systems show utility in the translation of in vitro cell culture to in vivo tissue engineering applications. Alginate has a history and a future in 3D cell culture. Historically, cells were encapsulated in alginate droplets cross-linked with calcium for the development of artificial organs. Now, several commercial products based on alginate are being used as 3D cell culture systems that also demonstrate the possibility of replacing or regenerating tissue. PMID:27600217

Self-disinfecting Alginate vs Conventional Alginate: Effect on Surface Hardness of Gypsum Cast-An in vitro Study.

PubMed

Madhavan, Ranjith; George, Navia; Thummala, Niharika R; Ravi, S V; Nagpal, Ajay

2017-11-01

For the construction of any dental prosthesis, accurate impressions are necessary. Hence, we undertook the present study to evaluate and compare the surface hardness of gypsum casts poured from impressions made using conventional alginate and self-disinfecting alginate. A total of 30 impressions of stainless steel die were made, out of which 15 impressions were made with conventional alginate and 15 were made with self-disinfecting alginate and poured using Type III dental stone. Thirty stone specimens were subjected for hardness testing. Data were analyzed using independent samples t-test to compare the mean surface hardness. Difference in surface hardness was statistically insignificant (p > 0.05). Surface hardness of gypsum casts poured using impressions made from self-disinfecting alginate and conventional alginates were comparable. Self-disinfecting alginates may be employed in clinical practice as safe and effective materials to overcome the infection control issues without compromising on the properties of the material.

Oxygen-dependent regulation of c-di-GMP synthesis by SadC controls alginate production in Pseudomonas aeruginosa.

PubMed

Schmidt, Annika; Hammerbacher, Anna Silke; Bastian, Mike; Nieken, Karen Jule; Klockgether, Jens; Merighi, Massimo; Lapouge, Karine; Poschgan, Claudia; Kölle, Julia; Acharya, K Ravi; Ulrich, Martina; Tümmler, Burkhard; Unden, Gottfried; Kaever, Volkhard; Lory, Stephen; Haas, Dieter; Schwarz, Sandra; Döring, Gerd

2016-10-01

Pseudomonas aeruginosa produces increased levels of alginate in response to oxygen-deprived conditions. The regulatory pathway(s) that links oxygen limitation to increased synthesis of alginate has remained elusive. In the present study, using immunofluorescence microscopy, we show that anaerobiosis-induced alginate production by planktonic PAO1 requires the diguanylate cyclase (DGC) SadC, previously identified as a regulator of surface-associated lifestyles. Furthermore, we found that the gene products of PA4330 and PA4331, located in a predicted operon with sadC, have a major impact on alginate production: deletion of PA4330 (odaA, for oxygen-dependent alginate synthesis activator) caused an alginate production defect under anaerobic conditions, whereas a PA4331 (odaI, for oxygen-dependent alginate synthesis inhibitor) deletion mutant produced alginate also in the presence of oxygen, which would normally inhibit alginate synthesis. Based on their sequence, OdaA and OdaI have predicted hydratase and dioxygenase reductase activities, respectively. Enzymatic assays using purified protein showed that unlike OdaA, which did not significantly affect DGC activity of SadC, OdaI inhibited c-di-GMP production by SadC. Our data indicate that SadC, OdaA and OdaI are components of a novel response pathway of P. aeruginosa that regulates alginate synthesis in an oxygen-dependent manner. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

3-Hydroxy-3-methylglutaryl CoA lyase (HL): Mouse and human HL gene (HMGCL) cloning and detection of large gene deletions in two unrelated HL-deficient patients

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

Wang, S.P.; Robert, M.F.; Mitchell, G.A.

1996-04-01

3-hydroxy-3-methylglutaryl CoA lyase (HL, EC 4.1.3.4) catalyzes the cleavage of 3-hydroxy-3-methylglutaryl CoA to acetoacetic acid and acetyl CoA, the final reaction of both ketogenesis and leucine catabolism. Autosomal-recessive HL deficiency in humans results in episodes of hypoketotic hypoglycemia and coma. Using a mouse HL cDNA as a probe, we isolated a clone containing the full-length mouse HL gene that spans about 15 kb of mouse chromosome 4 and contains nine exons. The promoter region of the mouse HL gene contains elements characteristic of a housekeeping gene: a CpG island containing multiple Sp1 binding sites surrounds exon 1, and neither amore » TATA nor a CAAT box are present. We identified multiple transcription start sites in the mouse HL gene, 35 to 9 bases upstream of the translation start codon. We also isolated two human HL genomic clones that include HL exons 2 to 9 within 18 kb. The mouse and human HL genes (HGMW-approved symbol HMGCL) are highly homologous, with identical locations of intron-exon junctions. By genomic Southern blot analysis and exonic PCR, was found 2 of 33 HL-deficient probands to be homozygous for large deletions in the HL gene. 26 refs., 4 figs., 2 tabs.« less

Alginate oligosaccharides enhanced Triticum aestivum L. tolerance to drought stress.

PubMed

Liu, Hang; Zhang, Yun-Hong; Yin, Heng; Wang, Wen-Xia; Zhao, Xiao-Ming; Du, Yu-Guang

2013-01-01

Alginate oligosaccharides (AOS) prepared from degradation of alginate is a potent plant elicitor. Hydroponic experiments were carried out to investigate the mechanism of AOS on improving Triticum aestivum L. resistant ability to drought stress. Drought model was simulated by exposing the roots of wheat to polyethylene glycol-6000 (PEG-6000) solution (150 g L(-1)) for 4 days and the growth of wheat treated with PEG was significantly decreased. However, after AOS application, seedling and root length, fresh weight and relative water content of wheat were increased by 18%, 26%, 43% and 33% under dehydration status compared with that of PEG group, respectively. Moreover, the antioxidative enzymes activities were obviously enhanced and malondialdehyde (MDA) content was reduced by 37.9% in samples treated by AOS. Additionally, the drought resistant related genes involved in ABA signal pathway, such as late embryogenesis abundant protein 1 gene (LEA1), psbA gene, Sucrose non-fermenting 1-related protein kinase 2 gene (SnRK2) and Pyrroline-5-Carboxylate Synthetase gene (P5CS) were up-regulated by AOS. Our results suggested that AOS might regulate ABA-dependent signal pathway to enhance drought stress resistance of wheat during growth period. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Nucleotide sequence and expression of a novel pectate lyase gene (pel-3) and a closely linked endopolygalacturonase gene (peh-1) of Erwinia carotovora subsp. carotovora 71.

PubMed Central

Liu, Y; Chatterjee, A; Chatterjee, A K

1994-01-01

Our previous genetic analysis (J. W. Willis, J. K. Engwall, and A. K. Chatterjee, Phytopathology 77:1199-1205, 1987) had revealed a tight linkage between pel-3 (pel, pectate lyase gene) and peh-1 (peh, polygalacturonase gene) within the chromosome of Erwinia carotovora subsp. carotovora 71. Nucleotide sequencing, transcript assays, and expression of enzymatic activities in Escherichia coli have now confirmed that a 3,500-bp segment contains the open reading frames (ORFs) for Pel-3 and Peh-1. The 1,041-bp pel-3 ORF and the 1,206-bp peh-1 ORF are separated by a 579-bp sequence. The genes are transcribed divergently from their own promoters. In E. coli and E. carotovora subsp. carotovora 71, peh-1 is better expressed than pel-3. However, plant signals activate the expression of both the genes in E. carotovora subsp. carotovora. A consensus integration host factor (IHF)-binding sequence upstream of pel-3 appears physiologically significant, since pel-3 promoter activity is higher in an E. coli IHF+ strain than in an IHF- strain. While peh-1 has extensive homology with plant and bacterial peh genes, pel-3 appears not to have significant homology with the pel genes belonging to the pelBC, pelADE, or periplasmic pel families. Pel-3 also is unusual in that it is predicted to contain an ATP- and GTP-binding site motif A (P-loop) not found in the other Pels. Images PMID:8074530

Alginate as a cell culture substrate for growth and differentiation of human retinal pigment epithelial cells.

PubMed

Heidari, Razeih; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Davari, Maliheh; Nazemroaya, Fatemeh; Bagheri, Abouzar; Deezagi, Abdolkhalegh

2015-03-01

The purpose of this study was to evaluate retinal pigment epithelium (RPE) cells' behavior in alginate beads that establish 3D environment for cellular growth and mimic extracellular matrix versus the conventional 2D monolayer culture. RPE cells were encapsulated in alginate beads by dripping alginate cell suspension into CaCl2 solution. Beads were suspended in three different media including Dulbecco's modified Eagle's medium (DMEM)/F12 alone, DMEM/F12 supplemented with 10 % fetal bovine serum (FBS), and DMEM/F12 supplemented with 30 % human amniotic fluid (HAF). RPE cells were cultivated on polystyrene under the same conditions as controls. Cell phenotype, cell proliferation, cell death, and MTT assay, immunocytochemistry, and real-time RT-PCR were performed to evaluate the effect of alginate on RPE cells characteristics and integrity. RPE cells can survive and proliferate in alginate matrixes. Immunocytochemistry analysis exhibited Nestin, RPE65, and cytokeratin expressions in a reasonable number of cultured cells in alginate beads. Real-time PCR data demonstrated high levels of Nestin, CHX10, RPE65, and tyrosinase gene expressions in RPE cells immobilized in alginate when compared to 2D monolayer culture systems. The results suggest that alginate can be used as a reliable scaffold for maintenance of RPE cells' integrity and in vitro propagation of human retinal progenitor cells for cell replacement therapies in retinal diseases.

Spontaneous gene transfection of human bone cells using 3D mineralized alginate-chitosan macrocapsules.

PubMed

Green, David W; Kim, Eun-Jung; Jung, Han-Sung

2015-09-01

The effectiveness of nonviral gene therapy remains uncertain because of low transfection efficiencies and high toxicities compared with viral-based strategies. We describe a simple system for transient transfection of continuous human cell lines, with low toxicity, using mineral-coated chitosan and alginate capsules. As proof-of-concept, we demonstrate transfection of Saos-2 and MG63 human osteosarcoma continuous cell lines with gfp, LacZ reporter genes, and a Sox-9 carrying plasmid, to illustrate expression of a functional gene with therapeutic relevance. We show that continuous cell lines transfect with significant efficiency of up to 65% possibly through the interplay between chitosan and DNA complexation and calcium/phosphate-induced translocation into cells entrapped within the 3D polysaccharide based environment, as evidenced by an absence of transfection in unmineralized and chitosan-free capsules. We demonstrated that our transfection system was equally effective at transfection of primary human bone marrow stromal cells. To illustrate, the Sox-9, DNA plasmid was spontaneously expressed in primary human bone marrow stromal cells at 7 days with up to 90% efficiency in two repeats. Mineralized polysaccharide macrocapsules are gene delivery vehicles with a number of biological and practical advantages. They are highly efficient at self-transfecting primary bone cells, with programmable spatial and temporal delivery prospects, premineralized bone-like environments, and have no cytotoxic effects, as compared with many other nonviral systems. © 2015 Wiley Periodicals, Inc.

21 CFR 582.7610 - Potassium alginate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Potassium alginate. 582.7610 Section 582.7610 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Potassium alginate. (a) Product. Potassium alginate. (b) Conditions of use. This substance is generally...

21 CFR 582.7610 - Potassium alginate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Potassium alginate. 582.7610 Section 582.7610 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Potassium alginate. (a) Product. Potassium alginate. (b) Conditions of use. This substance is generally...

21 CFR 582.7610 - Potassium alginate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Potassium alginate. 582.7610 Section 582.7610 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Potassium alginate. (a) Product. Potassium alginate. (b) Conditions of use. This substance is generally...

21 CFR 582.7187 - Calcium alginate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Calcium alginate. 582.7187 Section 582.7187 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Calcium alginate. (a) Product. Calcium alginate. (b) Conditions of use. This substance is generally...

21 CFR 582.7187 - Calcium alginate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Calcium alginate. 582.7187 Section 582.7187 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Calcium alginate. (a) Product. Calcium alginate. (b) Conditions of use. This substance is generally...

21 CFR 582.7187 - Calcium alginate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Calcium alginate. 582.7187 Section 582.7187 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Calcium alginate. (a) Product. Calcium alginate. (b) Conditions of use. This substance is generally...

Effect of alginate composition on profile release and characteristics of chitosan-alginate microparticles loaded with mangosteen extract

NASA Astrophysics Data System (ADS)

Mulia, Kamarza; Halimah, Nur; Krisanti, Elsa

2017-03-01

Preparation of mangostin-loaded chitosan-alginate microparticles, chemical and physical characterization of the particles, and mangostin release profiles, are described herein. Mangostin rich fraction was obtained from Garcinia mangostana L. pericarp by extraction followed by fractionation. Mangostin-loaded chitosan-alginate microparticles were prepared by ionic gelation method using tripolyphosphate as the linking agent and various concentration of alginate. Mangostin was effectively loaded in all microparticle formulations, resulting in ˜97% encapsulation efficiencies. The loading of mangostin and the in-vitro release profiles in simulated gastrointestinal fluids were affected by the chitosan to alginate ratios used in the preparation of the microparticles. Increased alginate concentration resulted in lowered release of mangostin from microparticles immersed in simulated gastric fluid (pH 1.2) up to two hours. Low release of mangostin in acidic fluid but high release in simulated colon fluid, indicated that the chitosan-alginate microparticles are prospective carrier for extended release of active compound in gastrointestinal system.

21 CFR 582.7724 - Sodium alginate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Sodium alginate. 582.7724 Section 582.7724 Food... DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Stabilizers § 582.7724 Sodium alginate. (a) Product. Sodium alginate. (b) Conditions of use. This substance is generally recognized as...

21 CFR 582.7724 - Sodium alginate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Sodium alginate. 582.7724 Section 582.7724 Food... DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Stabilizers § 582.7724 Sodium alginate. (a) Product. Sodium alginate. (b) Conditions of use. This substance is generally recognized as...

21 CFR 582.7724 - Sodium alginate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Sodium alginate. 582.7724 Section 582.7724 Food... DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Stabilizers § 582.7724 Sodium alginate. (a) Product. Sodium alginate. (b) Conditions of use. This substance is generally recognized as...

21 CFR 582.7724 - Sodium alginate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Sodium alginate. 582.7724 Section 582.7724 Food... DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Stabilizers § 582.7724 Sodium alginate. (a) Product. Sodium alginate. (b) Conditions of use. This substance is generally recognized as...

21 CFR 582.7724 - Sodium alginate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Sodium alginate. 582.7724 Section 582.7724 Food... DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Stabilizers § 582.7724 Sodium alginate. (a) Product. Sodium alginate. (b) Conditions of use. This substance is generally recognized as...

Isolation and genetic mapping of a Coffea canephora phenylalanine ammonia-lyase gene (CcPAL1) and its involvement in the accumulation of caffeoyl quinic acids.

PubMed

Mahesh, Venkataramaiah; Rakotomalala, Jean Jacques; Le Gal, Lénaïg; Vigne, Hélène; de Kochko, Alexandre; Hamon, Serge; Noirot, Michel; Campa, Claudine

2006-09-01

Biosynthesis of caffeoylquinic acids occurs via the phenylpropanoid pathway in which the phenylalanine ammonia-lyase (PAL) acts as a key-control enzyme. A full-length cDNA (pF6), corresponding to a PAL gene (CcPAL1), was isolated by screening a Coffea canephora fruit cDNA library and its corresponding genomic sequence was characterized. Amplification of total DNA from seven Coffea species revealed differences in intronic length. This interspecific polymorphism was used to locate the gene on a genetic map established for a backcross progeny between Coffea pseudozanguebariae and C. dewevrei. The CcPAL1 gene was found on the same linkage group, but genetically independent, as a caffeoyl-coenzyme A-O-methyltransferase gene, another gene intervening in the phenylpropanoid pathway. In the same backcross, a lower caffeoylquinic acid content was observed in seeds harvested from plants harbouring the C. pseudozanguebariae CcPAL1 allele. Involvement of the CcPAL1 allelic form in the differential accumulation of caffeoylquinic acids in coffee green beans is then discussed.

Chondroitin Lyase from a Marine Arthrobacter sp. MAT3885 for the Production of Chondroitin Sulfate Disaccharides.

PubMed

Kale, Varsha; Friðjónsson, Ólafur; Jónsson, Jón Óskar; Kristinsson, Hörður G; Ómarsdóttir, Sesselja; Hreggviðsson, Guðmundur Ó

2015-08-01

Chondroitin sulfate (CS) saccharides from cartilage tissues have potential application in medicine or as dietary supplements due to their therapeutic bioactivities. Studies have shown that depolymerized CS saccharides may display enhanced bioactivity. The objective of this study was to isolate a CS-degrading enzyme for an efficient production of CS oligo- or disaccharides. CS-degrading bacteria from marine environments were enriched using in situ artificial support colonization containing CS from shark cartilage as substrate. Subsequently, an Arthrobacter species (strain MAT3885) efficiently degrading CS was isolated from a CS enrichment culture. The genomic DNA from strain MAT3885 was pyro-sequenced by using the 454 FLX sequencing technology. Following assembly and annotation, an orf, annotated as family 8 polysaccharide lyase genes, was identified, encoding an amino acid sequence with a similarity to CS lyases according to NCBI blastX. The gene, designated choA1, was cloned in Escherichia coli and expressed downstream of and in frame with the E. coli malE gene for obtaining a high yield of soluble recombinant protein. Applying a dual-tag system (MalE-Smt3-ChoA1), the MalE domain was separated from ChoA1 with proteolytic cleavage using Ulp1 protease. ChoA1 was defined as an AC-type enzyme as it degraded chondroitin sulfate A, C, and hyaluronic acid. The optimum activity of the enzyme was at pH 5.5-7.5 and 40 °C, running a 10-min reaction. The native enzyme was estimated to be a monomer. As the recombinant chondroitin sulfate lyase (designated as ChoA1R) degraded chondroitin sulfate efficiently compared to a benchmark enzyme, it may be used for the production of chondroitin sulfate disaccharides for the food industry or health-promoting products.

Release of tissue inhibitor of metalloproteinase-2 from alginate microcapsule encapsulating genetically engineered cells

PubMed Central

Kim, Yeon Seong; Jeong, Young-II; Jin, Shu-Guang; Pei, Jian; Wen, Min; Kim, In-Young; Moon, Kyung-Sub; Jung, Tae-Young; Ryu, Hyang-Hwa; Jung, Shin

2013-01-01

Background In this study, 293T cells were genetically engineered to secrete tissue inhibitor of metalloproteinase-2 (TIMP2) and encapsulated into alginate microcapsules to continuously release TIMP2 protein. Methods The anti-invasive potential of the microcapsules was studied in vitro using brain tumor cells. The TIMP2 gene was transfected to 293T cells, and genetically engineered 293TIMP2 cells were encapsulated into alginate microcapsules. Release of TIMP2 protein was detected with Western blot analysis and the anti-invasive potential against U87MG cells was tested using gelatin zymography and a Matrigel assay. Results Cell viability within the alginate microcapsules was maintained at a cell density of 5 × 106. Because polycationic polymers are helpful for maintaining the mechanical strength of microcapsules with good cell viability, the alginate microcapsules were reinforced with chitosan (0.1% w/v). Expression of TIMP2 protein in cell lysates and secretion of TIMP2 into the conditioned medium was confirmed by Western blot analysis. Alginate microcapsules encapsulating 293TIMP2 cells released TIMP2 protein into the medium efficiently, where the TIMP2 protein participated in degradation of the matrix metalloproteinase-2 enzyme and inhibited invasion of U87MG cells. Conclusion Alginate microcapsules encapsulating 293TIMP2 cells are promising candidates for anti-invasive treatment of glioma. PMID:24231999

Cloning and Expression Analysis of Phenylalanine Ammonia-Lyase Gene in the Mycelium and Fruit Body of the Edible Mushroom Flammulina velutipes

PubMed Central

Yun, Yeo Hong; Koo, Ja Sun

2015-01-01

Phenylalanine ammonia-lyase (PAL) gene is known to be expressed in plants, and is involved in the differentiation, growth and synthesis of secondary metabolites. However, its expression in fungi remains to be explored. To understand its expression in mushroom fungi, the PAL gene of the edible mushroom Flammulina velutipes (Fvpal) was cloned and characterized. The cloned Fvpal consists of 2,175 bp, coding for a polypeptide containing 724 amino acids and having 11 introns. The translated amino acid sequence of Fvpal shares a high identity (66%) with that of ectomycorrhizal fungus Tricholoma matsutake. Distinctively, the Fvpal expression in the mycelium was higher in minimal medium supplemented with L-tyrosine than with other aromatic amino acids. During cultivation of the mushroom on sawdust medium, Fvpal expression in the fruit body correspondingly increased as the mushroom grew. In the fruiting body, Fvpal was expressed more in the stipe than in the pileus. These results suggest that F. velutipes PAL activity differs in the different organs of the mushroom. Overall, this is first report to show that the PAL gene expression is associated with mushroom growth in fungi. PMID:26539050

Lipoxygenase and Hydroperoxide Lyase in Germinating Watermelon Seedlings 1

PubMed Central

Vick, Brady A.; Zimmerman, Don C.

1976-01-01

Lipoxygenase (EC 1.13.1.13) was found in seedlings of Citrullus lanatus (Thunb.) Matsum. and Nakai (watermelon). The enzyme has pH optima of 4.4 and 5.5 and is inhibited by 0.2 mM nordihydroguaiaretic acid. It is present in two functional units with estimated molecular weights of 120,000 and 240,000, respectively. A new enzyme, tentatively termed hydroperoxide lyase, has been partially purified from watermelon seedlings. The enzyme, located principally in the region of the hypocotyl-root junction, catalyzes the conversion of 13-l-hydroperoxy-cis-9-trans-11-octadecadienoic acid to 12-oxo-trans-10-dodecenoic acid and hexanal. The hydroperoxide lyase enzyme from watermelon has a molecular weight in excess of 250,000, a pH optimum in the range of 6 to 6.5, and is inhibited by p-chloromercuribenzoic acid. Its presence has also been demonstrated in other cucurbits. The maximum activity of both enzymes occurs on the 6th day of germination. The identification of the products of the hydroperoxide lyase reaction suggests that lipoxygenase and hydroperoxide lyase may be involved in the conversion of certain polyunsaturated fatty acids to traumatic acid (trans-2-dodecenedioic acid). PMID:16659569

Transcriptome analysis revealed anti-obesity effects of the Sodium Alginate in high-fat diet -induced obese mice.

PubMed

Wang, Xiong; Liu, Fang; Gao, Yuan; Xue, Chang-Hu; Li, Robert W; Tang, Qing-Juan

2018-04-10

Human obesity and overweight, caused by accumulated of fat, is the most commonly phenomenon from all over the world, especially in Western countries and Chinese mainland during the past three decades. Sodium Alginate, a polysaccharide extracted from brown seaweeds, has been proved its strong ability on body weight loss and anti-inflammatory response. However, no studies have been explored the effects of Sodium Alginate on colonic transcriptome, especially in obese individuals. Therefore, the current study was designed to detect whether Sodium Alginate could remit obesity and ease chronic metabolism disease through strengthening the bio-functionality of the lower intestine, particularly in colon. The data showed after Sodium Alginate gavaged for four weeks, the body weight, fat accumulation, triglyceride and total cholesterol were ameliorated in high fat diet induced obese mice. Sodium Alginate also improved the blood glucose level and lipopolysaccharides in serum. Furthermore, data from RNA sequence indicated that there were significantly changes in several genes, which involved in lipid metabolism and carbohydrate metabolism. In conclusion, these results suggested that Sodium Alginate could effectively suppress obesity and obesity related metabolic syndromes, due to the colonic transcriptome changes. Copyright © 2018. Published by Elsevier B.V.

Efficient functionalization of alginate biomaterials.

PubMed

Dalheim, Marianne Ø; Vanacker, Julie; Najmi, Maryam A; Aachmann, Finn L; Strand, Berit L; Christensen, Bjørn E

2016-02-01

Peptide coupled alginates obtained by chemical functionalization of alginates are commonly used as scaffold materials for cells in regenerative medicine and tissue engineering. We here present an alternative to the commonly used carbodiimide chemistry, using partial periodate oxidation followed by reductive amination. High and precise degrees of substitution were obtained with high reproducibility, and without formation of by-products. A protocol was established using l-Tyrosine methyl ester as a model compound and the non-toxic pic-BH3 as the reducing agent. DOSY was used to indirectly verify covalent binding and the structure of the product was further elucidated using NMR spectroscopy. The coupling efficiency was to some extent dependent on alginate composition, being most efficient on mannuronan. Three different bioactive peptide sequences (GRGDYP, GRGDSP and KHIFSDDSSE) were coupled to 8% periodate oxidized alginate resulting in degrees of substitution between 3.9 and 6.9%. Cell adhesion studies of mouse myoblasts (C2C12) and human dental stem cells (RP89) to gels containing various amounts of GRGDSP coupled alginate demonstrated the bioactivity of the material where RP89 cells needed higher peptide concentrations to adhere. Copyright © 2015 Elsevier Ltd. All rights reserved.

Alginate: properties and biomedical applications

PubMed Central

Lee, Kuen Yong; Mooney, David J.

2011-01-01

Alginate is a biomaterial that has found numerous applications in biomedical science and engineering due to its favorable properties, including biocompatibility and ease of gelation. Alginate hydrogels have been particularly attractive in wound healing, drug delivery, and tissue engineering applications to date, as these gels retain structural similarity to the extracellular matrices in tissues and can be manipulated to play several critical roles. This review will provide a comprehensive overview of general properties of alginate and its hydrogels, their biomedical applications, and suggest new perspectives for future studies with these polymers. PMID:22125349

Molecular cloning and characterization of l-methionine γ-lyase from Streptomyces avermitilis.

PubMed

Kudou, Daizou; Yasuda, Eri; Hirai, Yoshiyuki; Tamura, Takashi; Inagaki, Kenji

2015-10-01

A pyridoxal 5'-phosphate-dependent methionine γ-lyase (MGL) was cloned from Streptomyces avermitilis catalyzed the degradation of methionine to α-ketobutyrate, methanethiol, and ammonia. The sav7062 gene (1,242 bp) was corresponded to 413 amino acid residues with a molecular mass of 42,994 Da. The deduced amino acid sequence showed a high degree of similarity to those of other MGL enzymes. The sav7062 gene was overexpressed in Escherichia coli. The enzyme was purified to homogeneity and exhibited the MGL catalytic activities. We cloned the enzyme that has the MGL activity in Streptomyces for the first time. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Optimization of alginate purification using polyvinylidene difluoride membrane filtration: Effects on immunogenicity and biocompatibility of three-dimensional alginate scaffolds

PubMed Central

Sondermeijer, Hugo P; Witkowski, Piotr; Woodland, David; Seki, Tetsunori; Aangenendt, Frank J; van der Laarse, Arnoud; Itescu, Silviu; Hardy, Mark A

2017-01-01

Sodium alginate is an effective biomaterial for tissue engineering applications. Non-purified alginate is contaminated with protein, lipopolysaccharide, DNA, and RNA, which could elicit adverse immunological reactions. We developed a purification protocol to generate biocompatible alginate based on (a) activated charcoal treatment, (b) use of hydrophobic membrane filtration (we used hydrophobic polyvinylidene difluoride membranes to remove organic contaminants), (c) dialysis, and finally (d) ethanol precipitation. Using this approach, we could omit pre-treatment with chloroform and significantly reduce the quantities of reagents used. Purification resulted in reduction of residual protein by 70% down to 0.315 mg/g, DNA by 62% down to 1.28 μg/g, and RNA by 61% down to less than 10 μg/g, respectively. Lipopolysaccharide levels were reduced by >90% to less than 125 EU/g. Purified alginate did not induce splenocyte proliferation in vitro. Three-dimensional scaffolds generated from purified alginate did not elicit a significant foreign body reaction, fibrotic overgrowth, or macrophage infiltration 4 weeks after implantation. This study describes a simplified and economical alginate purification method that results in alginate purity, which meets clinically useful criteria. PMID:27114440

Two pectin lyase genes, pnl-1 and pnl-2, from Colletotrichum gloeosporioides f. sp. malvae differ in a cellulose-binding domain and in their expression during infection of Malva pusilla.

PubMed

Wei, Yangdou; Shih, Jenny; Li, Jieran; Goodwin, Paul H

2002-07-01

Two pectin lyase genes, designated pnl-1 and pnl-2, were cloned from Colletotrichum gloeosporioides f. sp. malvae, a pathogen of round-leaved mallow (Malva pusilla). pnl-1 was isolated using cDNA from infected plant material; pnl-2 was isolated using cDNA from 3-day-old mycelia grown in mallow-cell-wall extract (MCWE) broth. pnl-1 is the first pectinase gene described thus far to encode a cellulose-binding domain (CBD), which is common in cellulases and xylanases, whereas pnl-2 encodes a pectin lyase that lacks a CBD. In pure culture, pnl-1 expression could be detected when purified pectin or glucose was the sole carbon source, but not when MCWE was the sole carbon source. The lack of pnl-1 expression appeared to be due to gene repression by some unknown factor(s) in the cell-wall extract. In contrast, expression of pnl-2 was detected in cultures when MCWE, but not when purified pectin or glucose, was the sole carbon source. In infected tissue, detection of pnl-1 expression by Northern-blot hybridization and by RT-PCR began with the onset of the necrotrophic phase of infection. Expression ofpnl-2 was not detectable by Northern-blot hybridization, but was observed byRT-PCR in both the biotrophic and necrotrophic phases of infection. The differences between pnl-1 and pnl-2 (i.e. pnl-1 encoding a CBD and differences in the expression patterns of both genes) may be related to the requirements of C. gloeosporioides f. sp. malvae to be able to grow in host tissue under the different conditions present during the biotrophic and necrotrophic phases of infection.

Pectate lyase affects pathogenicity in natural isolates of Colletotrichum coccodes and in pelA gene-disrupted and gene-overexpressing mutant lines.

PubMed

Ben-Daniel, Bat-Hen; Bar-Zvi, Dudy; Tsror Lahkim, Leah

2012-02-01

Colletotrichum coccodes (Wallr.) S. Hughes, the causal agent of black dot on potato and anthracnose on tomato, reduces yield and crop quality. We explored the role of secreted pectate lyase (PL), a cell wall-degrading enzyme, in the aggressiveness of C. coccodes. In vitro-cultivated highly aggressive isolates secreted immunologically detectable PL levels 6 h after transfer to secondary medium versus 12 h for mildly aggressive isolates, suggesting that secreted PL is a virulence factor. The gene encoding PL, CcpelA, was cloned and used for the genetic manipulation of highly (US-41 and Si-72) and mildly (Si-60) aggressive isolates. CcpelA gene-disrupted mutants showed reduced aggressiveness towards tomato fruits and impaired PL secretion and extracellular activity. Conversely, overexpression of CcpelA in the Si-60 isolate increased its aggressiveness and PL secretion. Comparison of CcpelA cloned from isolates US-41 and Si-60 revealed that both encode identical proteins, but differ in their promoters. Bioinformatics analysis for cis-acting elements suggested that the promoters of the US-41 and Si-60 isolates contain one and no AreA-binding site (GATA box), respectively. AreA has been suggested to be involved in fungal aggressiveness; therefore, CcpelA may be a key virulence factor in C. coccodes pathogenicity, and the differences in isolate aggressiveness might result from promoter activity. Quantitative reverse transcriptase-polymerase chain reaction analyses confirmed the higher level of CcpelA transcript in isolate US-41 versus Si-60. © 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.

Regulation of Pectate Lyase Synthesis in Pseudomonas fluorescens and Erwinia carotovora

PubMed Central

Zucker, Milton; Hankin, Lester

1970-01-01

Inducible synthesis of extracellular pectate lyase occurs in Erwinia carotovora, a bacterial soft-rot pathogen of plants, and, to a lesser extent, in a nonpathogenic isolate of Pseudomonas fluorescens. A combination of pectin and a heat-labile factor in fresh potato tissue or acetone powders of the tissue provided the best carbon source for induction. Yields of inducible pectate lyase were much greater than those usually reported. The pathogen, but not the saprophyte, produced a small amount of constitutive enzyme when grown on glucose. The relatively low level or absence of constitutive synthesis in these bacteria did not result from catabolite repression. Attempts were made to relieve any existing catabolite repression by restricting growth through slow feeding of glucose or by growing the organisms on glycerol. These conditions did not significantly alter the differential rate of lyase synthesis compared with changes observed in the presence of inducers. Previous growth history did not affect induction in the pathogen. However, P. fluorescens previously cultured on glucose required 10 to 20 generations of growth on inducing medium before appreciable lyase synthesis occurred. Differences between the pathogen and nonpathogen suggest that regulation of pectate lyase synthesis is related to pathogenicity of soft-rot bacteria. PMID:5473883

Clearance of phenylalanine ammonia-lyase from normal and tumor-bearing mice.

PubMed

Shen, R S; Fritz, R R; Abell, C W

1977-04-01

Yeast phenylalanine ammonia-lyase was administered i.p. to normal and tumor-bearing mice, and its clearance from plasma was studied. Single and multiple weekly injections at dosages of 10,20,50 and 100 units/kg were administered to C57BL female, C57BL X DBA/2F1 male, and A/J female mice. L5178Y murine lymphoblastic leukemia, B16 melanoma, BW10232 adenocarcinoma, and 15091A anaplastic carcinoma were implanted 7 to 11 days prior to enzyme injection in the appropriate host. After a single injection, the average plasma half-lives of phenylalanine ammonia-lyase were 18 to 24 hr in all groups studied. While the other tumors had no effect on the plasma level of phenylalanine ammonia-lyase after a single injection, L5178Y murine lymphoblastic leukemia and 15091A anaplastic carcinoma significantly depressed the maximal level of phenylalanine ammonia-lyase attained in the plasma. After repeated injections of phenylalanine ammonia-lyase, the initial plasma enzyme level was significantly reduced when 20 units/kg were administered, and the clearance of the enzyme from the plasma was greatly accelerated regardless of the amount administered. Furthermore, in tumor-bearing mice, the rate of clearance was significantly more rapid than in the appropriate non-tumor-bearing control.

Molecular and analysis of a phenylalanine ammonia-lyase gene (LrPAL2) from Lycoris radiata.

PubMed

Jiang, Yumei; Xia, Bing; Liang, Lijian; Li, Xiaodan; Xu, Sheng; Peng, Feng; Wang, Ren

2013-03-01

Phenylalanine ammonia-lyase (PAL), the first enzyme of phenylpropanoid biosynthesis, participates in the biosynthesis of flavonoids, lignins, stilbenes and many other compounds. In this study, we cloned a 2,326 bp full-length PAL2 gene from Lycoris radiata by using degenerate oligonucleotide primer PCR (DOP-PCR) and the rapid amplification of cDNA ends method. The cDNA contains a 2,124 bp coding region encoding 707 amino acids. The LrPAL2 shares about 77.0 % nucleic acid identity and 83 % amino acid identity with LrPAL1. Furthermore, genome sequence analysis demonstrated that LrPAL2 gene contains one intron and two exons. The 5' flanking sequence of LrPAL2 was also cloned by self-formed adaptor PCR (SEFA-PCR), and a group of putative cis-acting elements such as TATA box, CAAT box, G box, TC-rich repeats, CGTCA motif and TCA-element were identified. The LrPAL2 was detected in all tissues examined, with high abundance in bulbs at leaf sprouting stage and in petals at blooming stage. Besides, LrPAL2 drastically responded to MJ, SNP and UV, moderately responded to GA and SA, and a little increased under wounding. Comparison of LrPAL2 expression and LrPAL1 expression demonstrated that LrPAL2 can be more significantly induced than LrPAL1 under the above treatments, and LrPAL2 transcripts accumulated prominently at blooming stage, especially in petals, while LrPAL1 transcripts did not accumulated significantly at blooming stage. All these results suggested that LrPAL2 might play distinct roles in different branches of the phenylpropanoid pathway.

21 CFR 172.858 - Propylene glycol alginate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Propylene glycol alginate. 172.858 Section 172.858... Propylene glycol alginate. The food additive propylene glycol alginate (CAS Reg. No. 9005-37-2) may be used... the act: (1) The name of the additive, “propylene glycol alginate” or “propylene glycol ester of...

Distribution of Neuraminidase and N-Acetylneuraminate Lyase Activities Among Corynebacteria, Mycobacteria, and Nocardias

PubMed Central

Arden, Sheldon B.; Chang, Woo-Hyun; Barksdale, Lane

1972-01-01

In Corynebacterium diphtheriae and closely related neuraminidase-producing corynebacteria, we have found an N-acetylneuraminate (NAN) lyase activity which cleaves NAN into N-acetyl-d-mannosamine and, presumably, pyruvate. In vitro, these lyases can be shown to synthesize NAN. A survey of representative corynebacteria, “plant pathogenic corynebacteria,” mycobacteria, and nocardias revealed that only those corynebacteria closely related to C. diphtheriae exhibited both neuraminidase and NAN lyase activities. PMID:4629654

Enzyme immobilization in novel alginate-chitosan core-shell microcapsules.

PubMed

Taqieddin, Ehab; Amiji, Mansoor

2004-05-01

Alginate-chitosan core-shell microcapsules were prepared in order to develop a biocompatible matrix for enzyme immobilization, where the protein is retained either in a liquid or solid core and the shell allows permeability control over substrates and products. The permeability coefficients of different molecular weight compounds (vitamin B2, vitamin B12, and myoglobin) were determined through sodium tripolyphosphate (Na-TPP)-crosslinked chitosan membrane. The microcapsule core was formed by crosslinking sodium alginate with either calcium or barium ions. The crosslinked alginate core was uniformly coated with a chitosan layer and crosslinked with Na-TPP. In the case of calcium alginate, the phosphate ions of Na-TPP were able to extract the calcium ions from alginate and liquefy the core. A model enzyme, beta-galactosidase, was immobilized in the alginate core and the catalytic activity was measured with o-nitrophenyl-beta-D-galactopyranoside (ONPG). Change in the activity of free and immobilized enzyme was determined at three different temperatures. Na-TPP crosslinked chitosan membranes were found to be permeable to solutes of up to 17,000Da molecular weight. The enzyme loading efficiency was higher in the barium alginate core (100%) as compared to the calcium alginate core (60%). The rate of ONPG conversion to o-nitrophenol was faster in the case of calcium alginate-chitosan microcapsules as compared to barium alginate-chitosan microcapsules. Barium alginate-chitosan microcapsules, however, did improve the stability of the enzyme at 37 degrees C relative to calcium alginate-chitosan microcapsules or free enzyme. This study illustrates a new method of enzyme immobilization for biotechnology applications using liquid or solid core and shell microcapsule technology.

Purification, characterization and specificity of chondroitin lyases and glycuronidase from Flavobacterium heparinum.

PubMed Central

Gu, K; Linhardt, R J; Laliberté, M; Gu, K; Zimmermann, J

1995-01-01

The chondroitin lyases from Flavobacterium heparinum (Cytophaga heparinia) have been widely used in depolymerization of glycosaminoglycan and proteoglycan chondroitin sulphates. Oligosaccharide products derived from chondroitin sulphate can be further degraded by glycuronidases and sulphatases obtained from the same organism. There has been no reported purification of these enzymes to homogeneity nor is there any information on their physical and kinetic characteristics. The absence of pure enzymes has resulted in a lack of understanding of the optimal conditions for their catalytic activity and their substrate specificity. This has limited the use of these enzymes as reagents for preparation of oligosaccharides for structure and activity studies. Reproducible schemes to purify a chondroitin AC lyase, a glycuronidase and chondroitin B lyase from Flavobacterium heparinum to apparent homogeneity are described. Chondroitin AC lyase (chondroitinase AC, EC 4.2.2.5), glycuronidase [chondro-(1-->3)-glycuronidase, no EC number] and chondroitin B lyase (chondroitinase B, no EC number) have M(r) values (assessed by SDS/PAGE) of 74,000, 41,800 and 55,200 respectively, and isoelectric points (determined by isoelectric focusing) of 8.85, 9.28 and 9.05 respectively. Chondroitin lyase AC and B contain pyroglutamic acid at their N-termini precluding their analysis by Edman degradation. Deblocking with pyroglutamate aminopeptidase facilitated the determination of their N-terminal sequences. The kinetic properties of these enzymes have been determined as well as the optimum conditions for their catalytic activity. The specificity of the glycouronidase, determined using 17 different disaccharide substrates, shows that it only acts on unsulphated or 6-O-sulphated 1-->3 linkages. The chondroitin lyases are both endolytic enzymes, and oligosaccharide mapping shows their expected specificity towards the chondroitin and dermatan sulphate polymers. Images Figure 2 Figure 4 PMID:8526872

Raman probing of molecular interactions of alginate biopolymers with cells

NASA Astrophysics Data System (ADS)

Chourpa, Igor; Carpentier, Philippe; Maingault, Philippe; Fetissoff, Franck; Dubois, Pierre

2000-05-01

The biological polymers extracted from brown algae, alginates, are novel materials in biotechnology and biomedicine. Their ability to form viscous gels is used to immobilize or encapsulate yeast, enzymes, living cells and drugs. Calcium-alginate fibers are extensively used in wound dressings since exhibit antihaemostatic and healing properties. The problem with alginate-made dressings in surgery is their slow biodegradability: if entrapped within tissues, they can induce a local cellular recruitment with an inflammatory response contemporaneous to the resorption phase. In part, this problem is a consequence of poor solubility of the calcium alginates in water. Although calcium alginate fibers can exchange calcium ions with sodium ions from the wound exudate to create a calcium/sodium alginate fibers, the residual alginates are thought to be not totally degradable in vivo. Rapid and non- destructive characterization of series of the crude alginates and calcium alginate fibers has been performed using Raman spectroscopy with near IR excitation. Study of structural organization of the polymeric chains within calcium alginate fibers have been previously reported as made by confocal Raman multispectral imaging (CRMSI) in visible. Here, the Raman approach has been used to monitor the ion exchange reactions for different types of alginates and their salts in vitro. For in vivo evaluation, histological sections of alginate-treated rat tissue have been analyzed by light microscopy and CRMSI. The in vitro Raman modeling and the histochemical mapping were a necessary precursor for application of the Raman microprobe to follow in a non-invasive way the alginate-cell molecular interactions in rat tissue.

Abundance and Genetic Diversity of Microbial Polygalacturonase and Pectate Lyase in the Sheep Rumen Ecosystem

PubMed Central

Wang, Yaru; Luo, Huiying; Huang, Huoqing; Shi, Pengjun; Bai, Yingguo; Yang, Peilong; Yao, Bin

2012-01-01

Background Efficient degradation of pectin in the rumen is necessary for plant-based feed utilization. The objective of this study was to characterize the diversity, abundance, and functions of pectinases from microorganisms in the sheep rumen. Methodology/Principal Findings A total of 103 unique fragments of polygalacturonase (PF00295) and pectate lyase (PF00544 and PF09492) genes were retrieved from microbial DNA in the rumen of a Small Tail Han sheep, and 66% of the sequences of these fragments had low identities (<65%) with known sequences. Phylogenetic tree building separated the PF00295, PF00544, and PF09492 sequences into five, three, and three clades, respectively. Cellulolytic and noncellulolytic Butyrivibrio, Prevotella, and Fibrobacter species were the major sources of the pectinases. The two most abundant pectate lyase genes were cloned, and their protein products, expressed in Escherichia coli, were characterized. Both enzymes probably act extracellularly as their nucleotide sequences contained signal sequences, and they had optimal activities at the ruminal physiological temperature and complementary pH-dependent activity profiles. Conclusion/Significance This study reveals the specificity, diversity, and abundance of pectinases in the rumen ecosystem and provides two additional ruminal pectinases for potential industrial use under physiological conditions. PMID:22815874

O-acetylserine(thiol)lyase from spinach (Spinacia oleracea L.) leaf: cDNA cloning, characterization, and overexpression in Escherichia coli of the chloroplast isoform.

PubMed

Rolland, N; Droux, M; Lebrun, M; Douce, R

1993-01-01

The last enzymatic step for L-cysteine biosynthesis is catalyzed by O-acetylserine(thiol)lyase (OASTL, EC 4.2.99.8) which synthesizes L-cysteine from O-acetylserine and "sulfide." We have isolated and characterized a full-length cDNA (1432 bp) from a lambda gt11 library of spinach leaf encoding the complete precursor of the chloroplast isoform. The 1149-nucleotide open reading frame coding for O-acetylserine(thiol)lyase was in the direction opposite that of the lambda gt11 beta-galactosidase gene. The derived amino acid sequence indicates that the protein precursor consists of 383 amino acid residues including a N-terminal presequence peptide of 52 residues. The amino acid sequence of mature spinach chloroplast O-acetylserine(thiol)lyase shows 40 and 57% homology with its bacterial counterparts. Sequence comparison with several pyridoxal 5'-phosphate-containing proteins reveals the presence of a lysine residue assumed to be involved in cofactor binding. A synthetic cDNA was constructed, coding for the entire 331-amino-acid mature O-acetylserine(thiol)lyase and for an initiating methionine. A high level of expression of the active mature chloroplast isoform was achieved in an Escherichia coli strain carrying the T7 RNA polymerase system (F. W. Studier, A. H. Rosenberg, J. J. Dunn, and J. W. Dubendorff, 1990, in Methods in Enzymology, D. V. Goeddel, Ed., Vol. 185, pp. 60-89, Academic Press, San Diego, CA). Addition of pyridoxine to the bacterial growth medium enhanced the enzyme activity due to the recombinant protein. The extent of production is 25-fold higher than in chloroplast from spinach leaves and the recombinant protein presents the relative molecular mass and immunological properties of the natural enzyme from spinach leaf chloroplast. This work, together with our previous biochemical studies, are in accordance with a prokaryotic type enzyme for L-cysteine biosynthesis in higher plant chloroplasts. Southern blot analysis indicated that O-acetylserine(thiol)lyase

Cloning, expression and characterization of phenylalanine ammonia-lyase from Rhodotorula glutinis.

PubMed

Zhu, Longbao; Cui, Wenjing; Fang, Yueqin; Liu, Yi; Gao, Xinxing; Zhou, Zhemin

2013-05-01

The industrial-scale production of phenylalanine ammonia-lyase (PAL) mainly uses strains of Rhodotorula. However, the PAL gene from Rhodotorula has not been cloned. Here, the full-length gene of PAL from Rhodotorula glutinis was isolated. It was 2,121 bp, encoding a polypeptide with 706 amino acids and a calculated MW of 75.5 kDa. Though R. glutinis is an anamorph of Rhodosporium toruloides, the amino acid sequences of PALs them are not the same (about 74 % identity). PAL was expressed in E. coli and characterized. Its specific activity was 4.2 U mg(-1) and the k cat/K m was 1.9 × 10(4) mM(-1) s(-1), exhibiting the highest catalytic ability among the reported PALs. The genetic and biochemical information reported here should facilitate future application in industry.

Properties of alginate fiber spun-dyed with fluorescent pigment dispersion.

PubMed

Wang, Ping; Tawiah, Benjamin; Tian, Anli; Wang, Chunxia; Zhang, Liping; Fu, Shaohai

2015-03-15

Spun-dyed alginate fiber was prepared by the spun-dyeing method with the mixture of fluorescent pigment dispersion and sodium alginate fiber spinning solution, and its properties were characterized by SEM, TGA, DSC, and XRD. The results indicate that fluorescent pigment dispersion prepared with esterified poly (styrene-alt maleic acid) had excellent compatibility with sodium alginate fiber spinning solution, and small amount of fluorescent pigment could reduce the viscosity of spun-dyed spinning solutions. SEM photo of spun-dyed alginate fiber indicated that fewer pigment particles deposited on its surface. TGA, DSC, and XRD results suggested that thermal properties and crystal phase of spun-dyed alginate fibers had slight changes compared to the original alginate fibers. The fluorescence intensity of spun-dyed alginate fiber reached its maximum when the content of fluorescent pigment was 4%. The spun-dyed alginate fiber showed excellent rubbing and washing fastness. Copyright © 2014 Elsevier Ltd. All rights reserved.

Isolation and characterization of two hydroperoxide lyase genes from grape berries : HPL isogenes in Vitis vinifera grapes.

PubMed

Zhu, Bao-Qing; Xu, Xiao-Qing; Wu, Yu-Wen; Duan, Chang-Qing; Pan, Qiu-Hong

2012-07-01

C6 compounds are the major fraction of the volatile profiles of grape berries, contributing the typical 'green' aroma to the grape and wine. Hydroperoxide lyase (HPL) catalyzes the cleavage of fatty acid hydroperoxides to produce C6 compounds. Two hypothetical genes, VvHPL1 and VvHPL2 were cloned from grape berries (Vitis vinifera L. Cabernet Sauvignon). Bioinformatics analysis revealed that the proteins encoded by these two genes both belong to subfamily of cytochrome P450 and contain typical conserved domains of HPLs, and have high identity with HPLs from other plants. Prokaryotically-expressed VvHPL1 and VvHPL2 with thioredoxin-6xHis-fusion partner were confirmed to have enzymatic activity. VvHPL1 is specific for 13-HPOD (T) producing C6 aldehydes with relatively higher activity and VvHPL2 catalyzes the cleavage of both 9- and 13-hydroperoxides producing C6 aldehydes and C9 aldehydes respectively. Analysis of real time-PCR showed that VvHPL2 was highly expressed in the leaves and the flowers of the grapes, while relatively low transcript abundance was detected in the berries, tendril and stems; VvHPL1 had high expression in all detected tissues. During grape berry development, the expression of these two isogenes presented similar trends with a rapid increase after veraison and a decrease at full-ripen stage, which roughly corresponded to the accumulation of their volatile products. These data lay an essential foundation for further study on the accumulation and control of C6 volatiles in grape berries.

Reduced phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities and lignin synthesis in wheat grown under low pressure sodium lamps

NASA Technical Reports Server (NTRS)

Guerra, D.; Anderson, A. J.; Salisbury, F. B.

1985-01-01

Wheat (Triticum aestivum L. cv Fremont) grown in hydroponic culture under 24-hour continuous irradiation at 560 to 580 micromoles per square meter per second from either metalhalide (MH), high pressure sodium (HPS), or low pressure sodium (LPS) lamps reached maturity in 70 days. Grain yields were similar under all three lamps, although LPS-grown plants lodged at maturity. Phenylalanine ammonia-lyase (PAL) and a tyrosine ammonia lyase (TAL) with lesser activity were detected in all extracts of leaf, inflorescence, and stem. Ammonia-lyase activities increased with age of the plant, and plants grown under the LPS lamp displayed PAL and TAL activities lower than wheat cultured under MH and HPS radiation. Greenhouse solar-grown wheat had the highest PAL and TAL activities. Lignin content of LPS-grown wheat was also significantly reduced from that of plants grown under MH or HPS lamps or in the greenhouse, showing a correlation with the reduced PAL and TAL activities. Ratios of far red-absorbing phytochrome to total phytochrome were similar for all three lamps, but the data do not yet warrant a conclusion about specific wavelengths missing from the LPS lamps that might have induced PAL and TAL activities in plants under the other lamps.

Controlled release of metronidazole from composite poly-ε-caprolactone/alginate (PCL/alginate) rings for dental implants.

PubMed

Lan, Shih-Feng; Kehinde, Timilehin; Zhang, Xiangming; Khajotia, Sharukh; Schmidtke, David W; Starly, Binil

2013-06-01

Dental implants provide support for dental crowns and bridges by serving as abutments for the replacement of missing teeth. To prevent bacterial accumulation and growth at the site of implantation, solutions such as systemic antibiotics and localized delivery of bactericidal agents are often employed. The objective of this study was to demonstrate a novel method of controlled localized delivery of antibacterial agents to an implant site using a biodegradable custom fabricated ring. The study involved incorporating a model antibacterial agent (metronidazole) into custom designed poly-ε-caprolactone/alginate (PCL/alginate) composite rings to produce the intended controlled release profile. The rings can be designed to fit around the body of any root form dental implants of various diameters, shapes and sizes. In vitro release studies indicate that pure (100%) alginate rings exhibited an expected burst release of metronidazole in the first few hours, whereas Alginate/PCL composite rings produced a medium burst release followed by a sustained release for a period greater than 4 weeks. By varying the PCL/alginate weight ratios, we have shown that we can control the amount of antibacterial agents released to provide the minimal inhibitory concentration (MIC) needed for adequate protection. The fabricated composite rings have achieved a 50% antibacterial agent release profile over the first 48 h and the remaining amount slowly released over the remainder of the study period. The PCL/alginate agent release characteristic fits the Ritger-Peppas model indicating a diffusion-based mechanism during the 30-day study period. The developed system demonstrates a controllable drug release profile and the potential for the ring to inhibit bacterial biofilm growth for the prevention of diseases such as peri-implantitis resulting from bacterial infection at the implant site. Copyright © 2013 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Salt-specific regulation of the cytosolic O-acetylserine(thiol)lyase gene from Arabidopsis thaliana is dependent on abscisic acid.

PubMed

Barroso, C; Romero, L C; Cejudo, F J; Vega, J M; Gotor, C

1999-07-01

The expression of Atcys-3A gene coding for cytosolic O-acetylserine(thiol)lyase, a key enzyme in cysteine biosynthesis, from Arabidopsis thaliana is significantly induced by exposure to salt and heavy-metal stresses. Addition of NaCl to mature plants induced a rapid accumulation of the mRNA throughout the leaf lamina and roots, and later on in stems, being mainly restricted to vascular tissues. The salt-specific regulation of Atcys-3A was also mediated by abscisic acid (ABA) since: (1) exogenous addition of ABA to the culture medium mimicked the salt-induced plant response by raising the level of Atcys-3A transcript, and (2) Arabidopsis mutants aba-1 and abi2-1 were not able to respond to NaCl. Our results suggest that a high rate of cysteine biosynthesis is required in Arabidopsis under salt stress necessary for a plant protection or adaptation mechanism. This hypothesis was supported by the observation that intracellular levels of cysteine and glutathione increased up to 3-fold after salt treatment.

Antibacterial Performance of Alginic Acid Coating on Polyethylene Film

PubMed Central

Karbassi, Elika; Asadinezhad, Ahmad; Lehocký, Marian; Humpolíček, Petr; Vesel, Alenka; Novák, Igor; Sáha, Petr

2014-01-01

Alginic acid coated polyethylene films were examined in terms of surface properties and bacteriostatic performance against two most representative bacterial strains, that is, Escherichia coli and Staphylococcus aureus. Microwave plasma treatment followed by brush formation in vapor state from three distinguished precursors (allylalcohol, allylamine, hydroxyethyl methacrylate) was carried out to deposit alginic acid on the substrate. Surface analyses via various techniques established that alginic acid was immobilized onto the surface where grafting (brush) chemistry influenced the amount of alginic acid coated. Moreover, alginic acid was found to be capable of bacterial growth inhibition which itself was significantly affected by the brush type. The polyanionic character of alginic acid as a carbohydrate polymer was assumed to play the pivotal role in antibacterial activity. The cell wall composition of two bacterial strains along with the substrates physicochemical properties accounted for different levels of bacteriostatic performance. PMID:25196604

ATP-citrate lyase links cellular metabolism to histone acetylation.

PubMed

Wellen, Kathryn E; Hatzivassiliou, Georgia; Sachdeva, Uma M; Bui, Thi V; Cross, Justin R; Thompson, Craig B

2009-05-22

Histone acetylation in single-cell eukaryotes relies on acetyl coenzyme A (acetyl-CoA) synthetase enzymes that use acetate to produce acetyl-CoA. Metazoans, however, use glucose as their main carbon source and have exposure only to low concentrations of extracellular acetate. We have shown that histone acetylation in mammalian cells is dependent on adenosine triphosphate (ATP)-citrate lyase (ACL), the enzyme that converts glucose-derived citrate into acetyl-CoA. We found that ACL is required for increases in histone acetylation in response to growth factor stimulation and during differentiation, and that glucose availability can affect histone acetylation in an ACL-dependent manner. Together, these findings suggest that ACL activity is required to link growth factor-induced increases in nutrient metabolism to the regulation of histone acetylation and gene expression.

Characterization of recombinant pectate lyase refolded from inclusion bodies generated in E. coli BL21(DE3).

PubMed

Kumar, Sandeep; Jain, Kavish Kumar; Singh, Anupam; Panda, Amulya K; Kuhad, Ramesh Chander

2015-06-01

Pectate lyase (EC 4.2.2.2) gene from Bacillus subtilis RCK was cloned and expressed in Escherichia coli to maximize its production. In addition to soluble fraction, bioactive pectate lyase was also obtained from inclusion body aggregates by urea solubilization and refolding under in vitro conditions. Enzyme with specific activity ∼3194IU/mg and ∼1493IU/mg were obtained from soluble and inclusion bodies (IBs) fraction with recovery of 56% and 74% in terms of activity, respectively. The recombinant enzyme was moderately thermostable (t1/2 60min at 50°C) and optimally active in wider alkaline pH range (7.0-10.5). Interaction of protein with its cofactor CaCl2 was found to stimulate the change in tertiary structure as revealed by near UV CD spectra. Intrinsic tryptophan fluorescence spectra indicated that tryptophan is involved in substrate binding and there might be independent binding of Ca(2+) and polygalacturonic acid to the active site. The recombinant enzyme was found to be capable of degrading pectin and polygalacturonic acid. The work reports novel conditions for refolding to obtain active recombinant pectate lyase from inclusion bodies and elucidates the effect of ligand and substrate binding on protein conformation by circular dichroism (CD) and fluorescence spectrofluorometry. Copyright © 2014 Elsevier Inc. All rights reserved.

Alginate-based hybrid aerogel microparticles for mucosal drug delivery.

PubMed

Gonçalves, V S S; Gurikov, P; Poejo, J; Matias, A A; Heinrich, S; Duarte, C M M; Smirnova, I

2016-10-01

The application of biopolymer aerogels as drug delivery systems (DDS) has gained increased interest during the last decade since these structures have large surface area and accessible pores allowing for high drug loadings. Being biocompatible, biodegradable and presenting low toxicity, polysaccharide-based aerogels are an attractive carrier to be applied in pharmaceutical industry. Moreover, some polysaccharides (e.g. alginate and chitosan) present mucoadhesive properties, an important feature for mucosal drug delivery. This feature allows to extend the contact of DDS with biological membranes, thereby increasing the absorption of drugs through the mucosa. Alginate-based hybrid aerogels in the form of microparticles (<50μm) were investigated in this work as carriers for mucosal administration of drugs. Low methoxyl pectin and κ-carrageenan were co-gelled with alginate and further dried with supercritical CO2 (sc-CO2). Spherical mesoporous aerogel microparticles were obtained for alginate, hybrid alginate/pectin and alginate/κ-carrageenan aerogels, presenting high specific surface area (370-548m(2)g(-1)) and mucoadhesive properties. The microparticles were loaded with ketoprofen via adsorption from its solution in sc-CO2, and with quercetin via supercritical anti-solvent precipitation. Loading of ketoprofen was in the range between 17 and 22wt% whereas quercetin demonstrated loadings of 3.1-5.4wt%. Both the drugs were present in amorphous state. Loading procedure allowed the preservation of antioxidant activity of quercetin. Release of both drugs from alginate/κ-carrageenan aerogel was slightly faster compared to alginate/pectin. The results indicate that alginate-based aerogel microparticles can be viewed as promising matrices for mucosal drug delivery applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Chondroitin Sulfate (CS) Lyases: Structure, Function and Application in Therapeutics.

PubMed

Rani, Aruna; Patel, Seema; Goyal, Arun

2018-01-01

Glycosaminoglycans (GAGs) such as chondroitin sulfate (CS) are the chief natural polysaccharides which reside in biological tissues mainly in extracellular matrix. These CS along with adhesion molecules and growth factors are involved in central nervous system (CNS) development, cell progression and pathogenesis. The chondroitin lyases are the enzyme that degrade and alter the CS chains and hence modify various signalling pathways involving CS chains. These CS lyases are substrate specific, can precisely manipulate the CS polysaccharides and have various biotechnological, medical and therapeutic applications. These enzymes can be used to produce the unsaturated oligosaccharides, which have immune-modulatory, anti-inflammatory and neuroprotective properties. This review focuses on the major breakthrough of the chondroitin sulfate degrading enzymes, their structures and functioning mechanism. This also provides comprehensive information regarding production, purification, characterization of CS lyases and their major applications, both established as well as emerging ones such as neural development. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Effect of gamma-irradiation on degradation of alginate.

PubMed

Lee, Dong Wook; Choi, Won Seok; Byun, Myung Woo; Park, Hyun Jin; Yu, Yong-Man; Lee, Chong M

2003-07-30

The aqueous solution of alginate was irradiated by 60Co gamma-rays in the dose range of 10-500 kGy. To assess the effect of irradiation on the degradation of alginate, the irradiation-induced changes in the viscosity, molecular weight, color, monomer composition, and sequence were measured. The molecular weight of raw alginate was reduced from 300000 to 25000 when irradiated at 100 kGy. The degradation rate decreased and the chain breaks per molecule increased with increasing irradiation dose. The viscosity of irradiated alginate solution reached a near minimum as low as at 10 kGy. No appreciable color changes were observed in the samples irradiated at up to 100 kGy, but intense browning occurred beyond 200 kGy. The 13C NMR spectra showed that homopolymeric blocks, MM and GG, increased and the M/G ratio decreased with irradiation. Considering both the level of degradation and the color change of alginate, the optimum irradiation dose was found to be 100 kGy.

Bio-based barium alginate film: Preparation, flame retardancy and thermal degradation behavior.

PubMed

Liu, Yun; Zhang, Chuan-Jie; Zhao, Jin-Chao; Guo, Yi; Zhu, Ping; Wang, De-Yi

2016-03-30

A bio-based barium alginate film was prepared via a facile ionic exchange and casting approach. Its flammability, thermal degradation and pyrolysis behaviors, thermal degradation mechanism were studied systemically by limiting oxygen index (LOI), vertical burning (UL-94), microscale combustion calorimetry (MCC), thermogravimetric analysis (TGA) coupled with Fourier transform infrared analysis (FTIR) and pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS). It showed that barium alginate film had much higher LOI value (52.0%) than that of sodium alginate film (24.5%). Moreover, barium alginate film passed the UL-94 V-0 rating, while the sodium alginate film showed no classification. Importantly, peak of heat release rate (PHRR) of barium alginate film in MCC test was much lower than that of sodium alginate film, suggested that introduction of barium ion into alginate film significantly decreased release of combustible gases. TG-FTIR and Py-GC-MS results indicated that barium alginate produced much less flammable products than that of sodium alginate in whole thermal degradation procedure. Finally, a possible degradation mechanism of barium alginate had been proposed. Copyright © 2015 Elsevier Ltd. All rights reserved.

Purification and characterization of O-acetylserine (thiol) lyase from spinach chloroplasts.

PubMed

Droux, M; Martin, J; Sajus, P; Douce, R

1992-06-01

O-Acetylserine (thiol) lyase, the last enzyme in the cysteine biosynthetic pathway, was purified to homogeneity from spinach leaf chloroplasts. The enzyme has a molecular mass of 68,000 and consists of two identical subunits of Mr 35,000. The absorption spectrum obtained at pH 7.5 exhibited a peak at 407 nm due to pyridoxal phosphate, and addition of O-acetylserine induced a considerable modification of the spectrum. The pyridoxal phosphate content was found to be 1.1 per subunit of 35,000, and the chromophore was displaced from the enzyme by O-acetylserine, leading to a progressive inactivation of the holoenzyme. Upon gel filtration chromatography on Superdex 200, part of the chloroplastic O-acetylserine (thiol) lyase eluted in association with serine acetyltransferase at a position corresponding to a molecular mass of 310,000 (such a complex called cysteine synthase has been characterized in bacteria). The activity of O-acetylserine (thiol) lyase was optimum between pH 7.5 and 8.5. The apparent Km for O-acetylserine was 1.3 mM and for sulfide was 0.25 mM. The calculated activation energy was 12.6 kcal/mol at 10 mM O-acetylserine. The overall amino-acid composition of spinach chloroplast O-acetylserine (thiol) lyase was different than that determined for the same enzyme (cytosolic?) obtained from a crude extract of spinach leaves. A polyclonal antibody prepared against the chloroplastic O-acetylserine (thiol) lyase exhibited a very low cross-reactivity with a preparation of mitochondrial matrix and cytosolic proteins suggesting that the chloroplastic isoform was distinct from the mitochondrial and cytosolic counterparts.

Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel.

PubMed

Hoesli, Corinne A; Kiang, Roger L J; Raghuram, Kamini; Pedroza, René G; Markwick, Karen E; Colantuoni, Antonio M R; Piret, James M

2017-06-29

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet encapsulation has been studied extensively as a means to increase islet survival in allogeneic or xenogeneic transplants. Alginate encapsulation is commonly achieved by nozzle extrusion and external gelation. Using this method, cell-containing alginate droplets formed at the tip of nozzles fall into a solution containing divalent cations that cause ionotropic alginate gelation as they diffuse into the droplets. The requirement for droplet formation at the nozzle tip limits the volumetric throughput and alginate concentration that can be achieved. This video describes a scalable emulsification method to encapsulate mammalian cells in 0.5% to 10% alginate with 70% to 90% cell survival. By this alternative method, alginate droplets containing cells and calcium carbonate are emulsified in mineral oil, followed by a decrease in pH leading to internal calcium release and ionotropic alginate gelation. The current method allows the production of alginate beads within 20 min of emulsification. The equipment required for the encapsulation step consists in simple stirred vessels available to most laboratories.

Ag/alginate nanofiber membrane for flexible electronic skin

NASA Astrophysics Data System (ADS)

Hu, Wei-Peng; Zhang, Bin; Zhang, Jun; Luo, Wei-Ling; Guo, Ya; Chen, Shao-Juan; Yun, Mao-Jin; Ramakrishna, Seeram; Long, Yun-Ze

2017-11-01

Flexible electronic skin has stimulated significant interest due to its widespread applications in the fields of human-machine interactivity, smart robots and health monitoring. As typical elements of electrical skin, the fabrication process of most pressure sensors combined nanomaterials and PDMS films are redundant, expensive and complicated, and their unknown biological toxicity could not be widely used in electronic skin. Hence, we report a novel, cost-effective and antibacterial approach to immobilizing silver nanoparticles into-electrospun Na-alginate nanofibers. Due to the unique role of carboxyl and hydroxyl groups in Na-alginate, the silver nanopaticles with 30 nm size in diameter were uniformly distributed inside and outside the alginate nanofibers, which obtained pressure sensor shows stable response, including an ultralow detection limited (1 pa) and high durability (>1000 cycles). Notably, the pressure sensor fabricated by these Ag/alginate nanofibers could not only follow human respiration but also accurately distinguish words like ‘Nano’ and ‘Perfect’ spoke by a tester. Interestingly, the pixelated sensor arrays based on these Ag/alginate nanofibers could monitor distribution of objects and reflect their weight by measuring the different current values. Moreover, these Ag/alginate nanofibers exhibit great antibacterial activity, implying the great potential application in artificial electronic skin.

Injectable MMP-sensitive alginate hydrogels as hMSC delivery systems.

PubMed

Fonseca, Keila B; Gomes, David B; Lee, Kangwon; Santos, Susana G; Sousa, Aureliana; Silva, Eduardo A; Mooney, David J; Granja, Pedro L; Barrias, Cristina C

2014-01-13

Hydrogels with the potential to provide minimally invasive cell delivery represent a powerful tool for tissue-regeneration therapies. In this context, entrapped cells should be able to escape the matrix becoming more available to actively participate in the healing process. Here, we analyzed the performance of proteolytically degradable alginate hydrogels as vehicles for human mesenchymal stem cells (hMSC) transplantation. Alginate was modified with the matrix metalloproteinase (MMP)-sensitive peptide Pro-Val-Gly-Leu-Iso-Gly (PVGLIG), which did not promote dendritic cell maturation in vitro, neither free nor conjugated to alginate chains, indicating low immunogenicity. hMSC were entrapped within MMP-sensitive and MMP-insensitive alginate hydrogels, both containing cell-adhesion RGD peptides. Softer (2 wt % alginate) and stiffer (4 wt % alginate) matrices were tested. When embedded in a Matrigel layer, hMSC-laden MMP-sensitive alginate hydrogels promoted more extensive outward cell migration and invasion into the tissue mimic. In vivo, after 4 weeks of subcutaneous implantation in a xenograft mouse model, hMSC-laden MMP-sensitive alginate hydrogels showed higher degradation and host tissue invasion than their MMP-insensitive equivalents. In both cases, softer matrices degraded faster than stiffer ones. The transplanted hMSC were able to produce their own collagenous extracellular matrix, and were located not only inside the hydrogels, but also outside, integrated in the host tissue. In summary, injectable MMP-sensitive alginate hydrogels can act as localized depots of cells and confer protection to transplanted cells while facilitating tissue regeneration.

Three-dimensional bioprinting of complex cell laden alginate hydrogel structures.

PubMed

Tabriz, Atabak Ghanizadeh; Hermida, Miguel A; Leslie, Nicholas R; Shu, Wenmiao

2015-12-21

Different bioprinting techniques have been used to produce cell-laden alginate hydrogel structures, however these approaches have been limited to 2D or simple three-dimension (3D) structures. In this study, a new extrusion based bioprinting technique was developed to produce more complex alginate hydrogel structures. This was achieved by dividing the alginate hydrogel cross-linking process into three stages: primary calcium ion cross-linking for printability of the gel, secondary calcium cross-linking for rigidity of the alginate hydrogel immediately after printing and tertiary barium ion cross-linking for long-term stability of the alginate hydrogel in culture medium. Simple 3D structures including tubes were first printed to ensure the feasibility of the bioprinting technique and then complex 3D structures such as branched vascular structures were successfully printed. The static stiffness of the alginate hydrogel after printing was 20.18 ± 1.62 KPa which was rigid enough to sustain the integrity of the complex 3D alginate hydrogel structure during the printing. The addition of 60 mM barium chloride was found to significantly extend the stability of the cross-linked alginate hydrogel from 3 d to beyond 11 d without compromising the cellular viability. The results based on cell bioprinting suggested that viability of U87-MG cells was 93 ± 0.9% immediately after bioprinting and cell viability maintained above 88% ± 4.3% in the alginate hydrogel over the period of 11 d.

HMG-CoA lyase (HL) gene: Cloning and characterization of the 5{prime} end of the mouse gene, gene targeting in ES cells, and demonstration of large deletions in three HL-deficient patients

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

Wang, S.; Robert, M.F.; Mitchell, G.A.

1994-09-01

3-hydroxy-3-methylglutaryl CoA lyase (HL) is a mitochondrial matrix enzyme which catalyzes the last step of leucine catabolism and of ketogenesis. Autosomal recessive HL deficiency in humans results in episodes of hypoglycemia and coma. We are interested in the pathophysiology of HL deficiency as a model for both amino acid and fatty acid inborn errors. We have cloned the human and mouse HL genes. In order to analyze the 5{prime} nontranslated region of mouse HL gene, we cloned and sequenced a 1.8 kb fragment containing the 5{prime} extremity including exon 1 and about 1.6 kb of 5{prime} nontranslated sequence. The regionmore » surrounding exon 1 is CpG-rich (66.4%). Using the criteria of West, the Observed/Expected ratio for CpG dinucleotides is 0.7 ({ge}0.6 is consistent with a CpG island). We are carrying out primer extension and RNase protection experiments to determine the transcription initiation site. We constructed a gene targeting vector by introducing the neomycin resistance gene into exon 2 of a 7.5 kb genomic subclone of the mouse HL gene. Targeting was performed by electroporating 10 mg linearized vector into 10{sup 7} ES cells and selecting for 12 days with G418. 5/228 colonies (2.2%) had homologous recombination as shown by PCR screening and Southern analysis. We are microinjecting the 5 targeted clones into blastocysts to create an HL-deficient mouse. To date we have obtained two chimeras with contributions of 95% and 55% from 129, by coat color estimates. Three of 27 (11%) of the HL-deficient patients studied were suggested by genomic Southern analysis to be homozygous for large intragenic deletions. We confirmed this and defined the boundaries using exonic PCR.« less

The biofilm matrix polysaccharides cellulose and alginate both protect Pseudomonas putida mt-2 against reactive oxygen species generated under matric stress and copper exposure.

PubMed

Svenningsen, Nanna B; Martínez-García, Esteban; Nicolaisen, Mette H; de Lorenzo, Victor; Nybroe, Ole

2018-06-01

In natural environments most bacteria live in biofilms embedded in complex matrices of extracellular polymeric substances (EPS). This lifestyle is known to increase protection against environmental stress. Pseudomonas putida mt-2 harbours genes for the production of at least four different EPS polysaccharides, including alginate and cellulose. Little is known about the functional properties of cellulose, while alginate attenuates the accumulation of reactive oxygen species (ROS) caused by matric stress. By using mutants that are deficient in either alginate or cellulose production we show that even cellulose attenuates the accumulation of matric stress-induced ROS for cells in biofilms. Further, both cellulose and alginate attenuate ROS generated through exposure to copper. Interestingly, the two EPS polysaccharides protect cells in both liquid culture and in biofilms against ROS caused by matric stress, indicating that cellulose and alginate do not need to be produced as an integral part of the biofilm lifestyle to provide tolerance towards environmental stressors.

Magnesium Oxide Nanoparticles Reinforced Electrospun Alginate-Based Nanofibrous Scaffolds with Improved Physical Properties

PubMed Central

Mantilaka, M. M. M. G. P. G.; Goh, K. L.; Ratnayake, S. P.; Amaratunga, G. A. J.; de Silva, K. M. Nalin

2017-01-01

Mechanically robust alginate-based nanofibrous scaffolds were successfully fabricated by electrospinning method to mimic the natural extracellular matrix structure which benefits development and regeneration of tissues. Alginate-based nanofibres were electrospun from an alginate/poly(vinyl alcohol) (PVA) polyelectrolyte complex. SEM images revealed the spinnability of the complex composite nanofibrous scaffolds, showing randomly oriented, ultrafine, and virtually defects-free alginate-based/MgO nanofibrous scaffolds. Here, it is shown that an alginate/PVA complex scaffold, blended with near-spherical MgO nanoparticles (⌀ 45 nm) at a predetermined concentration (10% (w/w)), is electrospinnable to produce a complex composite nanofibrous scaffold with enhanced mechanical stability. For the comparison purpose, chemically cross-linked electrospun alginate-based scaffolds were also fabricated. Tensile test to rupture revealed the significant differences in the tensile strength and elastic modulus among the alginate scaffolds, alginate/MgO scaffolds, and cross-linked alginate scaffolds (P < 0.05). In contrast to cross-linked alginate scaffolds, alginate/MgO scaffolds yielded the highest tensile strength and elastic modulus while preserving the interfibre porosity of the scaffolds. According to the thermogravimetric analysis, MgO reinforced alginate nanofibrous scaffolds exhibited improved thermal stability. These novel alginate-based/MgO scaffolds are economical and versatile and may be further optimised for use as extracellular matrix substitutes for repair and regeneration of tissues. PMID:28694826

Alginate cryogel based glucose biosensor

NASA Astrophysics Data System (ADS)

Fatoni, Amin; Windy Dwiasi, Dian; Hermawan, Dadan

2016-02-01

Cryogel is macroporous structure provides a large surface area for biomolecule immobilization. In this work, an alginate cryogel based biosensor was developed to detect glucose. The cryogel was prepared using alginate cross-linked by calcium chloride under sub-zero temperature. This porous structure was growth in a 100 μL micropipette tip with a glucose oxidase enzyme entrapped inside the cryogel. The glucose detection was based on the colour change of redox indicator, potassium permanganate, by the hydrogen peroxide resulted from the conversion of glucose. The result showed a porous structure of alginate cryogel with pores diameter of 20-50 μm. The developed glucose biosensor was showed a linear response in the glucose detection from 1.0 to 5.0 mM with a regression of y = 0.01x+0.02 and R2 of 0.994. Furthermore, the glucose biosensor was showed a high operational stability up to 10 times of uninterrupted glucose detections.

Preparation and release characteristics of polymer-coated and blended alginate microspheres.

PubMed

Lee, D W; Hwang, S J; Park, J B; Park, H J

2003-01-01

To prevent a rapid drug release from alginate microspheres in simulated intestinal media, alginate microspheres were coated or blended with polymers. Three polymers were selected and evaluated such as HPMC, Eudragit RS 30D and chitosan, as both coating materials and additive polymers for controlling the drug release. This study focused on the release characteristics of polymer-coated and blended alginate microspheres, varying the type of polymer and its concentration. The alginate microspheres were prepared by dropping the mixture of drug and sodium alginate into CaCl(2) solution using a spray-gun. Polymer-coated microspheres were prepared by adding alginate microspheres into polymer solution with mild stirring. Polymer-blended microspheres were prepared by dropping the mixture of drug, sodium alginate and additive polymer with plasticizer into CaCl(2) solution. In vitro release test was carried out to investigate the release profiles in 500 ml of phosphate buffered saline (PBS, pH 7.4). As the amount of polymer in sodium alginate or coating solution increase, the drug release generally decreased. HPMC-blended microspheres swelled but withstood the disintegration, showing an ideal linear release profiles. Chitosan-coated microspheres showed smooth and round surface and extended the release of drug. In comparison with chitosan-coated microspheres, HPMC-blended alginate microspheres can be easily made and used for controlled drug delivery systems due to convenient process and controlled drug release.

Structural Basis for the Entrance into the Phenylpropanoid Metabolism Catalyzed by Phenylalanine Ammonia-Lyase

PubMed Central

Ritter, Holger; Schulz, Georg E.

2004-01-01

Because of its key role in secondary phenylpropanoid metabolism, Phe ammonia-lyase is one of the most extensively studied plant enzymes. To provide a basis for detailed structure–function studies, the enzyme from parsley (Petroselinum crispum) was crystallized, and the structure was elucidated at 1.7-Å resolution. It contains the unusual electrophilic 4-methylidene-imidazole-5-one group, which is derived from a tripeptide segment in two autocatalytic dehydration reactions. The enzyme resembles His ammonia-lyase from the general His degradation pathway but contains 207 additional residues, mainly in an N-terminal extension rigidifying a domain interface and in an inserted α-helical domain restricting the access to the active center. Presumably, Phe ammonia-lyase developed from His ammonia-lyase when fungi and plants diverged from the other kingdoms. A pathway of the catalyzed reaction is proposed in agreement with established biochemical data. The inactivation of the enzyme by a nucleophile is described in detail. PMID:15548745

Quantitation of heparosan with heparin lyase III and spectrophotometry.

PubMed

Huang, Haichan; Zhao, Yingying; Lv, Shencong; Zhong, Weihong; Zhang, Fuming; Linhardt, Robert J

2014-02-15

Heparosan is Escherichia coli K5 capsule polysaccharide, which is the key precursor for preparing bioengineered heparin. A rapid and effective quantitative method for detecting heparosan is important in the large-scale production of heparosan. Heparin lyase III (Hep III) effectively catalyzes the heparosan depolymerization, forming unsaturated disaccharides that are measurable using a spectrophotometer at 232 nm. We report a new method for the quantitative detection of heparosan with heparin lyase III and spectrophotometry that is safer and more specific than the traditional carbazole assay. In an optimized detection system, heparosan at a minimum concentration of 0.60 g/L in fermentation broth can be detected. Copyright © 2013 Elsevier Inc. All rights reserved.

21 CFR 172.858 - Propylene glycol alginate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Propylene glycol alginate. 172.858 Section 172.858... CONSUMPTION Multipurpose Additives § 172.858 Propylene glycol alginate. The food additive propylene glycol... information required by the act: (1) The name of the additive, “propylene glycol alginate” or “propylene...

21 CFR 172.858 - Propylene glycol alginate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Propylene glycol alginate. 172.858 Section 172.858... CONSUMPTION Multipurpose Additives § 172.858 Propylene glycol alginate. The food additive propylene glycol... information required by the act: (1) The name of the additive, “propylene glycol alginate” or “propylene...

Applications of Alginate-Based Bioinks in 3D Bioprinting.

PubMed

Axpe, Eneko; Oyen, Michelle L

2016-11-25

Three-dimensional (3D) bioprinting is on the cusp of permitting the direct fabrication of artificial living tissue. Multicellular building blocks (bioinks) are dispensed layer by layer and scaled for the target construct. However, only a few materials are able to fulfill the considerable requirements for suitable bioink formulation, a critical component of efficient 3D bioprinting. Alginate, a naturally occurring polysaccharide, is clearly the most commonly employed material in current bioinks. Here, we discuss the benefits and disadvantages of the use of alginate in 3D bioprinting by summarizing the most recent studies that used alginate for printing vascular tissue, bone and cartilage. In addition, other breakthroughs in the use of alginate in bioprinting are discussed, including strategies to improve its structural and degradation characteristics. In this review, we organize the available literature in order to inspire and accelerate novel alginate-based bioink formulations with enhanced properties for future applications in basic research, drug screening and regenerative medicine.

Applications of Alginate-Based Bioinks in 3D Bioprinting

PubMed Central

Axpe, Eneko; Oyen, Michelle L.

2016-01-01

Three-dimensional (3D) bioprinting is on the cusp of permitting the direct fabrication of artificial living tissue. Multicellular building blocks (bioinks) are dispensed layer by layer and scaled for the target construct. However, only a few materials are able to fulfill the considerable requirements for suitable bioink formulation, a critical component of efficient 3D bioprinting. Alginate, a naturally occurring polysaccharide, is clearly the most commonly employed material in current bioinks. Here, we discuss the benefits and disadvantages of the use of alginate in 3D bioprinting by summarizing the most recent studies that used alginate for printing vascular tissue, bone and cartilage. In addition, other breakthroughs in the use of alginate in bioprinting are discussed, including strategies to improve its structural and degradation characteristics. In this review, we organize the available literature in order to inspire and accelerate novel alginate-based bioink formulations with enhanced properties for future applications in basic research, drug screening and regenerative medicine. PMID:27898010

Localization of ATP Sulfurylase and O-Acetylserine(thiol)lyase in Spinach Leaves.

PubMed

Lunn, J E; Droux, M; Martin, J; Douce, R

1990-11-01

The intracellular compartmentation of ATP sulfurylase and O-acetylserine(thiol)lyase in spinach (Spinacia oleracea L.) leaves has been investigated by isolation of organelles and fractionation of protoplasts. ATP sulfurylase is located predominantly in the chloroplasts, but is also present in the cytosol. No evidence was found for ATP sulfurylase activity in the mitochondria. Two forms of ATP sulfurylase were separated by anion-exchange chromatography. The more abundant form is present in the chloroplasts, the second is cytosolic. O-Acetylserine(thiol)lyase activity is located primarily in the chloroplasts and cytosol, but is also present in the mitochondria. Three forms of O-acetylserine(thiol)lyase were separated by anion-exchange chromatography, and each was found to be specific to one intracellular compartment. The cytosolic ATP sulfurylase may not be active in vivo due to the unfavorable equilibrium constant of the reaction, and the presence of micromolar concentrations of inorganic pyrophosphate in the cytosol, therefore its role remains unknown. It is suggested that the plant cell may be unable to transport cysteine between the different compartments, so that the cysteine required for protein synthesis must be synthesized in situ, hence the presence of O-acetylserine(thiol)lyase in the three compartments where proteins are synthesized.

Localization of ATP Sulfurylase and O-Acetylserine(thiol)lyase in Spinach Leaves

PubMed Central

Lunn, John E.; Droux, Michel; Martin, Jacqueline; Douce, Roland

1990-01-01

The intracellular compartmentation of ATP sulfurylase and O-acetylserine(thiol)lyase in spinach (Spinacia oleracea L.) leaves has been investigated by isolation of organelles and fractionation of protoplasts. ATP sulfurylase is located predominantly in the chloroplasts, but is also present in the cytosol. No evidence was found for ATP sulfurylase activity in the mitochondria. Two forms of ATP sulfurylase were separated by anion-exchange chromatography. The more abundant form is present in the chloroplasts, the second is cytosolic. O-Acetylserine(thiol)lyase activity is located primarily in the chloroplasts and cytosol, but is also present in the mitochondria. Three forms of O-acetylserine(thiol)lyase were separated by anion-exchange chromatography, and each was found to be specific to one intracellular compartment. The cytosolic ATP sulfurylase may not be active in vivo due to the unfavorable equilibrium constant of the reaction, and the presence of micromolar concentrations of inorganic pyrophosphate in the cytosol, therefore its role remains unknown. It is suggested that the plant cell may be unable to transport cysteine between the different compartments, so that the cysteine required for protein synthesis must be synthesized in situ, hence the presence of O-acetylserine(thiol)lyase in the three compartments where proteins are synthesized. PMID:16667839

The importance of four histidine residues in isocitrate lyase from Escherichia coli.

PubMed Central

Diehl, P; McFadden, B A

1994-01-01

By site-directed mutagenesis, substitutions were made for His-184 (H-184), H-197, H-266, and H-306 in Escherichia coli isocitrate lyase. Of these changes, only mutations of H-184 and H-197 appreciably reduced enzyme activity. Mutation of H-184 to Lys, Arg, or Leu resulted in an inactive isocitrate lyase, and mutation of H-184 to Gln resulted in an enzyme with 0.28% activity. Nondenaturing polyacrylamide gel electrophoresis demonstrated that isocitrate lyase containing the Lys, Arg, Gln, and Leu substitutions at H-184 was assembled poorly into the tetrameric subunit complex. Mutation of H-197 to Lys, Arg, Leu, and Gln resulted in an assembled enzyme with less than 0.25% wild-type activity. Five substitutions for H-266 (Asp, Glu, Val, Ser, and Lys), four substitutions for H-306 (Asp, Glu, Val, and Ser), and a variant in which both H-266 and H-306 were substituted for showed little or no effect on enzyme activity. All the H-197, H-266, and H-306 mutants supported the growth of isocitrate lyase-deficient E. coli JE10 on acetate as the sole carbon source; however, the H-184 mutants did not. Images PMID:8300547

Phenylalanine ammonia-lyase. Induction and purification from yeast and clearance in mammals.

PubMed

Fritz, R R; Hodgins, D S; Abell, C W

1976-08-10

Yeast phenylalanine ammonia-lyase (EC 4.3.1.5) catalyzes the deamination of L-phenylalanine to form trans-cinnamic acid and tyrosine to trans-coumaric acid. Maximal enzyme activity in Rhodotorula glutinis (2 units/g, wet weight, of yeast) was induced in late-log phase (12 to 14 hours) of growth in a culture medium containing 1.0% malt extract, 0.1% yeast extract, and 0.1% L-phenylalanine. A highly purified enzyme was obtained by fractionation with ammonium sulfate and sodium citrate followed by chromatography on DEAE-cellulose and Sephadex G-200. The active preparation yielded a major component on three different polyacrylamide gel electrophoretic systems. Antisera to phenylalanine ammonia-lyase was raised in rabbits and detected by double immunodiffusion. The antigen-antibody complex was enzymatically active in vitro. The biological half-life of the enzyme was approximately 21 hours in several mammalian species (mice without and with BW10232 adenocarcinoma and B16 melanoma, rats, and monkeys) after a single injection; however, upon repeated administration, phenylalanine ammonia-lyase had a much shorter biological half-life. The onset of rapid clearance occurred earlier in tumor-bearing than in nontumor-bearing mice indicating a direct or indirect influence by the tumor on the biological half-life of phenylalanine ammonia-lyase.

Alginate: A Versatile Biomaterial to Encapsulate Isolated Ovarian Follicles.

PubMed

Vanacker, Julie; Amorim, Christiani A

2017-07-01

In vitro culture of ovarian follicles isolated or enclosed in ovarian tissue fragments and grafting of isolated ovarian follicles represent a potential alternative to restore fertility in cancer patients who cannot undergo cryopreservation of embryos or oocytes or transplantation of frozen-thawed ovarian tissue. In this regard, respecting the three-dimensional (3D) architecture of isolated follicles is crucial to maintaining their proper follicular physiology. To this end, alginate hydrogel has been widely investigated using follicles from numerous animal species, yielding promising results. The goal of this review is therefore to provide an overview of alginate applications utilizing the biomaterial as a scaffold for 3D encapsulation of isolated ovarian follicles. Different methods of isolated follicle encapsulation in alginate are discussed in this review, as its use of 3D alginate culture systems as a tool for in vitro follicle analysis. Possible improvements of this matrix, namely modification with arginine-glycine-aspartic acid peptide or combination with fibrin, are also summarized. Encouraging results have been obtained in different animal models, and particularly with isolated follicles encapsulated in alginate matrices and grafted to mice. This summary is designed to guide the reader towards development of next-generation alginate scaffolds, with enhanced properties for follicle encapsulation.

Guluronic acid content as a factor affecting turbidity removal potential of alginate.

PubMed

Kıvılcımdan Moral, Çiğdem; Ertesvåg, Helga; Sanin, F Dilek

2016-11-01

Alginates are natural polymers composed of mannuronic and guluronic acid residues. They are currently extracted from brown algae; however, alginate can also be synthesized by some species of Azotobacter and Pseudomonas. Alginates with different proportion of mannuronic and guluronic acids are known to have different characteristics and form gels at different extents in the presence of calcium ions. The aim of this work was to investigate the usefulness of alginate as a non-toxic coagulant used in purification of drinking water. This study utilized alginates from Azotobacter vinelandii having different guluronic acid levels. These were obtained partly by changing the cultivation parameters, partly by epimerizing a purified alginate sample in vitro using the A. vinelandii mannuronan C-5 epimerase AlgE1. The different alginates were then used for coagulation together with calcium. The results showed that turbidity removal capability was dependent on the content of guluronic acid residues. For the best performing samples, the turbidity decreased from 10 NTU to 1 NTU by the use of only 2 mg/L of alginate and 1.5 mM of calcium chloride.

Influence of hydrophobic modification in alginate-based hydrogels for biomedical applications

NASA Astrophysics Data System (ADS)

Choudhary, Soumitra

Alginate has been exploited commercially for decades in foods, textiles, paper, pharmaceutical industries, and also as a detoxifier for removing heavy metals. Alginate is also popular in cell encapsulation because of its relatively mild gelation protocol and simple chemistry with which biological active entities can be immobilized. Surface modification of alginate gels has been explored to induce desired cell interactions with the gel matrix. These modifications alter the bulk properties, which strongly determine on how cells feel and response to the three-dimensional microenvironment. However, there is a need to develop strategies to engineer functionalities into bulk alginate hydrogels that not only preserve their inherent qualities but are also less toxic. In this thesis, our main focus was to optimize the mechanical properties of alginate-based hydrogels, and by doing so control the performance of the biomaterials. In the first scheme, we used alginate and hydrophobically modified ethyl hydroxy ethyl cellulose as components in interpenetrating polymer network (IPN) gels. The second network was used to control gelation time and rheological properties. We believe these experiments also may provide insight into the mechanical and structural properties of more complex biopolymer gels and naturally-occurring IPNs. Next, we worked on incorporating a hydrophobic moiety directly into the alginate chain, resulting in materials for extended release of hydrophobic drugs. We successfully synthesized hydrophobically modified alginate (HMA) by attaching octylamine groups onto the alginate backbone by standard carbodiimide based amide coupling reaction. Solubility of several model hydrophobic drugs in dilute HMA solutions was found to be increased by more than an order of magnitude. HMA hydrogels, prepared by crosslinking the alginate chains with calcium ions, were found to exhibit excellent mechanical properties (modulus ˜100 kPa) with release extended upto 5 days. Ability

Role of alginate in antibacterial finishing of textiles.

PubMed

Li, Jiwei; He, Jinmei; Huang, Yudong

2017-01-01

Antibacterial finishing of textiles has been introduced as a necessary process for various purposes especially creating a fabric with antimicrobial activities. Currently, the textile industry continues to look for textiles antimicrobial finishing process based on sustainable biopolymers from the viewpoints of environmental friendliness, industrialization, and economic concerns. This paper reviews the role of alginate, a sustainable biopolymer, in the development of antimicrobial textiles, including both basic physicochemical properties of alginate such as preparation, chemical structure, molecular weight, solubility, viscosity, and sol-gel transformation property. Then different processing routes (e.g. nanocomposite coating, ionic cross-linking coating, and Layer-by-Layer coating) for the antibacterial finishing of textiles by using alginate are revised in some detail. The achievements in this area have increased our knowledge of alginate application in the field of textile industry and promoted the development of green textile finishing. Copyright © 2016 Elsevier B.V. All rights reserved.

Determinations of dimethylsulphoniopropionate (DMSP) lyase activity using headspace analysis of dimethylsulphide (DMS)

NASA Astrophysics Data System (ADS)

Steinke, M.; Malin, G.; Turner, S. M.; Liss, P. S.

2000-08-01

The osmolyte dimethylsulphoniopropionate (DMSP) can be enzymatically cleaved to dimethylsulphide (DMS), acrylate and a proton. The enzyme involved in this reaction is dimethylpropiothetin dethiomethylase (DMSP lyase; enzyme classification number 4.4.1.3.). Although the importance of this reaction for the global sulphur cycle, the influence of DMS on atmospheric acidity and the possible effect on climate regulation have been widely recognised, our knowledge of DMSP lyases is limited to just a few studies. Activity measurements of DMSP lyases offer an important step towards a better understanding of the conditions under which DMS is produced. In the available published data somewhat similar methods have been used before, but a critical examination of the method limitations has not been reported. To encourage further research on this enzyme, we suggest and detail two protocols for measurements of DMSP lyase activity: An in vitro assay for crude cell extracts or purified enzyme and an in vivo method for whole cells, which we recently started to use. After addition of DMSP, samples incubated in a gas tight vial may produce DMS from enzymatic cleavage under suitable conditions, and a DMS production rate can be estimated from time-series measurements of DMS in the headspace of the vial. Headspace analysis of DMS is a useful and rapid technique to estimate and compare DMSP lyase activities from different sources. The relative rates of DMS production in the liquid and of the gas transfer between liquid and headspace, determine the rate of DMS production measured via headspace analysis. If DMS production in the liquid is higher than the rate of transfer, headspace measurements will not reflect the actual amount of DMS produced in the liquid. In this case, extracts have to be diluted to a level that ensures linearity between dilution factor and reduction of enzyme activity. Additionally, incubation volumes and vials should be selected to provide a high surface-to-volume ratio

Identification and immunologic characterization of an allergen, alliin lyase, from garlic (Allium sativum).

PubMed

Kao, Shao-Hsuan; Hsu, Ching-Hsian; Su, Song-Nan; Hor, Wei-Ting; Chang T, Wen-Hong; Chow, Lu-Ping

2004-01-01

Garlic (Allium sativum) is one of the most common relishes used in cooking worldwide. Very few garlic allergens have been reported, and garlic allergy has been rarely studied. The aim of the study was to identify allergenic proteins in garlic and to investigate their importance in allergies to other Allium species (leek, shallot, and onion). A crude extract of garlic proteins was separated by SDS-PAGE and 2-dimensional electrophoresis; immunoblotting was then performed with the use of individual and pooled sera from patients with garlic allergy, and the major IgE-binding proteins were analyzed by amino acid sequencing and mass spectrometry. The putative allergens were further purified by chromatography; the antigenicity, allergenicity, and IgE-binding cross-reactivity of the purified protein were then studied by immunoblotting, periodate oxidation, skin tests, and IgE-binding inhibition assays. A major allergen, alliin lyase, was identified by mass spectrometry and Edman sequencing and purified to homogeneity through the use of a simple 2-step chromatographic method. Skin tests showed that the purified protein elicited IgE-mediated hypersensitive responses in patients with garlic allergy. Periodate oxidation showed that carbohydrate groups were involved in the antigenicity, allergenicity, and cross-reactivity. Garlic alliin lyase showed strong cross-reactivity with alliin lyases from other Allium species, namely leek, shallot, and onion. Alliin lyase was found to be a major garlic allergen in a garlic-allergic group of patients in Taiwan. The wide distribution of alliin lyase in Allium suggests it may be a new cross-reactive allergen.

The Alginate Demonstration: Polymers, Food Science, and Ion Exchange

NASA Astrophysics Data System (ADS)

Waldman, Amy Sue; Schechinger, Linda; Govindarajoo, Geeta; Nowick, James S.; Pignolet, Louis H.

1998-11-01

We have recently devised a polymer demonstration involving the crosslinking and decrosslinking of alginate, a polysaccharide isolated from seaweed. The polymer is composed of D-mannuronic acid and L-guluronic acid subunits and is a component of cell walls. It is commonly used as a thickener in foods such as ice cream and fruit-filled snacks. For the demonstration, a 2% solution of sodium alginate is poured into a 1% solution of calcium chloride. Nontoxic calcium alginate "worms" form due to crosslinking of the polymer. Alternatively, the commercially available antacid Gaviscon can be used as a source of sodium alginate. The crosslinks can then be broken by shaking the worms in brine. The demonstration is a fine addition to any chemical educator's repertoire of polymer experiments.

"Self-catabolite repression" of pectate lyase in Erwinia carotovora.

PubMed Central

Tsuyumu, S

1979-01-01

The induction of pectate lyase of Erwinia carotovora was repressed by a high concentration of its inducer. The concomitant addition of cyclic adenosine 3',5'-monophosphate reversed this repression. PMID:217862

Composite coating of alginate-olive oil enriched with antioxidants enhances postharvest quality and shelf life of Ber fruit (Ziziphus mauritiana Lamk. Var. Gola).

PubMed

Ramana Rao, T V; Baraiya, Nilanjana S; Vyas, Pinal B; Patel, Dhara M

2016-01-01

The present study has been carried out to evaluate the effect of a composite edible coating of 2 % Sodium alginate and 0.2 % Olive oil with combination of 1 % ascorbic acid and 1 % citric acid on the post harvest nutritional quality and shelf life of Ber fruit stored at 25 ± 2 °C and 65 % R.H. The coatings reduced the decay occurrence, weight loss, accumulation of total soluble solids (TSS) and total sugars in Ber fruit and enhanced the level of antioxidants. The delayed activity of polygalacturonase (PG), Pectate lyase (PL) and Pectin methyl esterase (PME) was noticed in coated fruits than that of the control fruit indicating the reduced softening and ripening process. These findings suggest that the composite edible coating tested under the current study has the potential to control decaying incidence of Ber fruit, extends its storage life and also improves its valuable nutritional characteristics.

Isolation of Inositol Hexaphosphate (IHP)-Degrading Bacteria from Arbuscular Mycorrhizal Fungal Hyphal Compartments Using a Modified Baiting Method Involving Alginate Beads Containing IHP

PubMed Central

Hara, Shintaro; Saito, Masanori

2016-01-01

Phytate (inositol hexaphosphate; IHP)-degrading microbes have been suggested to contribute to arbuscular mycorrhizal fungi (AMF)-mediated P transfer from IHP to plants; however, no IHP degrader involved in AMF-mediated P transfer has been isolated to date. We herein report the isolation of IHP-degrading bacteria using a modified baiting method. We applied alginate beads as carriers of IHP powder, and used them as recoverable IHP in the AM fungal compartment of plant cultivation experiments. P transfer from IHP in alginate beads via AMF was confirmed, and extracted DNA from alginate beads was analyzed by denaturing gradient gel electrophoresis targeting the 16S rRNA gene and a clone library method for the beta-propeller phytase (BPP) gene. The diversities of the 16S rRNA and BPP genes of microbes growing on IHP beads were simple and those of Sphingomonas spp. and Caulobacter spp. dominated. A total of 187 IHP-utilizing bacteria were isolated and identified, and they were consistent with the results of DNA analysis. Furthermore, some isolated Sphingomonas spp. and Caulobacter sp. showed IHP-degrading activity. Therefore, we successfully isolated dominant IHP-degrading bacteria from IHP in an AMF hyphal compartment. These strains may contribute to P transfer from IHP via AMF. PMID:27383681

Dental mesenchymal stem cells encapsulated in alginate hydrogel co-delivery microencapsulation system for cartilage regeneration

PubMed Central

Moshaverinia, Alireza; Xu, Xingtian; Chen, Chider; Akiyama, Kentaro; Snead, Malcolm L; Shi, Songtao

2013-01-01

Dental-derived MSCs are promising candidates for cartilage regeneration, with high chondrogenic differentiation capacity. This property contributes to making dental MSCs an advantageous therapeutic option compared to current treatment modalities. The MSC delivery vehicle is the principal determinant for the success of MSC-mediated cartilage regeneration therapies. The objectives of this study were to: (1) develop a novel co-delivery system based on TGF-β1 loaded RGD-coupled alginate microspheres encapsulating Periodontal Ligament Stem Cells (PDLSCs) or Gingival Mesenchymal Stem Cells (GMSCs); and (2) investigate dental MSC viability and chondrogenic differentiation in alginate microspheres. The results revealed the sustained release of TGF-β1 from the alginate microspheres. After 4 weeks of chondrogenic differentiation in vitro, PDLSCs, GMSCs as well as human bone marrow mesenchymal stem cells (hBMMSC) (as positive control) revealed chondrogenic gene expression markers (Col II and Sox-9) via qPCR, as well as matrix positively stained by toluidine blue and safranin-O. In animal studies, ectopic cartilage tissue regeneration was observed inside and around the transplanted microspheres, confirmed by histochemical and immunofluorescent staining. Interestingly, PDLSCs showed more chondrogenesis than GMSCs and hBMMSCs (P<0.05). Taken together, these results suggest that RGD-modified alginate microencapsulating dental MSCs make a promising candidate for cartilage regeneration. Our results highlight the vital role played by the microenvironment, as well as value of presenting inductive signals for viability and differentiation of MSCs. PMID:23891740

Alginate microparticles as oral colon drug delivery device: A review.

PubMed

Agüero, Lissette; Zaldivar-Silva, Dionisio; Peña, Luis; Dias, Marcos L

2017-07-15

The increase in the research interest on alginate microparticles in pharmaceutical and biomedical areas confirms its potential use as an effective matrix for drug and cell delivery. Among the well known alginate properties, pH sensitivity remains as an attractive option for targeting of drug in the colon region. This essential aspect is advantageous to enhance therapeutic efficacy of treatment of inflammatory bowel diseases, which require multi-drug administration frequently in a long period. As consequence, severe side effect appears leading to discontinuation of therapy and affecting quality of patient life. This review gives an overview of relevant properties of alginate as oral colon delivery systems and the recent innovative strategies of using alginate with other polymers as well as microencapsulation techniques. At the same time, it describes the several advantages of coating processes involving alginate over microparticles in order to design better material with sustained release characteristic for colon-targeted delivery. Copyright © 2017 Elsevier Ltd. All rights reserved.

Biodegradable Nanocomposite Films Based on Sodium Alginate and Cellulose Nanofibrils

PubMed Central

Deepa, B.; Abraham, Eldho; Pothan, Laly A.; Cordeiro, Nereida; Faria, Marisa; Thomas, Sabu

2016-01-01

Biodegradable nanocomposite films were prepared by incorporation of cellulose nanofibrils (CNF) into alginate biopolymer using the solution casting method. The effects of CNF content (2.5, 5, 7.5, 10 and 15 wt %) on mechanical, biodegradability and swelling behavior of the nanocomposite films were determined. The results showed that the tensile modulus value of the nanocomposite films increased from 308 to 1403 MPa with increasing CNF content from 0% to 10%; however, it decreased with further increase of the filler content. Incorporation of CNF also significantly reduced the swelling percentage and water solubility of alginate-based films, with the lower values found for 10 wt % in CNF. Biodegradation studies of the films in soil confirmed that the biodegradation time of alginate/CNF films greatly depends on the CNF content. The results evidence that the stronger intermolecular interaction and molecular compatibility between alginate and CNF components was at 10 wt % in CNF alginate films. PMID:28787850

Quantification of alginate by aggregation induced by calcium ions and fluorescent polycations.

PubMed

Zheng, Hewen; Korendovych, Ivan V; Luk, Yan-Yeung

2016-01-01

For quantification of polysaccharides, including heparins and alginates, the commonly used carbazole assay involves hydrolysis of the polysaccharide to form a mixture of UV-active dye conjugate products. Here, we describe two efficient detection and quantification methods that make use of the negative charges of the alginate polymer and do not involve degradation of the targeted polysaccharide. The first method utilizes calcium ions to induce formation of hydrogel-like aggregates with alginate polymer; the aggregates can be quantified readily by staining with a crystal violet dye. This method does not require purification of alginate from the culture medium and can measure the large amount of alginate that is produced by a mucoid Pseudomonas aeruginosa culture. The second method employs polycations tethering a fluorescent dye to form suspension aggregates with the alginate polyanion. Encasing the fluorescent dye in the aggregates provides an increased scattering intensity with a sensitivity comparable to that of the conventional carbazole assay. Both approaches provide efficient methods for monitoring alginate production by mucoid P. aeruginosa. Copyright © 2015 Elsevier Inc. All rights reserved.

3D Porous Chitosan-Alginate Scaffolds as an In Vitro Model for Evaluating Nanoparticle-Mediated Tumor Targeting and Gene Delivery to Prostate Cancer.

PubMed

Wang, Kui; Kievit, Forrest M; Florczyk, Stephen J; Stephen, Zachary R; Zhang, Miqin

2015-10-12

Cationic nanoparticles (NPs) for targeted gene delivery are conventionally evaluated using 2D in vitro cultures. However, this does not translate well to corresponding in vivo studies because of the marked difference in NP behavior in the presence of the tumor microenvironment. In this study, we investigated whether prostate cancer (PCa) cells cultured in three-dimensional (3D) chitosan-alginate (CA) porous scaffolds could model cationic NP-mediated gene targeted delivery to tumors in vitro. We assessed in vitro tumor cell proliferation, formation of tumor spheroids, and expression of marker genes that promote tumor malignancy in CA scaffolds. The efficacy of NP-targeted gene delivery was evaluated in PCa cells in 2D cultures, PCa tumor spheroids grown in CA scaffolds, and PCa tumors in a mouse TRAMP-C2 flank tumor model. PCa cells cultured in CA scaffolds grew into tumor spheroids and displayed characteristics of higher malignancy as compared to those in 2D cultures. Significantly, targeted gene delivery was only observed in cells cultured in CA scaffolds, whereas cells cultured on 2D plates showed no difference in gene delivery between targeted and nontarget control NPs. In vivo NP evaluation confirmed targeted gene delivery, indicating that only CA scaffolds correctly modeled NP-mediated targeted delivery in vivo. These findings suggest that CA scaffolds serve as a better in vitro platform than 2D cultures for evaluation of NP-mediated targeted gene delivery to PCa.

Alginate Polymerization and Modification Are Linked in Pseudomonas aeruginosa

PubMed Central

Moradali, M. Fata; Donati, Ivan; Sims, Ian M.; Ghods, Shirin

2015-01-01

ABSTRACT The molecular mechanisms of alginate polymerization/modification/secretion by a proposed envelope-spanning multiprotein complex are unknown. Here, bacterial two-hybrid assays and pulldown experiments showed that the catalytic subunit Alg8 directly interacts with the proposed copolymerase Alg44 while embedded in the cytoplasmic membrane. Alg44 additionally interacts with the lipoprotein AlgK bridging the periplasmic space. Site-specific mutagenesis of Alg44 showed that protein-protein interactions and stability were independent of conserved amino acid residues R17 and R21, which are involved in c-di-GMP binding, the N-terminal PilZ domain, and the C-terminal 26 amino acids. Site-specific mutagenesis was employed to investigate the c-di-GMP-mediated activation of alginate polymerization by the PilZAlg44 domain and Alg8. Activation was found to be different from the proposed activation mechanism for cellulose synthesis. The interactive role of Alg8, Alg44, AlgG (epimerase), and AlgX (acetyltransferase) on alginate polymerization and modification was studied by using site-specific deletion mutants, inactive variants, and overproduction of subunits. The compositions, molecular masses, and material properties of resulting novel alginates were analyzed. The molecular mass was reduced by epimerization, while it was increased by acetylation. Interestingly, when overproduced, Alg44, AlgG, and the nonepimerizing variant AlgG(D324A) increased the degree of acetylation, while epimerization was enhanced by AlgX and its nonacetylating variant AlgX(S269A). Biofilm architecture analysis showed that acetyl groups promoted cell aggregation while nonacetylated polymannuronate alginate promoted stigmergy. Overall, this study sheds new light on the arrangement of the multiprotein complex involved in alginate production. Furthermore, the activation mechanism and the interplay between polymerization and modification of alginate were elucidated. PMID:25968647

Development of nanocomposites based on hydroxyapatite/sodium alginate: Synthesis and characterisation

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

Rajkumar, M.; Meenakshisundaram, N.; Rajendran, V., E-mail: veerajendran@gmail.com

2011-05-15

In this study, a novel method was used to produce a nanostructured composite consisting of hydroxyapatite and sodium alginate by varying the composition of sodium alginate. The structure, morphology, simulated body fluid response and mechanical properties of the synthesised nanocomposites were characterised. From X-ray diffraction analysis, an increase in crystallite size and degree of crystallinity with an increase in the composition of sodium alginate up to 1.5 wt.% was observed. Further, it was found to decrease with an increase in the composition of sodium alginate. A notable peak shift from 1635 to 1607 cm{sup -1} and 1456 to 1418 cm{supmore » -1} in the Fourier transform infrared spectra of the nanocomposite was observed towards the lower wave number side when compared with pure hydroxyapatite. It reveals a strong interaction between the positively charged calcium (Ca{sup 2+}) and the negatively charged carboxyl group (COO{sup -}) in sodium alginate. Transmission electron microscopy images of pure hydroxyapatite showed a short nanorod-like morphology with an average particle size of 13 nm. Bioresorbability of the samples was observed by immersing them in simulated body fluid medium for 14 days to evaluate the changes in pH and Ca{sup 2+} ion strength. Microhardness shows an increasing trend with an increase in the composition of sodium alginate from 1.5 to 3.0 wt.%, which is similar to that in the density. - Research Highlights: {yields} We have prepared nanohydroxyapatite/sodium alginate as a composite. {yields} Effect of sodium alginate on the properties of nanohydrroxyapatite has been studied. {yields} The sodium alginate ranges from 0 to 3.75 wt.% has been used. {yields} Composites show improved biological and mechanical properties.« less

A Controlled Drug-Delivery Experiment Using Alginate Beads

ERIC Educational Resources Information Center

Farrell, Stephanie; Vernengo, Jennifer

2012-01-01

This paper describes a simple, cost-effective experiment which introduces students to drug delivery and modeling using alginate beads. Students produce calcium alginate beads loaded with drug and measure the rate of release from the beads for systems having different stir rates, geometries, extents of cross-linking, and drug molecular weight.…

Empirical study of alginate impression materials by customized proportioning system

PubMed Central

2016-01-01

PURPOSE Alginate mixers available in the market do not have the automatic proportioning unit. In this study, an automatic proportioning unit for the alginate mixer and controller software were designed and produced for a new automatic proportioning unit. With this device, it was ensured that proportioning operation could arrange weight-based alginate impression materials. MATERIALS AND METHODS The variation of coefficient in the tested groups was compared with the manual proportioning. Compression tension and tear tests were conducted to determine the mechanical properties of alginate impression materials. The experimental data were statistically analyzed using one way ANOVA and Tukey test at the 0.05 level of significance. RESULTS No statistically significant differences in modulus of elastisity (P>0.3), tensional/compresional strength (P>0.3), resilience (P>0.2), strain in failure (P>0.4), and tear energy (P>0.7) of alginate impression materials were seen. However, a decrease in the standard deviation of tested groups was observed when the customized machine was used. To verify the efficiency of the system, powder and powder/water mixing were weighed and significant decrease was observed. CONCLUSION It was possible to obtain more mechanically stable alginate impression materials by using the custom-made proportioning unit. PMID:27826387

Cysteine S-conjugate β-lyases: Important roles in the metabolism of naturally occurring sulfur and selenium-containing compounds, xenobiotics and anticancer agents

PubMed Central

Cooper, Arthur J. L.; Krasnikov, Boris F.; Niatsetskaya, Zoya V.; Pinto, John T.; Callery, Patrick S.; Villar, Maria T.; Artigues, Antonio; Bruschi, Sam A.

2010-01-01

Summary Cysteine S-conjugate β-lyases are pyridoxal 5′-phosphate-containing enzymes that catalyze β-elimination reactions with cysteine S-conjugates that possess a good leaving group in the β-position. The end products are aminoacrylate and a sulfur-containing fragment. The aminoacrylate tautomerizes and hydrolyzes to pyruvate and ammonia. The mammalian cysteine S-conjugate β-lyases thus far identified are enzymes involved in amino acid metabolism that catalyze β-lyase reactions as non-physiological side reactions. Most are aminotransferases. In some cases the lyase is inactivated by reaction products. The cysteine S-conjugate β-lyases are of much interest to toxicologists because they play an important key role in the bioactivation (toxication) of halogenated alkenes, some of which are produced on an industrial scale and are environmental contaminants. The cysteine S-conjugate β-lyases have been reviewed in this journal previously [Cooper and Pinto, 2006]. Here we focus on more recent findings regarding: 1) the identification of enzymes associated with high-Mr cysteine S-conjugate β-lyases in the cytosolic and mitochondrial fractions of rat liver and kidney; 2) the mechanism of syncatalytic inactivation of rat liver mitochondrial aspartate aminotransferase by the nephrotoxic β-lyase substrate S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (the cysteine S-conjugate of tetrafluoroethylene); 3) toxicant channeling of reactive fragments from the active site of mitochondrial aspartate aminotransferase to susceptible proteins in the mitochondria; 4) the involvement of cysteine S-conjugate β-lyases in the metabolism/bioactivation of drugs and natural products; and 5) the role of cysteine S-conjugate β-lyases in the metabolism of selenocysteine Se-conjugates. This review emphasizes the fact that the cysteine S-conjugate β-lyases are biologically more important than hitherto appreciated. PMID:20306345

Antimicrobial and anticancer activities of porous chitosan-alginate biosynthesized silver nanoparticles.

PubMed

Venkatesan, Jayachandran; Lee, Jin-Young; Kang, Dong Seop; Anil, Sukumaran; Kim, Se-Kwon; Shim, Min Suk; Kim, Dong Gyu

2017-05-01

The main aim of this study was to obtain porous antimicrobial composites consisting of chitosan, alginate, and biosynthesized silver nanoparticles (AgNPs). Chitosan and alginate were used owing to their pore-forming capacity, while AgNPs were used for their antimicrobial property. The developed porous composites of chitosan-alginate-AgNPs were characterized using Fourier transform infrared spectroscopy (FT-IR), ultraviolet-visible spectroscopy, X-ray diffraction (XRD) analysis, and scanning electron microscopy (SEM). The FT-IR results revealed the presence of a strong chemical interaction between chitosan and alginate due to polyelectrolyte complex; whereas, the XRD results confirmed the presence of AgNPs in the composites. The dispersion of AgNPs in the porous membrane was uniform with a pore size of 50-500μm. Antimicrobial activity of the composites was checked with Escherichia coli and Staphylococcus aureus. The developed composites resulted in the formation of a zone of inhibition of 11±1mm for the Escherichia coli, and 10±1mm for Staphylococcus aureus. The bacterial filtration efficiency of chitosan-alginate-AgNPs was 1.5-times higher than that of the chitosan-alginate composite. The breast cancer cell line MDA-MB-231 was used to test the anticancer activity of the composites. The IC 50 value of chitosan-alginate-AgNPs on MDA-MB-231 was 4.6mg. The developed chitosan-alginate-AgNPs composite showed a huge potential for its applications in antimicrobial filtration and cancer treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation of outgassing, tear strength, and detail reproduction in alginate substitute materials.

PubMed

Baxter, R T; Lawson, N C; Cakir, D; Beck, P; Ramp, L C; Burgess, J O

2012-01-01

To compare three alginate substitute materials to an alginate impression material for cast surface porosity (outgassing), tear strength, and detail reproduction. Detail reproduction tests were performed following American National Standards Institute/American Dental Association (ANSI/ADA) Specification No. 19. To measure tear strength, 12 samples of each material were made using a split mold, placed in a water bath until testing, and loaded in tension until failure at a rate of 500 mm/min using a universal testing machine. For cast surface porosity testing, five impressions of a Teflon mold with each material were placed in a water bath (37.8°C) for the in-mouth setting time and poured with vacuum-mixed Silky Rock die stone at 5, 10, 30, and 60 minutes from the start of mixing. The gypsum samples were analyzed with a digital microscope for surface porosity indicative of hydrogen gas release by comparing the surface obtained at each interval with four casts representing no, little, some, and significant porosity. Data analysis was performed using parametric and Kruskal-Wallis analysis of variance (ANOVA), Tukey/Kramer post-hoc tests (α=0.05), and individual Mann-Whitney U tests (α=0.0167). All alginate substitute materials passed the detail reproduction test. Tear strength of the alginate substitute materials was significantly better than alginate and formed three statistically different groups: AlgiNot had the lowest tear strength, Algin-X Ultra had the highest tear strength, and Position Penta Quick had intermediate tear strength. Significant variation in outgassing existed between materials and pouring times (p<0.05). All alginate substitute materials exhibited the least outgassing and cast porosity 60 minutes after mixing. Detail reproduction and tear strength of alginate substitute materials were superior to traditional alginate. The outgassing effect was minimal for most materials tested. Alginate substitute materials are superior replacements for

Structure-based functional annotation of putative conserved proteins having lyase activity from Haemophilus influenzae.

PubMed

Shahbaaz, Mohd; Ahmad, Faizan; Imtaiyaz Hassan, Md

2015-06-01

Haemophilus influenzae is a small pleomorphic Gram-negative bacteria which causes several chronic diseases, including bacteremia, meningitis, cellulitis, epiglottitis, septic arthritis, pneumonia, and empyema. Here we extensively analyzed the sequenced genome of H. influenzae strain Rd KW20 using protein family databases, protein structure prediction, pathways and genome context methods to assign a precise function to proteins whose functions are unknown. These proteins are termed as hypothetical proteins (HPs), for which no experimental information is available. Function prediction of these proteins would surely be supportive to precisely understand the biochemical pathways and mechanism of pathogenesis of Haemophilus influenzae. During the extensive analysis of H. influenzae genome, we found the presence of eight HPs showing lyase activity. Subsequently, we modeled and analyzed three-dimensional structure of all these HPs to determine their functions more precisely. We found these HPs possess cystathionine-β-synthase, cyclase, carboxymuconolactone decarboxylase, pseudouridine synthase A and C, D-tagatose-1,6-bisphosphate aldolase and aminodeoxychorismate lyase-like features, indicating their corresponding functions in the H. influenzae. Lyases are actively involved in the regulation of biosynthesis of various hormones, metabolic pathways, signal transduction, and DNA repair. Lyases are also considered as a key player for various biological processes. These enzymes are critically essential for the survival and pathogenesis of H. influenzae and, therefore, these enzymes may be considered as a potential target for structure-based rational drug design. Our structure-function relationship analysis will be useful to search and design potential lead molecules based on the structure of these lyases, for drug design and discovery.

Polygalacturonase production by calcium alginate immobilized Enterobacter aerogenes NBO2 cells.

PubMed

Darah, I; Nisha, M; Lim, Sheh-Hong

2015-03-01

Bacterial cells of Enterobacter aerogenes NBO2 were entrapped in calcium alginate beads in order to enhance polygalacturonase production compared to free cells. The optimized condition of 5 % (w/v) sodium alginate concentration, agitation speed of 250 rpm, and 15 beads of calcium alginate with inoculum size of 4 % (v/v; 5.4 × 10(7) cells/ml) produced 23.48 U/mL of polygalacturonase compared to free cells of 18.54 U/ml. There was about 26.6 % increment in polygalaturonase production. However, in this study, there was 296.6 % of increment in polygalacturonase production after improvement parameters compared to before improvement parameters of calcium alginate bead immobilization cells (5.92 U/ml). This research has indicated that optimized physical parameters of calcium alginate bead immobilization cells have significantly enhanced the production of polygalacturonase.

Alginate-Iron Speciation and Its Effect on In Vitro Cellular Iron Metabolism

PubMed Central

Horniblow, Richard D.; Dowle, Miriam; Iqbal, Tariq H.; Latunde-Dada, Gladys O.; Palmer, Richard E.

2015-01-01

Alginates are a class of biopolymers with known iron binding properties which are routinely used in the fabrication of iron-oxide nanoparticles. In addition, alginates have been implicated in influencing human iron absorption. However, the synthesis of iron oxide nanoparticles employs non-physiological pH conditions and whether nanoparticle formation in vivo is responsible for influencing cellular iron metabolism is unclear. Thus the aims of this study were to determine how alginate and iron interact at gastric-comparable pH conditions and how this influences iron metabolism. Employing a range of spectroscopic techniques under physiological conditions alginate-iron complexation was confirmed and, in conjunction with aberration corrected scanning transmission electron microscopy, nanoparticles were observed. The results infer a nucleation-type model of iron binding whereby alginate is templating the condensation of iron-hydroxide complexes to form iron oxide centred nanoparticles. The interaction of alginate and iron at a cellular level was found to decrease cellular iron acquisition by 37% (p < 0.05) and in combination with confocal microscopy the alginate inhibits cellular iron transport through extracellular iron chelation with the resulting complexes not internalised. These results infer alginate as being useful in the chelation of excess iron, especially in the context of inflammatory bowel disease and colorectal cancer where excess unabsorbed luminal iron is thought to be a driver of disease. PMID:26378798

Evaluation of setting time and flow properties of self-synthesize alginate impressions

NASA Astrophysics Data System (ADS)

Halim, Calista; Cahyanto, Arief; Sriwidodo, Harsatiningsih, Zulia

2018-02-01

Alginate is an elastic hydrocolloid dental impression materials to obtain negative reproduction of oral mucosa such as to record soft-tissue and occlusal relationships. The aim of the present study was to synthesize alginate and to determine the setting time and flow properties. There were five groups of alginate consisted of fifty samples self-synthesize alginate and commercial alginate impression product. Fifty samples were divided according to two tests, each twenty-five samples for setting time and flow test. Setting time test was recorded in the s unit, meanwhile, flow test was recorded in the mm2 unit. The fastest setting time result was in the group three (148.8 s) and the latest was group fours). The highest flow test result was in the group three (69.70 mm2) and the lowest was group one (58.34 mm2). Results were analyzed statistically by one way ANOVA (α= 0.05), showed that there was a statistical significance of setting time while no statistical significance of flow properties between self-synthesize alginate and alginate impression product. In conclusion, the alginate impression was successfully self-synthesized and variation composition gives influence toward setting time and flow properties. The most resemble setting time of control group is group three. The most resemble flow of control group is group four.

Promotive effects of alginate-derived oligosaccharides on the inducing drought resistance of tomato

NASA Astrophysics Data System (ADS)

Liu, Ruizhi; Jiang, Xiaolu; Guan, Huashi; Li, Xiaoxia; Du, Yishuai; Wang, Peng; Mou, Haijin

2009-09-01

In order to determine the role of alginate-derived oligosaccharides (ADO) in drought stress resistance of tomato ( Lycopersicon esculentum Miller) seedlings, the leaves were exposed to different concentrations of ADO (0.05%, 0.10%, 0.20%, 0.30% and 0.50%) after drought stress was simulated by exposing the roots to 0.6 molL-1 PEG-6000 solution for 6 h. Changes in biomass, electrolyte leakage and malondialdehyde (MDA), free proline, total soluble sugars (TSS) and abscisic acid (ABA), the enzyme activities of catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) and phenylalanine ammonia-lyase (PAL) were measured to investigate the effects of ADO treatment. The results showed that the treatment with an ADO concentration of 0.20% exhibited the highest performance of drought stress resistance in the tomato seedlings by decreasing the electrolyte leakage and the concentration of MDA, increasing the contents of free proline, TSS and ABA, and increasing the activities of CAT, SOD, POD and PAL after treatment with ADO. It is suggested that changes in electrolyte leakage, MDA, osmotic solutes, ABA, anti-oxidative enzyme and PAL activities were responsible for the increased drought stress resistance in tomato seedlings. To our best knowledge, this is the first report of the effect of ADO treatment on enhancing the drought stress resistance of tomato seedlings.

Cryptococcus neoformans ADS lyase is an enzyme essential for virulence whose crystal structure reveals features exploitable in antifungal drug design.

PubMed

Chitty, Jessica L; Blake, Kirsten L; Blundell, Ross D; Koh, Y Q Andre E; Thompson, Merinda; Robertson, Avril A B; Butler, Mark S; Cooper, Matthew A; Kappler, Ulrike; Williams, Simon J; Kobe, Bostjan; Fraser, James A

2017-07-14

There is significant clinical need for new antifungal agents to manage infections with pathogenic species such as Cryptococcus neoformans Because the purine biosynthesis pathway is essential for many metabolic processes, such as synthesis of DNA and RNA and energy generation, it may represent a potential target for developing new antifungals. Within this pathway, the bifunctional enzyme adenylosuccinate (ADS) lyase plays a role in the formation of the key intermediates inosine monophosphate and AMP involved in the synthesis of ATP and GTP, prompting us to investigate ADS lyase in C. neoformans. Here, we report that ADE13 encodes ADS lyase in C. neoformans. We found that an ade13 Δ mutant is an adenine auxotroph and is unable to successfully cause infections in a murine model of virulence. Plate assays revealed that production of a number of virulence factors essential for dissemination and survival of C. neoformans in a host environment was compromised even with the addition of exogenous adenine. Purified recombinant C. neoformans ADS lyase shows catalytic activity similar to its human counterpart, and its crystal structure, the first fungal ADS lyase structure determined, shows a high degree of structural similarity to that of human ADS lyase. Two potentially important amino acid differences are identified in the C. neoformans crystal structure, in particular a threonine residue that may serve as an additional point of binding for a fungal enzyme-specific inhibitor. Besides serving as an antimicrobial target, C. neoformans ADS lyase inhibitors may also serve as potential therapeutics for metabolic disease; rather than disrupt ADS lyase, compounds that improve the stability the enzyme may be used to treat ADS lyase deficiency disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The Global Regulators GacA and ςS Form Part of a Cascade That Controls Alginate Production in Azotobacter vinelandii

PubMed Central

Castañeda, Miguel; Sánchez, Judith; Moreno, Soledad; Núñez, Cinthia; Espín, Guadalupe

2001-01-01

Transcription of the Azotobacter vinelandii algD gene, which encodes GDP-mannose dehydrogenase (the rate-limiting enzyme of alginate synthesis), starts from three sites: p1, p2, and p3. The sensor kinase GacS, a member of the two-component regulatory system, is required for transcription of algD from its three sites during the stationary phase. Here we show that algD is expressed constitutively throughout the growth cycle from the p2 and p3 sites and that transcription from p1 started at the transition between the exponential growth phase and stationary phase. We constructed A. vinelandii strains that carried mutations in gacA encoding the cognate response regulator of GacS and in rpoS coding for the stationary-phase ςS factor. The gacA mutation impaired alginate production and transcription of algD from its three promoters. Transcription of rpoS was also abolished by the gacA mutation. The rpoS mutation impaired transcription of algD from the p1 promoter and increased it from the p2 ςE promoter. The results of this study provide evidence for the predominant role of GacA in a regulatory cascade controlling alginate production and gene expression during the stationary phase in A. vinelandii. PMID:11698366

Fabrication of micro-alginate gel tubes utilizing micro-gelatin fibers

NASA Astrophysics Data System (ADS)

Sakaguchi, Katsuhisa; Arai, Takafumi; Shimizu, Tatsuya; Umezu, Shinjiro

2017-05-01

Tissues engineered utilizing biofabrication techniques have recently been the focus of much attention, because these bioengineered tissues have great potential to improve the quality of life of patients with various hard-to-treat diseases. Most tissues contain micro-tubular structures including blood vessels, lymphatic vessels, and bile canaliculus. Therefore, we bioengineered a micro diameter tube using alginate gel to coat the core gelatin gel. Micro-gelatin fibers were fabricated by the coacervation method and then coated with a very thin alginate gel layer by dipping. A micro diameter alginate tube was produced by dissolving the core gelatin gel. Consequently, these procedures led to the formation of micro-alginate gel tubes of various shapes and sizes. This biofabrication technique should contribute to tissue engineering research fields.

Effect of Ammonia Production by Colletotrichum gloeosporioides on pelB Activation, Pectate Lyase Secretion, and Fruit Pathogenicity

PubMed Central

Kramer-Haimovich, H.; Servi, E.; Katan, T.; Rollins, J.; Okon, Y.; Prusky, D.

2006-01-01

The accumulation of ammonia and associated tissue alkalinization predispose avocado fruit to attack by Colletotrichum gloeosporioides. Secretion of ammonia by C. gloeosporioides in the presence of KNO3 was induced by decreasing the pH from 7.0 to 4.0. When the fungus was grown at pH 4.0 or 6.0 in the absence of a nitrogen source, ammonia did not accumulate, and neither pelB (encoding pectate lyase) transcription nor pectate lyase secretion was detected. Under these nitrogen starvation conditions, only transcriptional activation of areA, which encodes the global nitrogen regulator, was detected. pelB transcription and pectate lyase secretion were both detected when C. gloeosporioides was grown at pH 6.0 in the presence of ammonia accumulated from different nitrogen sources. The early accumulation of ammonia induced early pelB expression and pectate lyase secretion. As the external pH increased from 4.0 to 6.0, transcripts of pac1, the C. gloeosporioides pacC homolog, also could be detected. Nit mutants of C. gloeosporioides, which cannot utilize KNO3 as a nitrogen source, did not secrete ammonia, alkalinize the medium, or secrete pectate lyase. If Nit mutants were grown at pH 6.0 in the presence of glutamate, then pectate lyase secretion was induced. Infiltration of 0.1 M ammonium hydroxide at pH 10 into ripening avocado fruits enhanced the activation of quiescent infection and symptom development by C. gloeosporioides. These results suggest that ambient pH alkalinization resulting from ammonia accumulation and the availability of ammonia as a nitrogen source independently regulate pelB expression, pectate lyase secretion, and virulence of C. gloeosporioides. These data suggest that alkalinization during C. gloeosporioides infection is important for its transformation from the quiescent biotrophic stage to the necrotrophic stage of fungal colonization in the fruit host. PMID:16461646

Floating dosage forms to prolong gastro-retention--the characterisation of calcium alginate beads.

PubMed

Stops, Frances; Fell, John T; Collett, John H; Martini, Luigi G

2008-02-28

Floating calcium alginate beads, designed to improve drug bioavailability from oral preparations compared with that from many commercially available and modified release products, have been investigated as a possible gastro-retentive dosage form. A model drug, riboflavin, was also incorporated into the formula. The aims of the current work were (a) to obtain information regarding the structure, floating ability and changes that occurred when the dosage form was placed in aqueous media, (b) to investigate riboflavin release from the calcium alginate beads in physiologically relevant media prior to in vivo investigations. Physical properties of the calcium alginate beads were investigated. Using SEM and ESEM, externally the calcium alginate beads were spherical in shape, and internally, air filled cavities were present thereby enabling floatation of the beads. The calcium alginate beads remained buoyant for times in excess of 13h, and the density of the calcium alginate beads was <1.000gcm(-3). Riboflavin release from the calcium alginate beads showed that riboflavin release was slow in acidic media, whilst in more alkali media, riboflavin release was more rapid. The characterisation studies showed that the calcium alginate beads could be considered as a potential gastro-retentive dosage form.

In vivo evaluation of EPO-secreting cells immobilized in different alginate-PLL microcapsules.

PubMed

Ponce, S; Orive, G; Hernández, R M; Gascón, A R; Canals, J M; Muñoz, M T; Pedraz, J L

2006-11-01

Alginates are the most employed biomaterials for cell encapsulation due to their abundance, easy gelling properties and apparent biocompatibility. However, as natural polymers different impurities including endotoxins, proteins and polyphenols can be found in their composition. Several purification protocols as well as different batteries of assays to prove the biocompatibility of the alginates in vitro have been recently developed. However, little is known about how the use of alginates with different purity grade may affect the host immune response after their implantation in vivo. The present paper investigates the long-term functionality and biocompatibility of murine erythropoietin (EPO) secreting C2C12 cells entrapped in microcapsules elaborated with alginates with different properties (purity, composition and viscosity). Results showed that independently of the alginate type employed, the animals presented elevated hematocrit levels until day 130, remaining at values between 70-87%. However, histological analysis of the explanted devices showed higher overgrowth around non-biomedical grade alginate microcapsules which could be directly related with higher impurity content of this type of alginate. Although EPO delivery may be limited by the formation of a fibrotic layer around non-biomedical grade alginate microcapsules, the high EPO secretion of the encapsulated cells together with the pharmacodynamic behaviour and the angiogenic and immune-modulatory properties of EPO result in no direct correlation between the biocompatibility of the alginate and the therapeutic response obtained.

Preparation of aminated chitosan/alginate scaffold containing halloysite nanotubes with improved cell attachment.

PubMed

Amir Afshar, Hamideh; Ghaee, Azadeh

2016-10-20

The chemical nature of biomaterials play important role in cell attachment, proliferation and migration in tissue engineering. Chitosan and alginate are biodegradable and biocompatible polymers used as scaffolds for various medical and clinical applications. Amine groups of chitosan scaffolds play an important role in cell attachment and water adsorption but also associate with alginate carboxyl groups via electrostatic interactions and hydrogen bonding, consequently the activity of amine groups in the scaffold decreases. In this study, chitosan/alginate/halloysite nanotube (HNTs) composite scaffolds were prepared using a freeze-drying method. Amine treatment on the scaffold occurred through chemical methods, which in turn caused the hydroxyl groups to be replaced with carboxyl groups in chitosan and alginate, after which a reaction between ethylenediamine, 1-ethyl-3,(3-dimethylaminopropyl) carbodiimide (EDC) and scaffold triggered the amine groups to connect to the carboxyl groups of chitosan and alginate. The chemical structure, morphology and mechanical properties of the composite scaffolds were investigated by FTIR, CHNS, SEM/EDS and compression tests. The electrostatic attraction and hydrogen bonding between chitosan, alginate and halloysite was confirmed by FTIR spectroscopy. Chitosan/alginate/halloysite scaffolds exhibit significant enhancement in compressive strength compared with chitosan/alginate scaffolds. CHNS and EDS perfectly illustrate that amine groups were effectively introduced in the aminated scaffold. The growth and cell attachment of L929 cells as well as the cytotoxicity of the scaffolds were investigated by SEM and Alamar Blue (AB). The results indicated that the aminated chitosan/alginate/halloysite scaffold has better cell growth and cell adherence in comparison to that of chitosan/alginate/halloysite samples. Aminated chitosan/alginate/halloysite composite scaffolds exhibit great potential for applications in tissue engineering, ideally in

Encapsulation optimization of lemon balm antioxidants in calcium alginate hydrogels.

PubMed

Najafi-Soulari, Samira; Shekarchizadeh, Hajar; Kadivar, Mahdi

2016-11-01

Calcium alginate hydrogel beads were used to encapsulate lemon balm extract. Chitosan layer was used to investigate the effect of hydrogel coating. To determine the interactions of antioxidant compounds of extract with encapsulation materials and its stability, microstructure of hydrogel beads was thoroughly monitored using scanning electron microscopy and Fourier transform infrared (FTIR). Total polyphenols content and antiradical activity of lemon balm extract were also evaluated before and after encapsulation. Three significant parameters (lemon balm extract, sodium alginate, and calcium chloride concentrations) were optimized by response surface methodology to obtain maximum encapsulation efficiency. The FTIR spectra showed no interactions between extract and polymers as there were no new band in spectra of alginate hydrogel after encapsulation of active compounds of lemon balm extract. The antioxidant activity of lemon balm extract did not change after encapsulation. Therefore, it was found that alginate is a suitable material for encapsulation of natural antioxidants. Sodium alginate solution concentration, 1.84%, lemon balm extract concentration, 0.4%, and calcium chloride concentration, 0.2% was determined to be the optimum condition to reach maximum encapsulation efficiency.

Utilization of Glyphosate as Phosphate Source: Biochemistry and Genetics of Bacterial Carbon-Phosphorus Lyase

PubMed Central

Zechel, David L.; Jochimsen, Bjarne

2014-01-01

SUMMARY After several decades of use of glyphosate, the active ingredient in weed killers such as Roundup, in fields, forests, and gardens, the biochemical pathway of transformation of glyphosate phosphorus to a useful phosphorus source for microorganisms has been disclosed. Glyphosate is a member of a large group of chemicals, phosphonic acids or phosphonates, which are characterized by a carbon-phosphorus bond. This is in contrast to the general phosphorus compounds utilized and metabolized by microorganisms. Here phosphorus is found as phosphoric acid or phosphate ion, phosphoric acid esters, or phosphoric acid anhydrides. The latter compounds contain phosphorus that is bound only to oxygen. Hydrolytic, oxidative, and radical-based mechanisms for carbon-phosphorus bond cleavage have been described. This review deals with the radical-based mechanism employed by the carbon-phosphorus lyase of the carbon-phosphorus lyase pathway, which involves reactions for activation of phosphonate, carbon-phosphorus bond cleavage, and further chemical transformation before a useful phosphate ion is generated in a series of seven or eight enzyme-catalyzed reactions. The phn genes, encoding the enzymes for this pathway, are widespread among bacterial species. The processes are described with emphasis on glyphosate as a substrate. Additionally, the catabolism of glyphosate is intimately connected with that of aminomethylphosphonate, which is also treated in this review. Results of physiological and genetic analyses are combined with those of bioinformatics analyses. PMID:24600043

Expression and Properties of the Highly Alkalophilic Phenylalanine Ammonia-Lyase of Thermophilic Rubrobacter xylanophilus

PubMed Central

Kovács, Klaudia; Bánóczi, Gergely; Varga, Andrea; Szabó, Izabella; Holczinger, András; Hornyánszky, Gábor; Zagyva, Imre

2014-01-01

The sequence of a phenylalanine ammonia-lyase (PAL; EC: 4.3.1.24) of the thermophilic and radiotolerant bacterium Rubrobacter xylanophilus (RxPAL) was identified by screening the genomes of bacteria for members of the phenylalanine ammonia-lyase family. A synthetic gene encoding the RxPAL protein was cloned and overexpressed in Escherichia coli TOP 10 in a soluble form with an N-terminal His6-tag and the recombinant RxPAL protein was purified by Ni-NTA affinity chromatography. The activity assay of RxPAL with l-phenylalanine at various pH values exhibited a local maximum at pH 8.5 and a global maximum at pH 11.5. Circular dichroism (CD) studies showed that RxPAL is associated with an extensive α-helical character (far UV CD) and two distinctive near-UV CD peaks. These structural characteristics were well preserved up to pH 11.0. The extremely high pH optimum of RxPAL can be rationalized by a three-dimensional homology model indicating possible disulfide bridges, extensive salt-bridge formation and an excess of negative electrostatic potential on the surface. Due to these properties, RxPAL may be a candidate as biocatalyst in synthetic biotransformations leading to unnatural l- or d-amino acids or as therapeutic enzyme in treatment of phenylketonuria or leukemia. PMID:24475062

The influence of storage duration on the setting time of type 1 alginate impression material

NASA Astrophysics Data System (ADS)

Rahmadina, A.; Triaminingsih, S.; Irawan, B.

2017-08-01

Alginate is one of the most commonly used dental impression materials; however, its setting time is subject to change depending on storage conditions and duration. This creates problems because consumer carelessness can affect alginate shelf life and quality. In the present study, the setting times of two groups of type I alginate with different expiry dates was tested. The first group consisted of 11 alginate specimens that had not yet passed the expiry date, and the second group consisted of alginates that had passed the expiry date. The alginate powder was mixed with distilled water, poured into a metal ring, and tested with a polished rod of poly-methyl methacrylate. Statistical analysis showed a significant difference (p<0.05) between the setting times of the alginate that had not passed the expiry date (157 ± 3 seconds) and alginate that had passed the expiry date (144 ± 2 seconds). These findings indicate that storage duration can affect alginate setting time.

Lactic acid bacteria involved in cocoa beans fermentation from Ivory Coast: Species diversity and citrate lyase production.

PubMed

Ouattara, Hadja D; Ouattara, Honoré G; Droux, Michel; Reverchon, Sylvie; Nasser, William; Niamke, Sébastien L

2017-09-01

Microbial fermentation is an indispensable process for high quality chocolate from cocoa bean raw material. lactic acid bacteria (LAB) are among the major microorganisms responsible for cocoa fermentation but their exact role remains to be elucidated. In this study, we analyzed the diversity of LAB in six cocoa producing regions of Ivory Coast. Ribosomal 16S gene sequence analysis showed that Lactobacillus plantarum and Leuconostoc mesenteroides are the dominant LAB species in these six regions. In addition, other species were identified as the minor microbial population, namely Lactobacillus curieae, Enterococcus faecium, Fructobacillus pseudoficulneus, Lactobacillus casei, Weissella paramesenteroides and Weissella cibaria. However, in each region, the LAB microbial population was composed of a restricted number of species (maximum 5 species), which varied between the different regions. LAB implication in the breakdown of citric acid was investigated as a fundamental property for a successful cocoa fermentation process. High citrate lyase producer strains were characterized by rapid citric acid consumption, as revealed by a 4-fold decrease in citric acid concentration in the growth medium within 12h, concomitant with an increase in acetic acid and lactic acid concentration. The production of citrate lyase was strongly dependent on environmental conditions, with optimum production at acidic pH (pH<5), and moderate temperature (30-40°C), which corresponds to conditions prevailing in the early stage of natural cocoa fermentation. This study reveals that one of the major roles of LAB in the cocoa fermentation process involves the breakdown of citric acid during the early stage of cocoa fermentation through the activity of citrate lyase. Copyright © 2017 Elsevier B.V. All rights reserved.

Three-dimensional alginate spheroid culture system of murine osteosarcoma.

PubMed

Akeda, Koji; Nishimura, Akinobu; Satonaka, Haruhiko; Shintani, Ken; Kusuzaki, Katsuyuki; Matsumine, Akihiko; Kasai, Yuichi; Masuda, Koichi; Uchida, Atsumasa

2009-11-01

Osteosarcoma (OS) is the most common primary malignant tumor of the bone and often forms pulmonary metastases, which are the most important prognostic factor. For further elucidation of the mechanism underlying the progression and metastasis of human OS, a culture system mimicking the microenvironment of the tumor in vivo is needed. We report a novel three-dimensional (3D) alginate spheroid culture system of murine osteosarcoma. Two different metastatic clones, the parental Dunn and its derivative line LM8, which has a higher metastatic potential to the lungs, were encapsulated in alginate beads to develop the 3D culture system. The beads containing murine OS cells were also transplanted into mice to determine their metastatic potential in vivo. In this culture system, murine OS cells encapsulated in alginate beads were able to grow in a 3D structure with cells detaching from the alginate environment. The number of detaching cells was higher in the LM8 cell line than the Dunn cell line. In the in vivo alginate bead transplantation model, the rate of pulmonary metastasis was higher with LM8 cells compared with that of Dunn cells. The cell characteristics and kinetics in this culture system closely reflect the original malignant potential of the cells in vivo.

Magneto-responsive alginate capsules.

PubMed

Degen, Patrick; Zwar, Elena; Schulz, Imke; Rehage, Heinz

2015-05-20

Upon incorporation of magnetic nanoparticles (mNPs) into gels, composite materials called ferrogels are obtained. These magneto-responsive systems have a wide range of potential applications including switches and sensors as well as drug delivery systems. In this article, we focus on the properties of calcium alginate capsules, which are widely used as carrier systems in medicine and technology. We studied the incorporation of different kinds of mNPs in matrix capsules and in the core and the shell of hollow particles. We found out that not all particle-alginate or particle-CaCl2 solution combinations were suitable for a successful capsule preparation on grounds of a destabilization of the nanoparticles or the polymer. For those systems allowing the preparation of switchable beads or capsules, we systematically studied the size and microscopic structure of the capsules, their magnetic behavior and mechanical resistance.

Identification of one of the apurinic/apyrimidinic lyase active sites of topoisomerase V by structural and functional studies

PubMed Central

Rajan, Rakhi; Prasad, Rajendra; Taneja, Bhupesh; Wilson, Samuel H.; Mondragón, Alfonso

2013-01-01

Topoisomerase V (Topo-V) is the only member of a novel topoisomerase subtype. Topo-V is unique because it is a bifunctional enzyme carrying both topoisomerase and DNA repair lyase activities within the same protein. Previous studies had shown that the topoisomerase domain spans the N-terminus of the protein and is followed by 12 tandem helix–hairpin–helix [(HhH)2] domains. There are at least two DNA repair lyase active sites for apurinic/apyrimidinic (AP) site processing, one within the N-terminal region and the second within the C-terminal domain of Topo-V, but their exact locations and characteristics are unknown. In the present study, the N-terminal 78-kDa fragment of Topo-V (Topo-78), containing the topoisomerase domain and one of the lyase DNA repair domains, was characterized by structural and biochemical studies. The results show that an N-terminal 69-kDa fragment is the minimal fragment with both topoisomerase and AP lyase activities. The lyase active site of Topo-78 is at the junction of the fifth and sixth (HhH)2 domains. From the biochemical and structural data, it appears that Lys571 is the most probable nucleophile responsible for the lyase activity. Our experiments also suggest that Topo-V most likely acts as a Class I AP endonuclease in vivo. PMID:23125368

Enzymatic saccharification of brown seaweed for production of fermentable sugars.

PubMed

Sharma, Sandeep; Horn, Svein Jarle

2016-08-01

This study shows that high drying temperatures negatively affect the enzymatic saccharification yield of the brown seaweed Saccharina latissima. The optimal drying temperature of the seaweed in terms of enzymatic sugar release was found to be 30°C. The enzymatic saccharification process was optimized by investigating factors such as kinetics of sugar release, enzyme dose, solid loading and different blend ratios of cellulases and an alginate lyase. It was found that the seaweed biomass could be efficiently hydrolysed to fermentable sugars using a commercial cellulase cocktail. The inclusion of a mono-component alginate lyase was shown to improve the performance of the enzyme blend, in particular at high solid loadings. At 25% dry matter loading a combined glucose and mannitol concentration of 74g/L was achieved. Copyright © 2016 Elsevier Ltd. All rights reserved.

Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy

PubMed Central

Rolando, Monica; Escoll, Pedro; Nora, Tamara; Botti, Joëlle; Boitez, Valérie; Daniels, Craig; Abraham, Gilu; Stogios, Peter J.; Skarina, Tatiana; Christophe, Charlotte; Dervins-Ravault, Delphine; Cazalet, Christel; Hilbi, Hubert; Rupasinghe, Thusitha W. T.; Tull, Dedreia; McConville, Malcolm J.; Ong, Sze Ying; Hartland, Elizabeth L.; Codogno, Patrice; Levade, Thierry; Naderer, Thomas; Savchenko, Alexei; Buchrieser, Carmen

2016-01-01

Autophagy is an essential component of innate immunity, enabling the detection and elimination of intracellular pathogens. Legionella pneumophila, an intracellular pathogen that can cause a severe pneumonia in humans, is able to modulate autophagy through the action of effector proteins that are translocated into the host cell by the pathogen’s Dot/Icm type IV secretion system. Many of these effectors share structural and sequence similarity with eukaryotic proteins. Indeed, phylogenetic analyses have indicated their acquisition by horizontal gene transfer from a eukaryotic host. Here we report that L. pneumophila translocates the effector protein sphingosine-1 phosphate lyase (LpSpl) to target the host sphingosine biosynthesis and to curtail autophagy. Our structural characterization of LpSpl and its comparison with human SPL reveals high structural conservation, thus supporting prior phylogenetic analysis. We show that LpSpl possesses S1P lyase activity that was abrogated by mutation of the catalytic site residues. L. pneumophila triggers the reduction of several sphingolipids critical for macrophage function in an LpSpl-dependent and -independent manner. LpSpl activity alone was sufficient to prevent an increase in sphingosine levels in infected host cells and to inhibit autophagy during macrophage infection. LpSpl was required for efficient infection of A/J mice, highlighting an important virulence role for this effector. Thus, we have uncovered a previously unidentified mechanism used by intracellular pathogens to inhibit autophagy, namely the disruption of host sphingolipid biosynthesis. PMID:26831115

Enzyme-entrapping behaviors in alginate fibers and their papers

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

Kobayashi, Y.; Matsuo, R.; Ohya, T.

1987-01-01

Enzyme immobilization in the form of fiber and paper was easily achieved by wet spinning of aqueous admixture of sodium alginate and enzymes into divalent metallic ion solution as a coagulating bath, followed by paper making of resultant shortly cut fibers. Entrapment yields of enzymes used, e.g., glucoamylase, cyclodextrin glucanotransferase, endo-polygalacturonase, and protease, were always higher in calcium alginate fibers and their papers than those in corresponding beads. It was found that the yields increased with an increase of the discharge rate through the spinning nozzle because the higher discharge rate could provide more highly oriented metal-chelate linear polymer moleculesmore » along the fiber axis for preventing leakage of entrapped enzymes. Divalent metallic ions affected greatly the entrapment of glucoamylase in alginate fibers, the order of which followed rougly the ionotropic series of Thiele. Entrapment of glucoamylase in bicomponent systems comprising alginate and other water-soluble polymers was also investigated. (Refs. 41).« less

Fundamental Characteristics of Bioprint on Calcium Alginate Gel

NASA Astrophysics Data System (ADS)

Umezu, Shinjiro; Hatta, Tatsuru; Ohmori, Hitoshi

2013-05-01

The goal of this study is to fabricate precision three-dimensional (3D) biodevices those are micro fluidics and artificial organs utilizing digital fabrication. Digital fabrication is fabrication method utilizing inkjet technologies. Electrostatic inkjet is one of the inkjet technologies. The electrostatic inkjet method has following two merits; those are high resolution to print and ability to eject highly viscous liquid. These characteristics are suitable to print biomaterials precisely. We are now applying for bioprint. In this paper, the electrostatic inkjet method is applied for fabrication of 3D biodevices that has cave like blood vessel. When aqueous solution of sodium alginate is printed to aqueous solution of calcium chloride, calcium alginate is produced. 3D biodevices are fabricated in case that calcium alginate is piled.

Adenylosuccinate lyase (ADSL) and infantile autism: Absence of previously reported point mutation

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

Fon, E.A.; Sarrazin, J.; Rouleau, G.A.

Autism is a heterogeneous neuropsychiatric syndrome of unknown etiology. There is evidence that a deficiency in the enzyme adenylosuccinate lyase (ADSL), essential for de novo purine biosynthesis, could be involved in the pathogenesis of certain cases. A point mutation in the ADSL gene, resulting in a predicted serine-to-proline substitution and conferring structural instability to the mutant enzyme, has been reported previously in 3 affected siblings. In order to determine the prevalence of the mutation, we PCR-amplified the exon spanning the site of this mutation from the genomic DNA of patients fulfilling DSM-III-R criteria for autistic disorder. None of the 119more » patients tested were found to have this mutation. Furthermore, on preliminary screening using single-strand conformation polymorphism (SSCP), no novel mutations were detected in the coding sequence of four ADSL exons, spanning approximately 50% of the cDNA. In light of these findings, it appears that mutations in the ADSL gene represent a distinctly uncommon cause of autism. 12 refs., 2 figs.« less

Developmental role of phenylalanine-ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) genes during adventitious rooting of Juglans regia L. microshoots.

PubMed

Cheniany, Monireh; Ganjeali, Ali

2016-12-01

Phenylalanine-ammonia-lyase and cinnamate-4-hydroxylase play important role in the phenylpropanoid pathway, which produces many biologically important secondary metabolites participating in normal plant development. Flavonol quercetin is the main representant of these compounds that has been identified in numerous Juglans spp. In this survey, the developmental expression patterns of PAL and C4H genes during in vitro rooting of two walnut cultivars 'Sunland' and 'Howard' was examined by RT-PCR. To understand the potential role in rooting, the changing pattern of endogenous content of quercetin was also analyzed by HPLC. The 'Sunland' with better capacity to root had more quercetin content during the "inductive phase" of rooting than 'Howard'. In each cultivar, the level of PAL transcripts showed the same behavior with the changing patterns of quercetin during root formation of microshoots. The positive correlation between the changes of quercetin and PAL-mRNA indicated that PAL gene may have an immediate effect on flavonoid pathway metabolites including quercetin. Although the behavioral change of C4H expression was similar in both cultivars during root formation (with significantly more level for 'Howard'), it was not coincide with the changes of quercerin concentrations. Our results showed that C4H function is important for the normal development, but its transcriptional regulation does not correlate with quercetin as an efficient phenolic compound for walnut rhizogenesis.

Benzaldehyde lyase, a novel thiamine PPi-requiring enzyme, from Pseudomonas fluorescens biovar I.

PubMed Central

González, B; Vicuña, R

1989-01-01

Pseudomonas fluorescens biovar I can grow on benzoin as the sole carbon and energy source. This ability is due to benzaldehyde lyase, a new type of enzyme that irreversibly cleaves the acyloin linkage of benzoin, producing two molecules of benzaldehyde. Benzaldehyde lyase was purified 70-fold and found to require catalytic amounts of thiamine PPi (TPP) and a divalent cation as cofactors. Optimal activity was obtained with a 1.0 mM concentration of Mn2+, Mg2+, or Ca2+. Gel permeation chromatography indicated a native molecular weight of 80,000, whereas the enzyme migrated in sodium dodecyl sulfate-containing polyacrylamide gels as a single polypeptide with a molecular weight of 53,000. Benzaldehyde lyase is highly specific; of a variety of structurally related compounds tested, only benzoin and anisoin (4,4'-dimethoxybenzoin) acted as substrates, their apparent Kms being 9.0 x 10(-3) and 3.25 x 10(-2) mM, respectively. A catalytic mechanism for the enzyme is proposed. Images PMID:2496105

Review: peripheral nerve regeneration using non-tubular alginate gel crosslinked with covalent bonds.

PubMed

Hashimoto, Tadashi; Suzuki, Yoshihisa; Suzuki, Kyoko; Nakashima, Toshihide; Tanihara, Masao; Ide, Chizuka

2005-06-01

We have developed a nerve regeneration material consisting of alginate gel crosslinked with covalent bonds. in the first part of this study, we attempted to analyze nerve regeneration through alginate gel in the early stages within 2 weeks. in the second part, we tried to regenerate cat peripheral nerve by using alginate tubular or non-tubular nerve regeneration devices, and compared their efficacies. Four days after surgery, regenerating axons grew without Schwann cell investment through the partially degraded alginate gel, being in direct contact with the alginate without a basal lamina covering. One to 2 weeks after surgery, regenerating axons were surrounded by common Schwann cells, forming small bundles, with some axons at the periphery being partly in direct contact with alginate. At the distal stump, numerous Schwann cells had migrated into the alginate 8-14 days after surgery. Remarkable restorations of the 50-mm gap in cat sciatic nerve were obtained after a long term by using tubular or non-tubular nerve regeneration material consisting mainly of alginate gel. However, there was no significant difference between both groups at electrophysiological and morphological evaluation. Although, nowadays, nerve regeneration materials being marketed mostly have a tubular structure, our results suggest that the tubular structure is not indispensable for peripheral nerve regeneration.

Selective digestion of Ba2+/Ca2+ alginate gel microdroplets for single-cell handling

NASA Astrophysics Data System (ADS)

Odaka, Masao; Hattori, Akihiro; Matsuura, Kenji; Yasuda, Kenji

2018-06-01

Cells encapsuled by polymer microdroplets are an effective platform for the identification and separation of individual cells for single-cell-based analysis. However, a key challenge is to maintain and release the captured cells in the microdroplets selectively, nondestructively, and noninvasively. We developed a simple method of encapsulating cells in alginate microdroplets having different digestion characteristics. Cells were diluted with an alginate polymer of sol state and encapsulated into microdroplets with Ba2+ and Ca2+ by a spray method. When a chelating buffer was applied, alginate gel microdroplets were digested according to the difference in chelating efficiency of linkage-divalent cations; hence, two types of alginate microdroplets were formed. Moreover, we examined the capability of the alginate gel to exchange linkage-divalent cations and found that both Ca2+ exchange in Ba-alginate microdroplets and Ba2+ exchange in Ca-alginate microdroplets occurred. These results indicate that the potential applications of a mixture of alginate microdroplets with different divalent cations control the selective digestion of microdroplets to improve the high-throughput, high-content microdroplet-based separation, analysis, or storage of single cells.

Host-Pathogen interactions. 25. Endopolygalacturonic acid lyase from Erwinia carotovora elicits phytoalexin accumulation by releasing plant cell wall fragments

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

Davis, K.R.; Lyon, G.D.; Darvill, A.G.

1984-01-01

Heat-labile elicitors of phytoalexin accumulation in soybeans (Glycine max L. Merr. cv Wayne) were detected in culture filtrates of Erwinia carotovora grown on a defined medium containing citrus pectin as the sole carbon source. The heat-labile elicitors were highly purified by cation-exchange chromatography on a CM-Sephadex (C-50) column, followed by agarose-affinity chromatography on a Bio-Gel A-0.5m gel filtration column. The heat-labile elicitor activity co-purified with two ..cap alpha..-1,4-endopolygalacturonic acid lyases (EC 4 x 2 x 2 x 2). Endopolygalacturonic acid lyase activity appeared to be necessary for elicitor activity because heat-inactivated enzyme preparations did not elicit phytoalexins. The purified endopolygalacturonicmore » acid lyases elicited pterocarpan phytoalexins at microbial-inhibitory concentrations in the soybean-cotyledon bioassay when applied at a concentration of 55 nanograms per milliliter (1 x 10/sup -9/ molar). One of these lyases released heat-stable elicitors from soybean cell walls, citrus pectin, and sodium polypectate. The heat-stable elicitor-active material solubilized from soybean cell walls by the lyase was composed of at least 90% (w/v) uronosyl residues. These results demonstrate that endopolygalacturonic acid lyase elicits phytoalexin accumulation by releasing fragments from pectic polysaccharides in plant cell walls.« less

Identification of amino acid residues involved in the dRP-lyase activity of human Pol ι.

PubMed

Miropolskaya, Nataliya; Petushkov, Ivan; Kulbachinskiy, Andrey; Makarova, Alena V

2017-08-31

Besides X-family DNA polymerases (first of all, Pol β) several other human DNA polymerases from Y- and A- families were shown to possess the dRP-lyase activity and could serve as backup polymerases in base excision repair (Pol ι, Rev1, Pol γ and Pol θ). However the exact position of the active sites and the amino acid residues involved in the dRP-lyase activity in Y- and A- family DNA polymerases are not known. Here we carried out functional analysis of fifteen amino acid residues possibly involved in the dRP-lyase activity of human Pol ι. We show that substitutions of residues Q59, K60 and K207 impair the dRP-lyase activity of Pol ι while residues in the HhH motif of the thumb domain are dispensable for this activity. While both K60G and K207A substitutions decrease Schiff-base intermediate formation during dRP group cleavage, the latter substitution also strongly affects the DNA polymerase activity of Pol ι, suggesting that it may impair DNA binding. These data are consistent with an important role of the N-terminal region in the dRP-lyase activity of Pol ι, with possible involvement of residues from the finger domain in the dRP group cleavage.

Effect of gamma radiation on the physico-chemical properties of alginate-based films and beads

NASA Astrophysics Data System (ADS)

Huq, Tanzina; Khan, Avik; Dussault, Dominic; Salmieri, Stephane; Khan, Ruhul A.; Lacroix, Monique

2012-08-01

Alginate solution (3%, w/v) was prepared using deionized water from its powder. Then the solution was exposed to gamma radiation (0.1-25 kGy). The alginate films were prepared by solution casting. It was found that gamma radiation has strong effect on alginate solution. At low doses, mechanical strength of the alginate films improved but after 5 kGy dose, the strength started to decrease. The mechanism of alginate radiolysis in aqueous solution is discussed. Film formation was not possible from alginate solution at doses >5 kGy. The mechanical properties such as puncture strength (PS), puncture deformation (PD), viscoelasticity (Y) coefficient of the un-irradiated films were investigated. The values of PS, PD and Y coefficient of the films were 333 N/mm, 3.20 mm and 27%, respectively. Alginate beads were prepared from 3% alginate solution (w/v) by ionotropic gelation method in 5% CaCl2 solution. The rate of gel swelling improved in irradiated alginate-based beads at low doses (up to 0.5 kGy).

Preparation, characterisation and viability of encapsulated Trichoderma harzianum UPM40 in alginate-montmorillonite clay.

PubMed

Adzmi, Fariz; Meon, Sariah; Musa, Mohamed Hanafi; Yusuf, Nor Azah

2012-01-01

Microencapsulation is a process by which tiny parcels of an active ingredient are packaged within a second material for the purpose of shielding the active ingredient from the surrounding environment. This study aims to determine the ability of the microencapsulation technique to improve the viability of Trichoderma harzianum UPM40 originally isolated from healthy groundnut roots as effective biological control agents (BCAs). Alginate was used as the carrier for controlled release, and montmorillonite clay (MMT) served as the filler. The encapsulated Ca-alginate-MMT beads were characterised using Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). The FTIR results showed the interaction between the functional groups of alginate and MMT in the Ca-alginate-MMT beads. Peaks at 1595, 1420 and 1020 cm(-1) characterised alginate, and peaks at 1028 and 453 cm(-1) characterised MMT; both sets of peaks appeared in the Ca-alginate-MMT FTIR spectrum. The TGA analysis showed an improvement in the thermal stability of the Ca-alginate-MMT beads compared with the alginate beads alone. SEM analysis revealed a homogeneous distribution of the MMT particles throughout the alginate matrix. T. harzianum UPM40 was successfully encapsulated in the Ca-alginate-MMT beads. Storage analysis of the encapsulated T. harzianum UPM40 showed that the low storage temperature of 5°C resulted in significantly (p < 0.05) better storage compared with room temperature (30°C).

Bilateral PLA/alginate membranes for the prevention of postsurgical adhesions.

PubMed

Kessler, Martina; Esser, Eva; Groll, Jürgen; Tessmar, Jörg

2016-11-01

A bilateral barrier membrane for the prevention of postsurgical adhesions was developed. Thereby, a smooth PLA side was supposed to keep the affected tissues glidingly separated, while a mucoadhesive side made of alginate was meant to keep the barrier resident on the site of injury so that suturing becomes redundant or at least the membrane stays long enough to facilitate surgical handling. Because hydrophilic alginate and lipophilic PLA films show only low cohesion, solution electrospun meshes of PLA and PLA-PEG-PLA triblock copolymers with varying poly(ethylene glycol) [PEG] content were investigated as cohesion promoter to avoid an easy separation of the functionally different layers. Using direct electrospinning onto the PLA film, a modified contact surface of the mesh was created, which allowed the tested alginate solutions (3%, 5%) to infiltrate to different extents. Thereby, an increasing content of hydrophilic PEG within the mesh copolymer and a lower alginate concentration facilitated the infiltration. As a result, the PLA film with a PLA35k-PEG10k-PLA35k (racemic PLA chains) mesh and an alginate layer cast from a 3% alginate solution appeared to be the most effective combination as examined by means of a t peel test, a mucoadhesion test, a tensile test and optical evaluations. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1563-1570, 2016. © 2015 Wiley Periodicals, Inc.

Silk sericin loaded alginate nanoparticles: Preparation and anti-inflammatory efficacy.

PubMed

Khampieng, Thitikan; Aramwit, Pornanong; Supaphol, Pitt

2015-09-01

In this study, silk sericin loaded alginate nanoparticles were prepared by the emulsification method followed by internal crosslinking. The effects of various silk sericin loading concentration on particle size, shape, thermal properties, and release characteristics were investigated. The initial silk sericin loadings of 20, 40, and 80% w/w to polymer were incorporated into these alginate nanoparticles. SEM images showed a spherical shape and small particles of about 71.30-89.50 nm. TGA analysis showed that thermal stability slightly increased with increasing silk sericin loadings. FTIR analysis suggested interactions between alginate and silk sericin in the nanoparticles. The release study was performed in acetate buffer at normal skin conditions (pH 5.5; 32 °C). The release profiles of silk sericin exhibited initial rapid release, consequently with sustained release. These silk sericin loaded alginate nanoparticles were further incorporated into topical hydrogel and their anti-inflammatory properties were studied using carrageenan-induced paw edema assay. The current study confirms the hypothesis that the application of silk sericin loaded alginate nanoparticle gel can inhibit inflammation induced by carrageenan. Copyright © 2015 Elsevier B.V. All rights reserved.

Purification and Kinetic Properties of Serine Acetyltransferase Free of O-Acetylserine(thiol)lyase from Spinach Chloroplasts.

PubMed

Ruffet, M. L.; Droux, M.; Douce, R.

1994-02-01

Serine acetyltransferase, a key enzyme in the L-cysteine biosynthetic pathway, was purified over 300,000-fold from the stroma of spinach (Spinacia oleracea) leaf chloroplasts. The purification procedure consisted of ammonium sulfate precipitation, anion-exchange chromatography (Trisacryl M DEAE and Mono Q HR10/10), hydroxylapatite chromatography, and gel filtration (Superdex 200). The purified enzyme exhibited a specific activity higher than 200 units mg-1 and a subunit molecular mass of about 33 kD upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Moreover, the purified serine acetyltransferase appeared to be essentially free of O-acetyleserine(thiol)lyase, another enzyme component in the L-cysteine biosynthetic pathway. A steady-state kinetic analysis indicated that the mechanism of the enzyme-catalyzed reaction involves a double displacement. The apparent Km for the two substrates, L-serine and acetyl-coenzyme A, were 2.29 [plus or minus] 0.43 and 0.35 [plus or minus] 0.02 mM, respectively. The rate of L-cysteine synthesis in vitro was measured in a coupled enzyme assay using extensively purified O-acetylserine(thiol)lyase and serine acetyltransferase. This rate was maximum when the assay contained approximately a 400-fold excess of O-acetylserine(thiol)lyase over serine acetyltransferase. Measurements of the relative level of O-acetylserine(thiol)lyase and serine acetyltransferase activities in the stroma indicated that the former enzyme was present in much larger quantities than the latter. Thus, the activity ratio for these two enzymes [O-acetylserine(thiol)lyase activity/serine acetyltransferase activity] measured in the stromal protein extract was 345. This strongly suggested that all the O-acetylserine(thiol)lyase and serine acetyltransferase activities in the stroma are involved in bringing a full synthesis of L-cysteine in the chloroplast.

Purification and Kinetic Properties of Serine Acetyltransferase Free of O-Acetylserine(thiol)lyase from Spinach Chloroplasts.

PubMed Central

Ruffet, M. L.; Droux, M.; Douce, R.

1994-01-01

Serine acetyltransferase, a key enzyme in the L-cysteine biosynthetic pathway, was purified over 300,000-fold from the stroma of spinach (Spinacia oleracea) leaf chloroplasts. The purification procedure consisted of ammonium sulfate precipitation, anion-exchange chromatography (Trisacryl M DEAE and Mono Q HR10/10), hydroxylapatite chromatography, and gel filtration (Superdex 200). The purified enzyme exhibited a specific activity higher than 200 units mg-1 and a subunit molecular mass of about 33 kD upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Moreover, the purified serine acetyltransferase appeared to be essentially free of O-acetyleserine(thiol)lyase, another enzyme component in the L-cysteine biosynthetic pathway. A steady-state kinetic analysis indicated that the mechanism of the enzyme-catalyzed reaction involves a double displacement. The apparent Km for the two substrates, L-serine and acetyl-coenzyme A, were 2.29 [plus or minus] 0.43 and 0.35 [plus or minus] 0.02 mM, respectively. The rate of L-cysteine synthesis in vitro was measured in a coupled enzyme assay using extensively purified O-acetylserine(thiol)lyase and serine acetyltransferase. This rate was maximum when the assay contained approximately a 400-fold excess of O-acetylserine(thiol)lyase over serine acetyltransferase. Measurements of the relative level of O-acetylserine(thiol)lyase and serine acetyltransferase activities in the stroma indicated that the former enzyme was present in much larger quantities than the latter. Thus, the activity ratio for these two enzymes [O-acetylserine(thiol)lyase activity/serine acetyltransferase activity] measured in the stromal protein extract was 345. This strongly suggested that all the O-acetylserine(thiol)lyase and serine acetyltransferase activities in the stroma are involved in bringing a full synthesis of L-cysteine in the chloroplast. PMID:12232109

A conformational landscape for alginate secretion across the outer membrane of Pseudomonas aeruginosa

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

Tan, Jingquan; Rouse, Sarah L.; Li, Dianfan

2014-08-01

Crystal structures of the β-barrel porin AlgE reveal a mechanism whereby alginate is exported from P. aeruginosa for biofilm formation. The exopolysaccharide alginate is an important component of biofilms produced by Pseudomonas aeruginosa, a major pathogen that contributes to the demise of cystic fibrosis patients. Alginate exits the cell via the outer membrane porin AlgE. X-ray structures of several AlgE crystal forms are reported here. Whilst all share a common β-barrel constitution, they differ in the degree to which loops L2 and T8 are ordered. L2 and T8 have been identified as an extracellular gate (E-gate) and a periplasmic gatemore » (P-gate), respectively, that reside on either side of an alginate-selectivity pore located midway through AlgE. Passage of alginate across the membrane is proposed to be regulated by the sequential opening and closing of the two gates. In one crystal form, the selectivity pore contains a bound citrate. Because citrate mimics the uronate monomers of alginate, its location is taken to highlight a route through AlgE taken by alginate as it crosses the pore. Docking and molecular-dynamics simulations support and extend the proposed transport mechanism. Specifically, the P-gate and E-gate are flexible and move between open and closed states. Citrate can leave the selectivity pore bidirectionally. Alginate docks stably in a linear conformation through the open pore. To translate across the pore, a force is required that presumably is provided by the alginate-synthesis machinery. Accessing the open pore is facilitated by complex formation between AlgE and the periplasmic protein AlgK. Alginate can thread through a continuous pore in the complex, suggesting that AlgK pre-orients newly synthesized exopolysaccharide for delivery to AlgE.« less

Alginate Beads Containing Lactase: Stability and Microstructure.

PubMed

Traffano-Schiffo, Maria Victoria; Aguirre Calvo, Tatiana R; Castro-Giraldez, Marta; Fito, Pedro J; Santagapita, Patricio R

2017-06-12

β-Galactosidase (lactase) is a widely used enzyme in the food industry; however, it has low stability against thermal and mechanical treatments. Due to this, the purpose of the present research was to analyze the encapsulation of lactase in alginate-Ca(II) beads in order to maintain its enzymatic activity toward freezing, freezing/thawing, and storage. Also, the effect of the addition of trehalose, and arabic and guar gums and their influence on the microstructure as well as on thermal properties and molecular mobility were studied. Lactase was successfully encapsulated in alginate-Ca(II) beads, and the inclusion of trehalose was critical for activity preservation toward treatments, being improved in guar gum-containing systems. The gums increased the T m ' values, which represents a valuable technological improvement. Finally, the presence of secondary excipients affected the microstructure, showing rods with smaller outer diameter and with lower compactness than alginate-Ca(II) beads. Also, bead composition greatly affects the size, shape, and relaxation times.

Physicochemical characterization and biocompatibility of alginate-polycation microcapsules designed for islet transplantation

NASA Astrophysics Data System (ADS)

Tam, Susan Kimberly

Microencapsulation represents a method for immunoprotecting transplanted therapeutic cells or tissues from graft rejection using a physical barrier. This approach is advantageous in that it eliminates the need to induce long-term immunosuppression and allows the option of transplanting non-cadaveric cell sources, such as animal cells and stem cell-derived tissues. The microcapsules that we have investigated are designed to immunoprotect islets of Langerhans (i.e. clusters of insulin-secreting cells), with the goal of treating insulin-dependent diabetes. With the aid of techniques for physicochemical analysis, this research focused on understanding which properties of the microcapsule are the most important for determining its biocompatibility. The objective of this work was to elucidate correlations between the chemical make-up, physicochemical properties, and in vivo biocompatibility of alginate-based microcapsules. Our approach was based on the hypothesis that the immune response to the microcapsules is governed by, and can therefore be controlled by, specific physicochemical properties of the microcapsule and its material components. The experimental work was divided into five phases, each associated with a specific aim : (1) To prove that immunoglobulins adsorb to the surface of alginate-polycation microcapsules, and to correlate this adsorption with the microcapsule chemistry. (2) To test interlaboratory reproducibility in making biocompatible microcapsules, and evaluate the suitability of our materials and fabrication protocols for subsequent studies. (3) To determine which physicochemical properties of alginates affect the in vivo biocompatibility of their gels. (4) To determine which physiochemical properties of alginate-polycation microcapsules are most important for determining their in vivo biocompatibility (5) To determine whether a modestly immunogenic membrane hinders or helps the ability of the microcapsule to immunoprotect islet xenografts in

Link between alginate reaction front propagation and general reaction diffusion theory.

PubMed

Braschler, Thomas; Valero, Ana; Colella, Ludovica; Pataky, Kristopher; Brugger, Jürgen; Renaud, Philippe

2011-03-15

We provide a common theoretical framework reuniting specific models for the Ca(2+)-alginate system and general reaction diffusion theory along with experimental validation on a microfluidic chip. As a starting point, we use a set of nonlinear, partial differential equations that are traditionally solved numerically: the Mikkelsen-Elgsaeter model. Applying the traveling-wave hypothesis as a major simplification, we obtain an analytical solution. The solution indicates that the fundamental properties of the alginate reaction front are governed by a single dimensionless parameter λ. For small λ values, a large depletion zone accompanies the reaction front. For large λ values, the alginate reacts before having the time to diffuse significantly. We show that the λ parameter is of general importance beyond the alginate model system, as it can be used to classify known solutions for second-order reaction diffusion schemes, along with the novel solution presented here. For experimental validation, we develop a microchip model system, in which the alginate gel formation can be carried out in a highly controlled, essentially 1D environment. The use of a filter barrier enables us to rapidly renew the CaCl(2) solution, while maintaining flow speeds lower than 1 μm/s for the alginate compartment. This allows one to impose an exactly known bulk CaCl(2) concentration and diffusion resistance. This experimental model system, taken together with the theoretical development, enables the determination of the entire set of physicochemical parameters governing the alginate reaction front in a single experiment.

PECTATE LYASE-LIKE 9 from Brassica campestris is associated with intine formation.

PubMed

Jiang, Jingjing; Yao, Lina; Yu, Youjian; Liang, Ying; Jiang, Jianxia; Ye, Nenghui; Miao, Ying; Cao, Jiashu

2014-12-01

Brassica campestris pectate lyase-like 9 (BcPLL9) was previously identified as a differentially expressed gene both in buds during late pollen developmental stage and in pistils during fertilization in Chinese cabbage. To characterize the gene's function, antisense-RNA lines of BcPLL9 (bcpll9) were constructed in Chinese cabbage. Self- and cross-fertilization experiments harvested half seed yields when bcpll9 lines were used as pollen donors. In vivo and in vitro pollen germination assays showed that nearly half of the pollen tubes in bcpll9 were irregular with shorter length and uneven surface. Aniline blue staining identified abnormal accumulation of a specific bright blue unknown material in the bcpll9 pollen portion. Scanning electron microscopy observation verified the abnormal outthrust material to be near the pollen germinal furrows. Transmission electron microscopy observation revealed the internal endintine layer was overdeveloped and predominantly occupied the intine. This abnormally formed intine likely induced the wavy structure and growth arrest of the pollen tube in half of the bcpll9 pollen grains, which resulted in less seed yields. Collectively, this study presented a novel PLL gene that has an important function in B. campestris intine formation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Macrophage Repolarization with Targeted Alginate Nanoparticles Containing IL-10 Plasmid DNA for the Treatment of Experimental Arthritis

PubMed Central

Jain, Shardool; Tran, Thanh-Huyen; Amiji, Mansoor

2015-01-01

In this study, we have shown for the first time the effectiveness of a non-viral gene transfection strategy to re-polarize macrophages from M1 to M2 functional sub-type for the treatment of rheumatoid arthritis (RA). An anti-inflammatory (IL-10) cytokine encoding plasmid DNA was successfully encapsulated into non-condensing alginate based nanoparticles and the surface of the nano-carriers was modified with tuftsin peptide to achieve active macrophage targeting. Enhanced localization of tuftsin-modified alginate nanoparticles was observed in the inflamed paws of arthritic rats upon intraperitoneal administration. Importantly, targeted nanoparticle treatment was successful in reprogramming macrophage phenotype balance as ~66% of total synovial macrophages from arthritic rats treated with the IL-10 plasmid DNA loaded tuftsin/alginate nanoparticles were in the M2 state compared to ~9% of macrophages in the M2 state from untreated arthritic rats. Treatment significantly reduced systemic and joint tissue pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) expression and prevented the progression of inflammation and joint damage as revealed by magnetic resonance imaging and histology. Treatment enabled animals to retain their mobility throughout the course of study, whereas untreated animals suffered from impaired mobility. Overall, this study demonstrates that targeted alginate nanoparticles loaded with IL-10 plasmid DNA can efficiently re-polarize macrophages from an M1 to an M2 state, offering a novel treatment paradigm for treatment of chronic inflammatory diseases. PMID:26004232

Controlling the size of alginate gel beads by use of a high electrostatic potential.

PubMed

Klokk, T I; Melvik, J E

2002-01-01

The effect of several parameters on the size of alginate beads produced by use of an electrostatic potential bead generator was examined. Parameters studied included needle diameter, electrostatic potential, alginate solution flow rate, gelling ion concentration and alginate concentration and viscosity, as well as alginate composition. Bead size was found to decrease with increasing electrostatic potential, but only down to a certain level. Minimum bead size was reached at between 2-4 kV/cm for the needles tested. The smallest alginate beads produced (using a needle with inner diameter 0.18 mm) had a mean diameter of approximately 300 microm. Bead size was also found to be dependent upon the flow rate of the fed alginate solution. Increasing the gelling ion concentration resulted in a moderate decrease in bead size. The concentration and viscosity of the alginate solution also had an effect on bead size as demonstrated by an increased bead diameter when the concentration or viscosity was increased. This effect was primarily an effect of the viscosity properties of the solution, which led to changes in the rate of droplet formation in the bead generator. Lowering the flow rate of the alginate solution could partly compensate for the increase in bead size with increased viscosity. For a constant droplet size, alginates with a low G block content (F(GG) approximately 0.20) resulted in approximately 30% smaller beads than alginates with a high G block content (F(GG) approximately 0.60). This is explained as a result of differences in the shrinking properties of the beads.

Dispensing of very low volumes of ultra high viscosity alginate gels: a new tool for encapsulation of adherent cells and rapid prototyping of scaffolds and implants.

PubMed

Gepp, Michael M; Ehrhart, Friederike; Shirley, Stephen G; Howitz, Steffen; Zimmermann, Heiko

2009-01-01

We present a tool for dispensing very low volumes (20 nL or more) of ultra high viscosity (UHV) medical-grade alginate hydrogels. It uses a modified piezo-driven micrometering valve, integrated into a versatile system that allows fast prototyping of encapsulation procedures and scaffold production. Valves show excellent dispensing properties for UHV alginate in concentrations of 0.4% and 0.7% and also for aqueous liquids. An optimized process flow provides excellent handling of biological samples under sterile conditions. This technique allows the encapsulation of adherent cells and structuring of substrates for biotechnology and regenerative medicine. A variety of cell lines showed at least 70% viability after encapsulation (including cell lines that are relevant in regenerative medicine like Hep G2), and time-lapse analysis revealed cells proliferating and showing limited motility under alginate spots. Cells show metabolic activity, gene product expression, and physiological function. Encapsulated cells have contact with the substrate and can exchange metabolites while being isolated from macromolecules in the environment. Contactless dispensing allows structuring of substrates with alginate, isolation and transfer of cell-alginate complexes, and the dispensing of biological active hydrogels like extracellular matrix-derived gels.

Crystal structure and characterization of a novel L-serine ammonia-lyase from Rhizomucor miehei

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

Qin, Zhen; Yan, Qiaojuan; Ma, Qingjun

L-serine ammonia-lyase, as a member of the β-family of pyridoxal-5′-phosphate (PLP) dependent enzymes, catalyzes the conversion of L-serine (L-threonine) to pyruvate (α-ketobutyrate) and ammonia. The crystal structure of L-serine ammonia-lyase from Rhizomucor miehei (RmSDH) was solved at 1.76 Å resolution by X-ray diffraction method. The overall structure of RmSDH had the characteristic β-family PLP dependent enzyme fold. It consisted of two distinct domains, both of which show the typical open twisted α/β structure. A PLP cofactor was located in the crevice between the two domains, which was attached to Lys52 by a Schiff-base linkage. Unique residue substitutions (Gly78, Pro79, Ser146, Ser147more » and Thr312) were discovered at the catalytic site of RmSDH by comparison of structures of RmSDH and other reported eukaryotic L-serine ammonia-lyases. Optimal pH and temperature of the purified RmSDH were 7.5 and 40 °C, respectively. It was stable in the pH range of 7.0–9.0 and at temperatures below 40 °C. This is the first crystal structure of a fungal L-serine ammonia-lyase. It will be useful to study the catalytic mechanism of β-elimination enzymes and will provide a basis for further enzyme engineering. - Highlights: • The crystal structure of a fungal L-serine ammonia-lyase (RmSDH) was solved. • Five unique residue substitutions are found at the catalytic site of RmSDH. • RmSDH was expressed in Pichia. pastoris and biochemically characterized. • RmSDH has potential application in splitting D/L-serine.« less

Induction of L-phenylalanine ammonia-lyase during utilization of phenylalanine as a carbon or nitrogen source in Rhodotorula glutinis

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

Marusich, W.C.; Jensen, R.A.; Zamir, L.O.

Rhodotorula glutinis is a convenient source of L-phenylalanine ammonia-lyase, an enzyme that is useful as a biochemical reagent in the assay of L-phenylalanine. There have been previous descriptions of induced lyase production in complex medium where induction occurs late in exponential growth, suggesting a role in secondary metabolism such as is the case in higher plants. A higher specific activity of L-phenylalanine ammonia-lyase (sixfold higher than in complex medium) can be obtained during midexponential growth in a defined medium containing L-phenylalanine as the sole source of carbon. L-phenylalanine will also induce lyase synthesis during exponential growth in minimal medium inmore » which L-phenylalanine is the sole source of nitrogen. The appearance of lyase in complex medium supplemented with L-phenylalanine is probably triggered fortuitously by exhaustion late in growth of a prime source of nitrogen. In this study, R. glutinis appeared to express a single lyase enzyme, regardless of whether induction was nitrogen signaled or carbon signaled. Thin-layer chromatographic analysis of ether extracts prepared fom cultures induced with doubly labeled (U-/sup 14/C; ring-4-/sup 3/H) L-phenylalanine provided evidence of a catabolic sequence containing cinnamic acid, benzoic acid, and 4-hydroxybenzoic acid as degradative intermediates. 3,4-Dihydroxybenzoic acid was not identified as a catabolic intermediate.« less

Induction of L-phenylalanine ammonia-lyase during utilization of phenylalanine as a carbon or nitrogen source in Rhodotorula glutinis.

PubMed Central

Marusich, W C; Jensen, R A; Zamir, L O

1981-01-01

Rhodotorula glutinis is a convenient source of L-phenylalanine ammonia-lyase, an enzyme that is useful as a biochemical reagent in the assay of L-phenylalanine. There have been previous descriptions of induced lyase production in complex medium where induction occurs late in exponential growth, suggesting a role in secondary metabolism such as is the case in higher plants. A higher specific activity of L-phenylalanine ammonia-lyase (sixfold higher than a complex medium) can be obtained during midexponential growth in a defined medium containing L-phenylalanine as the sole source of carbon. L-Phenylalanine will also induce lyase synthesis during exponential growth in minimal in which L-phenylalanine is the sole source of nitrogen. The appearance of lyase in complex medium supplemented with L-phenylalanine is probably triggered fortuitously by exhaustion late in growth of a prime source of nitrogen. In this study, R. glutinis appeared to express a single lyase enzyme, regardless of whether induction was nitrogen signaled or carbon signaled. Thin-layer chromatographic analysis of ether extracts prepared from cultures induced with doubly labeled (U-14C; ring-4-3H) L-phenylalanine provided evidence of a catabolic sequence containing cinnamic acid, benzoic acid, and 4-hydroxybenzoic acid as degradative intermediates. 3,4-Dihydroxybenzoic acid was not identified as a catabolic intermediate. PMID:7195398

Production of polyhydroxybutyrate and alginate from glycerol by Azotobacter vinelandii under nitrogen-free conditions.

PubMed

Yoneyama, Fuminori; Yamamoto, Mayumi; Hashimoto, Wataru; Murata, Kousaku

2015-01-01

Glycerol is an interesting feedstock for biomaterials such as biofuels and bioplastics because of its abundance as a by-product during biodiesel production. Here we demonstrate glycerol metabolism in the nitrogen-fixing species Azotobacter vinelandii through metabolomics and nitrogen-free bacterial production of biopolymers, such as poly-d-3-hydroxybutyrate (PHB) and alginate, from glycerol. Glycerol-3-phosphate was accumulated in A. vinelandii cells grown on glycerol to the exponential phase, and its level drastically decreased in the cells grown to the stationary growth phase. A. vinelandii also overexpressed the glycerol-3-phosphate dehydrogenase gene when it was grown on glycerol. These results indicate that glycerol was first converted to glycerol-3-phosphate by glycerol kinase. Other molecules with industrial interests, such as lactic acid and amino acids including γ-aminobutyric acid, have also been accumulated in the bacterial cells grown on glycerol. Transmission electron microscopy revealed that glycerol-grown A. vinelandii stored PHB within the cells. The PHB production level reached 33% per dry cell weight in nitrogen-free glycerol medium. When grown on glycerol, alginate-overproducing mutants generated through chemical mutagenesis produced 2-fold the amount of alginate from glycerol than the parental wild-type strain. To the best of our knowledge, this is the first report on bacterial production of biopolymers from glycerol without addition of any nitrogen source.

Production, deformation and mechanical investigation of magnetic alginate capsules

NASA Astrophysics Data System (ADS)

Zwar, Elena; Kemna, Andre; Richter, Lena; Degen, Patrick; Rehage, Heinz

2018-02-01

In this article we investigated the deformation of alginate capsules in magnetic fields. The sensitivity to magnetic forces was realised by encapsulating an oil in water emulsion, where the oil droplets contained dispersed magnetic nanoparticles. We solved calcium ions in the aqueous emulsion phase, which act as crosslinking compounds for forming thin layers of alginate membranes. This encapsulating technique allows the production of flexible capsules with an emulsion as the capsule core. It is important to mention that the magnetic nanoparticles were stable and dispersed throughout the complete process, which is an important difference to most magnetic alginate-based materials. In a series of experiments, we used spinning drop techniques, capsule squeezing experiments and interfacial shear rheology in order to determine the surface Young moduli, the surface Poisson ratios and the surface shear moduli of the magnetically sensitive alginate capsules. In additional experiments, we analysed the capsule deformation in magnetic fields. In spinning drop and capsule squeezing experiments, water droplets were pressed out of the capsules at elevated values of the mechanical load. This phenomenon might be used for the mechanically triggered release of water-soluble ingredients. After drying the emulsion-filled capsules, we produced capsules, which only contained a homogeneous oil phase with stable suspended magnetic nanoparticles (organic ferrofluid). In the dried state, the thin alginate membranes of these particles were rather rigid. These dehydrated capsules could be stored at ambient conditions for several months without changing their properties. After exposure to water, the alginate membranes rehydrated and became flexible and deformable again. During this swelling process, water diffused back in the capsule. This long-term stability and rehydration offers a great spectrum of different applications as sensors, soft actuators, artificial muscles or drug delivery systems.

Effect of alginate in patients with GERD hiatal hernia matters.

PubMed

Vardar, R; Keskin, M; Valitova, E; Bayrakci, B; Yildirim, E; Bor, S

2017-10-01

Alginate-based formulations are frequently used as add-on proton pump inhibitor (PPI) therapy to help control of heartburn and regurgitation. There are limited data regarding the mechanisms and effects of alginate-based formulations. We aimed to evaluate the effects of the sodium alginate intake and its likely temporal relations on intraesophageal reflux events by MII-pH in patients with and without hiatal hernia (HH). Fifty GERD patients (18 with HH, 32 without HH) with heartburn or regurgitation once a week or more common were included. After combined multichannel intraluminal impedance and pH-metry (MII-pH) had been performed, all patients were asked to eat the same standard meal (double cheeseburger, 1 banana, 100 g regular yoghurt, and 200 mL water with total energy value of 744 kcal: 37.6% of carbohydrates, 21.2% of proteins, and 41.2% of lipids) during two consecutive days. On separate random two consecutive days, all patients took 10 mL of sodium alginate (GA; Gaviscon Advance; Reckitt Benckiser Healthcare, Hull, UK) or 10 mL of water, 30 minutes after the refluxogenic meal. After eating refluxogenic meal, patients were examined ½ hour for basal conditions, 1 hour in upright, and 1 hour in supine positions. Alginate significantly decreased acid reflux after intake at the first hour in comparison to water in patients with HH (6.1 vs. 13.7, P = 0.004) and without HH (3.5 vs. 5.5, P = 0.001). Weakly acid reflux were increased at the first hour in patients with HH (3.4 vs. 1.3, P = 0.019) and without HH (1.7 vs. 5, P = 0.02) compared to water. There was no distinctive effect of alginate on the height of proximal migration of reflux events in patients with HH and without HH. Alginate decreases acid reflux events within a limited time period, especially at the first hour both in patients with and without HH. Alginate has no effect on the height of reflux events along the esophagus both in patients with and without HH. © The Authors 2017. Published by Oxford

Cloning, expression and characterization of a pectate lyase from Paenibacillus sp. 0602 in recombinant Escherichia coli

PubMed Central

2014-01-01

Background Biotechnological applications of microbial pectate lyases (Pels) in plant fiber processing are considered as environmentally friendly. As such, they become promising substitutes for conventional chemical degumming process. Since applications of Pels in various fields are widening, it is necessary to explore new pectolytic microorganisms and enzymes for efficient and effective usage. Here, we describe the cloning, expression, characterization and application of the recombinant Pel protein from a pectolytic bacterium of the genus Paenibacillus in Escherichia coli. Results A Pel gene (pelN) was cloned using degenerate PCR and inverse PCR from the chromosomal DNA of Paenibacillus sp. 0602. The open reading frame of pelN encodes a 30 amino acid signal peptide and a 445 amino acid mature protein belonging to the polysaccharide lyase family 1. The maximum Pel activity produced by E. coli in shake flasks reached 2,467.4 U mL−1, and the purified recombinant enzyme exhibits a specific activity of 2,060 U mg−1 on polygalacturonic acid (PGA). The maximum activity was observed in a buffer with 5 mM Ca2+ at pH 9.8 and 65°C. PelN displays a half-life of around 9 h and 42 h at 50°C and 45°C, respectively. The biochemical treatment achieved the maximal reduction of percentage weight (30.5%) of the ramie bast fiber. Conclusions This work represents the first study that describes the extracellular expression of a Pel gene from Paenibacillus species in E. coli. The high yield of the extracellular overexpression, relevant thermostability and efficient degumming using combined treatments indicate its strong potential for large-scale industrial production. PMID:24612647

Cloning and Characterization of the Gene Encoding Alpha-Pinene Oxide Lyase Enzyme (Prα-POL) from Pseudomonas rhodesiae CIP 107491 and Production of the Recombinant Protein in Escherichia coli.

PubMed

Dubessay, Pascal; Larroche, Christian; Fontanille, Pierre

2017-12-28

The alpha-pinene oxide lyase (Prα-POL) from Pseudomonas rhodesiae CIP107491 belongs to catabolic alpha-pinene degradation pathway. In this study, the gene encoding Prα-POL has been identified using mapping approach combined to inverse PCR (iPCR) strategy. The Prα-POL gene included a 609-bp open reading frame encoding 202 amino acids and giving rise to a 23.7 kDa protein, with a theoretical isoelectric point (pI) of 5.23. The amino acids sequence analysis showed homologies with those of proteins with unknown function from GammaProteobacteria group. Identification of a conserved domain in amino acid in positions 18 to 190 permitted to classify Prα-POL among the nuclear transport factor 2 (NTF2) protein superfamily. Heterologous expression of Prα-POL, both under its native form and with a histidin tag, was successfully performed in Escherichia coli, and enzymatic kinetics were analyzed. Bioconversion assay using recombinant E. coli strain allowed to reach a rate of isonovalal production per gramme of biomass about 40-fold higher than the rate obtained with P. rhodesiae.

3D porous calcium-alginate scaffolds cell culture system improved human osteoblast cell clusters for cell therapy.

PubMed

Chen, Ching-Yun; Ke, Cherng-Jyh; Yen, Ko-Chung; Hsieh, Hui-Chen; Sun, Jui-Sheng; Lin, Feng-Huei

2015-01-01

Age-related orthopedic disorders and bone defects have become a critical public health issue, and cell-based therapy is potentially a novel solution for issues surrounding bone tissue engineering and regenerative medicine. Long-term cultures of primary bone cells exhibit phenotypic and functional degeneration; therefore, culturing cells or tissues suitable for clinical use remain a challenge. A platform consisting of human osteoblasts (hOBs), calcium-alginate (Ca-Alginate) scaffolds, and a self-made bioreactor system was established for autologous transplantation of human osteoblast cell clusters. The Ca-Alginate scaffold facilitated the growth and differentiation of human bone cell clusters, and the functionally-closed process bioreactor system supplied the soluble nutrients and osteogenic signals required to maintain the cell viability. This system preserved the proliferative ability of cells and cell viability and up-regulated bone-related gene expression and biological apatite crystals formation. The bone-like tissue generated could be extracted by removal of calcium ions via ethylenediaminetetraacetic acid (EDTA) chelation, and exhibited a size suitable for injection. The described strategy could be used in therapeutic application and opens new avenues for surgical interventions to correct skeletal defects.

Site-directed mutagenesis of lysine 193 in Escherichia coli isocitrate lyase by use of unique restriction enzyme site elimination.

PubMed Central

Diehl, P; McFadden, B A

1993-01-01

By a newly developed double-stranded mutagenesis technique, histidine (H), glutamate (E), arginine (R) and leucine (L) have been substituted for the lysyl 193 residue (K-193) in isocitrate lyase from Escherichia coli. The substitutions for this residue, which is present in a highly conserved, cationic region, significantly affect both the Km for Ds-isocitrate and the apparent kcat of isocitrate lyase. Specifically, the conservative substitutions, K-193-->H (K193H) and K193R, reduce catalytic activity by ca. 50- and 14-fold, respectively, and the nonconservative changes, K193E and K193L, result in assembled tetrameric protein that is completely inactive. The K193H and K193R mutations also increase the Km of the enzyme by five- and twofold, respectively. These results indicate that the cationic and/or acid-base character of K193 is essential for isocitrate lyase activity. In addition to the noted effects on enzyme activity, the effects of the mutations on growth of JE10, an E. coli strain which does not express isocitrate lyase, were observed. Active isocitrate lyase is necessary for E. coli to grow on acetate as the sole carbon source. It was found that a mutation affecting the activity of isocitrate lyase similarly affects the growth of E. coli JE10 on acetate when the mutated plasmid is expressed in this organism. Specifically, the lag time before growth increases over sevenfold and almost twofold for E. coli JE10 expressing the K193H and K193R isocitrate lyase variants, respectively. In addition, the rate of growth decreases by almost 40-fold for E. coli JE10 cells expressing form K193H and ca. 2-fold for those expressing the K193R variants. Thus, the onset and rate of E. coli growth on acetate appears to depend on isocitrate lyase activity. Images PMID:8385665

Heavy metal removal by caustic-treated yeast immobilized in alginate

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

Lu, Y.; Wilkins, E.

1995-12-31

Saccharomyces cerevisiae yeast biomass was treated with hot alkali to increase its biosorption capacity for heavy metals and then was immobilized in alginate gel. Biosorption capacities for Cu{sup 2+}, Cd{sup 2+}, and Zn{sup 2+} on alginate gel, native yeast, native yeast immobilized in alginate gel, and caustic-treated yeast immobilized in alginate gel were all compared. Immobilized yeasts could be reactivated and reused in a manner similar to the ion exchange resins. Immobilized caustic-treated yeast has high heavy metal biosorption capacity and high metal removal efficiency in a rather wide acidic pH region. The biosorption isotherm of immobilized caustic-treated yeast wasmore » studied, and empirical equations were obtained. The initial pH of polluted water affected the metal removal efficiency significantly, and the equilibrium biosorption capacity seemed to be temperature independent at lower initial metal concentrations.« less

Chondrocyte Culture in Three Dimensional Alginate Sulfate Hydrogels Promotes Proliferation While Maintaining Expression of Chondrogenic Markers

PubMed Central

Mhanna, Rami; Kashyap, Aditya; Palazzolo, Gemma; Vallmajo-Martin, Queralt; Becher, Jana; Möller, Stephanie; Schnabelrauch, Matthias

2014-01-01

The loss of expression of chondrogenic markers during monolayer expansion remains a stumbling block for cell-based treatment of cartilage lesions. Here, we introduce sulfated alginate hydrogels as a cartilage biomimetic biomaterial that induces cell proliferation while maintaining the chondrogenic phenotype of encapsulated chondrocytes. Hydroxyl groups of alginate were converted to sulfates by incubation with sulfur trioxide–pyridine complex (SO3/pyridine), yielding a sulfated material cross-linkable with calcium chloride. Passage 3 bovine chondrocytes were encapsulated in alginate and alginate sulfate hydrogels for up to 35 days. Cell proliferation was five-fold higher in alginate sulfate compared with alginate (p=0.038). Blocking beta1 integrins in chondrocytes within alginate sulfate hydrogels significantly inhibited proliferation (p=0.002). Sulfated alginate increased the RhoA activity of chondrocytes compared with unmodified alginate, an increase that was blocked by β1 blocking antibodies (p=0.017). Expression and synthesis of type II collagen, type I collagen, and proteoglycan was not significantly affected by the encapsulation material evidenced by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry. Alginate sulfate constructs showed an opaque appearance in culture, whereas the unmodified alginate samples remained translucent. In conclusion, alginate sulfate provides a three dimensional microenvironment that promotes both chondrocyte proliferation and maintenance of the chondrogenic phenotype and represents an important advance for chondrocyte-based cartilage repair therapies providing a material in which cell expansion can be done in situ. PMID:24320935

Dental mesenchymal stem cells encapsulated in an alginate hydrogel co-delivery microencapsulation system for cartilage regeneration.

PubMed

Moshaverinia, Alireza; Xu, Xingtian; Chen, Chider; Akiyama, Kentaro; Snead, Malcolm L; Shi, Songtao

2013-12-01

Dental-derived mesenchymal stem cells (MSCs) are promising candidates for cartilage regeneration, with a high capacity for chondrogenic differentiation. This property helps make dental MSCs an advantageous therapeutic option compared to current treatment modalities. The MSC delivery vehicle is the principal determinant for the success of MSC-mediated cartilage regeneration therapies. The objectives of this study were to: (1) develop a novel co-delivery system based on TGF-β1 loaded RGD-coupled alginate microspheres encapsulating periodontal ligament stem cells (PDLSCs) or gingival mesenchymal stem cells (GMSCs); and (2) investigate dental MSC viability and chondrogenic differentiation in alginate microspheres. The results revealed the sustained release of TGF-β1 from the alginate microspheres. After 4 weeks of chondrogenic differentiation in vitro, PDLSCs and GMSCs as well as human bone marrow mesenchymal stem cells (hBMMSCs) (as positive control) revealed chondrogenic gene expression markers (Col II and Sox-9) via qPCR, as well as matrix positively stained by Toluidine Blue and Safranin-O. In animal studies, ectopic cartilage tissue regeneration was observed inside and around the transplanted microspheres, confirmed by histochemical and immunofluorescent staining. Interestingly, PDLSCs showed more chondrogenesis than GMSCs and hBMMSCs (p<0.05). Taken together, these results suggest that RGD-modified alginate microencapsulating dental MSCs make a promising candidate for cartilage regeneration. Our results highlight the vital role played by the microenvironment, as well as value of presenting inductive signals for viability and differentiation of MSCs. Copyright © 2013 Acta Materialia Inc. All rights reserved.

Mineralized alginate hydrogels using marine carbonates for bone tissue engineering applications.

PubMed

Diaz-Rodriguez, P; Garcia-Triñanes, P; Echezarreta López, M M; Santoveña, A; Landin, M

2018-09-01

The search for an ideal bone tissue replacement has led to the development of new composite materials designed to simulate the complex inorganic/organic structure of bone. The present work is focused on the development of mineralized calcium alginate hydrogels by the addition of marine derived calcium carbonate biomineral particles. Following a novel approach, we were able to obtain calcium carbonate particles of high purity and complex micro and nanostructure dependent on the source material. Three different types of alginates were selected to develop inorganic/organic scaffolds in order to correlate alginate composition with scaffold properties and cell behavior. The incorporation of calcium carbonates into alginate networks was able to promote extracellular matrix mineralization and osteoblastic differentiation of mesenchymal stem cells when added at 7 mg/ml. We demonstrated that the selection of the alginate type and calcium carbonate origin is crucial to obtain adequate systems for bone tissue engineering as they modulate the mechanical properties and cell differentiation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Binding and Leakage of Barium in Alginate Microbeads

PubMed Central

Mørch, Yrr A.; Qi, Meirigeng; Gundersen, Per Ole M.; Formo, Kjetil; Lacik, Igor; Skjåk-Bræk, Gudmund; Oberholzer, Jose; Strand, Berit L.

2013-01-01

Microbeads of alginate cross-linked with Ca2+ and/or Ba2+ are popular matrices in cell-based therapy. The aim of this study was to quantify the binding of barium in alginate microbeads and its leakage under in vitro and accumulation under in vivo conditions. Low concentrations of barium (1 mM) in combination with calcium (50 mM) and high concentrations of barium (20 mM) in gelling solutions were used for preparation of microbeads made of high-G and high-M alginates. High-G microbeads accumulated barium from gelling solution and contained higher concentrations of divalent ions for both low- and high-Ba exposure compared to high-G microbeads exposed to calcium solely and to high-M microbeads for all gelling conditions. Although most of the unbound divalent ions were removed during the wash and culture steps, leakage of barium was still detected during storage. Barium accumulation in blood and femur bone of mice implanted with high-G beads was found to be dose-dependent. Estimated barium leakage relevant to transplantation to diabetic patients with islets in alginate microbeads showed that the leakage was 2.5 times lower than the tolerable intake value given by WHO for high-G microbeads made using low barium concentration. The similar estimate gave 1.5 times higher than is the tolerable intake value for the high-G microbeads made using high barium concentration. In order to reduce the risk of barium accumulation that may be of safety concern, the microbeads made of high-G alginate gelled with a combination of calcium and low concentration of barium ions is recommended for islet transplantation. PMID:22700168

Binding and leakage of barium in alginate microbeads.

PubMed

Mørch, Yrr A; Qi, Meirigeng; Gundersen, Per Ole M; Formo, Kjetil; Lacik, Igor; Skjåk-Braek, Gudmund; Oberholzer, Jose; Strand, Berit L

2012-11-01

Microbeads of alginate crosslinked with Ca(2+) and/or Ba(2+) are popular matrices in cell-based therapy. The aim of this study was to quantify the binding of barium in alginate microbeads and its leakage under in vitro and accumulation under in vivo conditions. Low concentrations of barium (1 mM) in combination with calcium (50 mM) and high concentrations of barium (20 mM) in gelling solutions were used for preparation of microbeads made of high-G and high-M alginates. High-G microbeads accumulated barium from gelling solution and contained higher concentrations of divalent ions for both low- and high-Ba exposure compared with high-G microbeads exposed to calcium solely and to high-M microbeads for all gelling conditions. Although most of the unbound divalent ions were removed during the wash and culture steps, leakage of barium was still detected during storage. Barium accumulation in blood and femur bone of mice implanted with high-G beads was found to be dose-dependent. Estimated barium leakage relevant to transplantation to diabetic patients with islets in alginate microbeads showed that the leakage was 2.5 times lower than the tolerable intake value given by WHO for high-G microbeads made using low barium concentration. The similar estimate gave 1.5 times higher than is the tolerable intake value for the high-G microbeads made using high barium concentration. To reduce the risk of barium accumulation that may be of safety concern, the microbeads made of high-G alginate gelled with a combination of calcium and low concentration of barium ions is recommended for islet transplantation. Copyright © 2012 Wiley Periodicals, Inc.

Microfluidic Synthesis of Ca-Alginate Microcapsules for Self-Healing of Bituminous Binder.

PubMed

Shu, Benan; Wu, Shaopeng; Dong, Lijie; Wang, Qing; Liu, Quantao

2018-04-19

This work aims to develop an original alginate micro-emulsion combining with droplets microfluidic method to produce multinuclear Ca-alginate microcapsules containing rejuvenator for the self-healing of bituminous binder. The sizes of the Ca-alginate microcapsules could be easily controlled by tuning flow rates of the continuous and dispersed phases. The addition of a surfactant Tween80 not only improved the stability of the emulsion, but it also effectively reduced the size of the microcapsules. Size predictive mathematical model of the microcapsules was proposed through the analysis of fluid force. Optical microscope and remote Fourier infrared test confirmed the multinuclear structure of Ca-alginate microcapsules. Thermogravimetric analysis showed that the microcapsules coated with nearly 40% rejuvenator and they remained intact during the preparation of bitumen specimen at 135 °C. Micro self-healing process of bituminous binder with multinuclear Ca-alginate microcapsules containing rejuvenator was monitored and showed enhanced self-healing performance. Tensile stress-recovery test revealed that the recovery rate increased by 32.08% (in the case of 5% microcapsules), which meant that the Ca-alginate microcapsules containing rejuvenator could effectively enhance the self-healing property of bituminous binder.

Antifungal Effect of a Dental Tissue Conditioner Containing Nystatin-Loaded Alginate Microparticles.

PubMed

Kim, Hyun-Jin; Son, Jun Sik; Kwon, Tae-Yub

2018-02-01

In this in vitro study, nystatin-alginate microparticles were successfully fabricated to control the release of nystatin from a commercial dental tissue conditioner. These nystatin-alginate microparticles were spherical and had a slightly rough surface. The microparticles incorporated into the tissue conditioner were distributed homogeneously throughout the tissue conditioner matrix. The incorporation of the microparticles did not deteriorate the mechanical properties of the original material. The agar diffusion test results showed that the tissue conditioner containing the microparticles had a good antifungal effect against Candida albicans. The nystatin-alginate microparticles efficiently controlled the release of nystatin from the tissue conditioner matrix over the experimental period of 14 days. Moreover, the nystatin-alginate microparticles incorporated in the tissue conditioner showed effective antifungal function even at lower concentrations of nystatin. The current study suggests that the tissue conditioner containing the nystatin-alginate microparticle carrier system has potential as an effective antifungal material.

Bacterial alginate production: an overview of its biosynthesis and potential industrial production.

PubMed

Urtuvia, Viviana; Maturana, Nataly; Acevedo, Fernando; Peña, Carlos; Díaz-Barrera, Alvaro

2017-10-07

Alginate is a linear polysaccharide that can be used for different applications in the food and pharmaceutical industries. These polysaccharides have a chemical structure composed of subunits of (1-4)-β-D-mannuronic acid (M) and its C-5 epimer α-L-guluronic acid (G). The monomer composition and molecular weight of alginates are known to have effects on their properties. Currently, these polysaccharides are commercially extracted from seaweed but can also be produced by Azotobacter vinelandii and Pseudomonas spp. as an extracellular polymer. One strategy to produce alginates with different molecular weights and with reproducible physicochemical characteristics is through the manipulation of the culture conditions during fermentation. This mini-review provides a comparative analysis of the metabolic pathways and molecular mechanisms involved in alginate polymerization from A. vinelandii and Pseudomonas spp. Different fermentation strategies used to produce alginates at a bioreactor laboratory scale are described.

Preferential localization of Lactococcus lactis cells entrapped in a caseinate/alginate phase separated system.

PubMed

Léonard, Lucie; Gharsallaoui, Adem; Ouaali, Fahima; Degraeve, Pascal; Waché, Yves; Saurel, Rémi; Oulahal, Nadia

2013-09-01

This study aimed to entrap bioprotective lactic acid bacteria in a sodium caseinate/sodium alginate aqueous two-phase system. Phase diagram at pH=7 showed that sodium alginate and sodium caseinate were not miscible when their concentrations exceeded 1% (w/w) and 6% (w/w), respectively. The stability of the caseinate/alginate two-phase system was also checked at pH values of 6.0 and 5.5. Lactococcus lactis subsp. lactis LAB3 cells were added in a 4% (w/w) caseinate/1.5% (w/w) alginate two-phase system at pH=7. Fluorescence microscopy allowed to observe that the caseinate-rich phase formed droplets dispersed in a continuous alginate-rich phase. The distribution of bacteria in such a system was observed by epifluorescence microscopy: Lc. lactis LAB3 cells stained with Live/Dead(®) Baclight kit™ were located exclusively in the protein phase. Since zeta-potential measurements indicated that alginate, caseinate and bacterial cells all had an overall negative charge at pH 7, the preferential adhesion of LAB cells was assumed to be driven by hydrophobic effect or by depletion phenomena in such biopolymeric systems. Moreover, LAB cells viability was significantly higher in the ternary mixture obtained in the presence of both caseinate and alginate than in single alginate solution. Caseinate/alginate phase separated systems appeared thus well suited for Lc. lactis LAB3 cells entrapment. Copyright © 2013 Elsevier B.V. All rights reserved.

Microfabrication of proangiogenic cell-laden alginate-g-pyrrole hydrogels.

PubMed

DeVolder, Ross J; Zill, Andrew T; Jeong, Jae H; Kong, Hyunjoon

2012-11-01

Cells have been extensively studied for their uses in various therapies because of their capacities to produce therapeutic proteins and recreate new tissues. It has often been suggested that the efficacy of cell therapies can greatly be improved through the ability to localize and regulate cellular activities at a transplantation site; however, the technologies for this control are lacking. Therefore, this study reports a cell-Laden hydrogel patch engineered to support the proliferation and angiogenic growth factor expression of cells adhered to their surfaces, and to further promote neovascularization. Hydrogels consisting of alginate chemically linked with pyrrole units, termed alginate-g-pyrrole, were prepared through an oxidative cross-linking reaction between pyrrole units. Fibroblasts adhered to the alginate-g-pyrrole hydrogels, and exhibited increased proliferation and overall vascular endothelial growth factor (VEGF) expression, compared to those on pyrrole-free hydrogels. Furthermore, the alginate-g-pyrrole hydrogel surfaces were modified to present microposts, subsequently increasing the amount of pyrrole units on their surfaces. Cells adhered to the microfabricated gel surfaces exhibited increased proliferation and overall VEGF expression proportional to the density of the microposts. The resulting micropatterned alginate-g-pyrrole hydrogels exhibited increases in the size and density of mature blood vessels when implanted on chick chorioallantoic membranes (CAMs). The hydrogel system developed in this study will be broadly useful for improving the efficacy of a wide array of cell-based wound healing and tissue regenerative therapies. Copyright © 2012 Elsevier Ltd. All rights reserved.

A Facile Stable-Isotope Dilution Method for Determination of Sphingosine Phosphate Lyase Activity

PubMed Central

Suh, Jung H.; Eltanawy, Abeer; Rangan, Apoorva; Saba, Julie D.

2015-01-01

A new technique for quantifying sphingosine phosphate lyase activity in biological samples is described. In this procedure, 2-hydrazinoquinoline is used to convert (2E)-hexadecenal into the corresponding hydrazone derivative to improve ionization efficiency and selectivity of detection. Combined utilization of liquid chromatographic separation and multiple reaction monitoring-mass spectrometry allows for simultaneous quantification of the substrate S1P and product (2E)-hexadecenal. Incorporation of (2E)-d5-hexadecenal as an internal standard improves detection accuracy and precision. A simple one-step derivatization procedure eliminates the need for further extractions. Limits of quantification for (2E)-hexadecenal and sphingosine-1-phosphate are 100 and 50 fmol, respectively. The assay displays a wide dynamic detection range useful for detection of low basal sphingosine phosphate lyase activity in wild type cells, SPL-overexpressing cell lines, and wild type mouse tissues. Compared to current methods, the capacity for simultaneous detection of sphingosine-1-phosphate and (2E)-hexadecenal greatly improves the accuracy of results and shows excellent sensitivity and specificity for sphingosine phosphate lyase activity detection. PMID:26408264

Hematopoietic Sphingosine 1-Phosphate Lyase Deficiency Decreases Atherosclerotic Lesion Development in LDL-Receptor Deficient Mice

PubMed Central

Bot, Martine; Van Veldhoven, Paul P.; de Jager, Saskia C. A.; Johnson, Jason; Nijstad, Niels; Van Santbrink, Peter J.; Westra, Marijke M.; Van Der Hoeven, Gerd; Gijbels, Marion J.; Müller-Tidow, Carsten; Varga, Georg; Tietge, Uwe J. F.; Kuiper, Johan; Van Berkel, Theo J. C.; Nofer, Jerzy-Roch

2013-01-01

Aims Altered sphingosine 1-phosphate (S1P) homeostasis and signaling is implicated in various inflammatory diseases including atherosclerosis. As S1P levels are tightly controlled by S1P lyase, we investigated the impact of hematopoietic S1P lyase (Sgpl1−/−) deficiency on leukocyte subsets relevant to atherosclerosis. Methods and Results LDL receptor deficient mice that were transplanted with Sgpl1−/− bone marrow showed disrupted S1P gradients translating into lymphopenia and abrogated lymphocyte mitogenic and cytokine response as compared to controls. Remarkably however, Sgpl1−/− chimeras displayed mild monocytosis, due to impeded stromal retention and myelopoiesis, and plasma cytokine and macrophage expression patterns, that were largely compatible with classical macrophage activation. Collectively these two phenotypic features of Sgpl1 deficiency culminated in diminished atherogenic response. Conclusions Here we not only firmly establish the critical role of hematopoietic S1P lyase in controlling S1P levels and T cell trafficking in blood and lymphoid tissue, but also identify leukocyte Sgpl1 as critical factor in monocyte macrophage differentiation and function. Its, partly counterbalancing, pro- and anti-inflammatory activity spectrum imply that intervention in S1P lyase function in inflammatory disorders such as atherosclerosis should be considered with caution. PMID:23700419

Isolation of Nicotiana plumbaginifolia cDNAs encoding isoforms of serine acetyltransferase and O-acetylserine (thiol) lyase in a yeast two-hybrid system with Escherichia coli cysE and cysK genes as baits.

PubMed

Liszewska, Frantz; Gaganidze, Dali; Sirko, Agnieszka

2005-01-01

We applied the yeast two-hybrid system for screening of a cDNA library of Nicotiana plumbaginifolia for clones encoding plant proteins interacting with two proteins of Escherichia coli: serine acetyltransferase (SAT, the product of cysE gene) and O-acetylserine (thiol)lyase A, also termed cysteine synthase (OASTL-A, the product of cysK gene). Two plant cDNA clones were identified when using the cysE gene as a bait. These clones encode a probable cytosolic isoform of OASTL and an organellar isoform of SAT, respectively, as indicated by evolutionary trees. The second clone, encoding SAT, was identified independently also as a "prey" when using cysK as a bait. Our results reveal the possibility of applying the two-hybrid system for cloning of plant cDNAs encoding enzymes of the cysteine synthase complex in the two-hybrid system. Additionally, using genome walking sequences located upstream of the sat1 cDNA were identified. Subsequently, in silico analyses were performed aiming towards identification of the potential signal peptide and possible location of the deduced mature protein encoded by sat1.

Characterization of C-S lyase from Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365 and its potential role in food flavour applications.

PubMed

Allegrini, Alessandra; Astegno, Alessandra; La Verde, Valentina; Dominici, Paola

2017-04-01

Volatile thiols have substantial impact on the aroma of many beverages and foods. Thus, the control of their formation, which has been linked to C-S lyase enzymatic activities, is of great significance in industrial applications involving food flavours. Herein, we have carried out a spectroscopic and functional characterization of a putative pyridoxal 5'-phosphate (PLP)-dependent C-S lyase from the lactic acid bacterium Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365 (LDB C-S lyase). Recombinant LDB C-S lyase exists as a tetramer in solution and shows spectral properties of enzymes containing PLP as cofactor. The enzyme has a broad substrate specificity toward sulphur-containing amino acids with aminoethyl-L-cysteine and L-cystine being the most effective substrates over L-cysteine and L-cystathionine. Notably, the protein also reveals cysteine-S-conjugate β-lyase activity in vitro, and is able to cleave a cysteinylated substrate precursor into the corresponding flavour-contributing thiol, with a catalytic efficiency higher than L-cystathionine. Contrary to similar enzymes of other lactic acid bacteria however, LDB C-S lyase is not capable of α,γ-elimination activity towards L-methionine to produce methanethiol, which is a significant compound in flavour development. Based on our results, future developments can be expected regarding the flavour-forming potential of Lactobacillus C-S lyase and its use in enhancing food flavours. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Structural and Molecular Basis for the Novel Catalytic Mechanism and Evolution of DddP, an Abundant Peptidase-Like Bacterial Dimethylsulfoniopropionate Lyase: A New Enzyme from an Old Fold

NASA Astrophysics Data System (ADS)

Zhang, Y. Z.; Wang, P.; Chen, X. L.; Li, C. Y.; Gao, X.; Zhu, D.; Xie, B. B.; Qin, Q. L.; Zhang, X. Y.; Su, H. N.; Zhou, B. C.; Xun, L.

2015-12-01

The microbial cleavage of dimethylsulfoniopropionate (DMSP) generates volatile dimethyl sulfide (DMS) and is an important step in global sulfur and carbon cycles. DddP is a DMSP lyase in marine bacteria and the deduced dddP gene product is abundant in marine metagenomic data sets. However, DddP belongs to the M24 peptidase family according to sequence alignment. Peptidases hydrolyze C-N bonds but DddP is deduced to cleave C-S bonds. Mechanisms responsible for this striking functional shift are currently unknown. We determined the structures of DMSP lyase RlDddP (the DddP from Ruegeria lacuscaerulensis ITI_1157) bound to inhibitory 2-(N-morpholino) ethanesulfonic acid or PO43- and of two mutants of RlDddP bound to acrylate. Based on structural, mutational and biochemical analyses, we characterized a new ion-shift catalytic mechanism of RlDddP for DMSP cleavage. Further, we suggested the structural mechanism leading to the loss of peptidase activity and the subsequent development of DMSP lyase activity in DddP. This study sheds light on the catalytic mechanism and the divergent evolution of DddP, leading to a better understanding of marine bacterial DMSP catabolism and global DMS production.

Genetics Home Reference: 17 alpha-hydroxylase/17,20-lyase deficiency

MedlinePlus

... Center Frequency 17α-hydroxylase/17,20-lyase deficiency accounts for about 1 percent of congenital adrenal hyperplasia cases. It is estimated to occur in 1 in 1 million people worldwide. Related Information What information about a genetic condition can statistics ...

Fabrication of large size alginate beads for three-dimensional cell-cluster culture

NASA Astrophysics Data System (ADS)

Zhang, Zhengtao; Ruan, Meilin; Liu, Hongni; Cao, Yiping; He, Rongxiang

2017-08-01

We fabricated large size alginate beads using a simple microfluidic device under a co-axial injection regime. This device was made by PDMS casting with a mold formed by small diameter metal and polytetrafluorothylene tubes. Droplets of 2% sodium alginate were generated in soybean oil through the device and then cross-linked in a 2% CaCl2 solution, which was mixed tween80 with at a concentration of 0.4 to 40% (w/v). Our results showed that the morphology of the produced alginate beads strongly depends on the tween80 concentration. With the increase of concentration of tween80, the shape of the alginate beads varied from semi-spherical to tailed-spherical, due to the decrease of interface tension between oil and cross-link solution. To access the biocompatibility of the approach, MCF-7 cells were cultured with the alginate beads, showing the formation of cancer cells clusters which might be useful for future studies.

Effect of Oxygen Tension and Medium Components on Monomer Distribution of Alginate.

PubMed

Kıvılcımdan Moral, Çiğdem; Doğan, Özdemir; Sanin, Faika Dilek

2015-06-01

Alginate is a natural biopolymer composed of mannuronic and guluronic acid monomers. It is produced by algae and some species of Azotobacter and Pseudomonas. This study aims to investigate the effect of dissolved oxygen tension (DOT) and growth medium substrate and calcium concentrations on the monomeric composition of alginate produced by Azotobacter vinelandii ATCC® 9046 in a fermenter. Results showed that alginate production increased with increasing DOT from 1 to 5 %. The highest alginate production was obtained as 4.51 g/L under 20 g/L of sucrose and 50 mg/L of calcium at 5 % DOT. At these conditions, alginate was rich in mannuronic acid (up to 61 %) and it was particularly high at low calcium concentration. On the other hand, at extreme conditions such as high DOT level (10 % DOT) and low sucrose concentration (10 g/L), guluronic acid was dominant (ranging between 65 and 100 %).

Granular gel support-enabled extrusion of three-dimensional alginate and cellular structures.

PubMed

Jin, Yifei; Compaan, Ashley; Bhattacharjee, Tapomoy; Huang, Yong

2016-06-03

Freeform fabrication of soft structures has been of great interest in recent years. In particular, it is viewed as a critical step toward the grand vision of organ printing--the on-demand design and fabrication of three-dimensional (3D) human organ constructs for implantation and regenerative medicine. The objective of this study is to develop a novel granular gel support material-enabled, two-step gelation-based 'printing-then-gelation' approach to fabricate 3D alginate structures using filament extrusion. Specifically, a granular Carbopol microgel bath holds the ungelled alginate structure being extruded, avoiding the instantaneous gelation of each printed layer as well as resultant surface tension-induced nozzle clogging. Since Carbopol microgels react with multivalent cations, which are needed for alginate crosslinking, gelatin is introduced as a sacrificial material to make an alginate and gelatin bioink for extrusion, which gels thermally (step-one gelation) to initially stabilize the printed structure for removal from Carbopol. Then gelatin is melted and diffused away while alginate is ionically crosslinked in a 37 °C calcium chloride bath (step-two gelation), resulting in an alginate structure. The proposed 'printing-then-gelation' approach works for alginate structure fabrication, and it is also applicable for the printing of cellular constructs and other similar homogeneous soft structures using a two-step or even multi-step approach. The main conclusions are: (1) 0.8% (w/v) Carbopol bath with a neutral pH value may be most suitable for soft structure printing; (2) it is most effective to use a 0.9% (w/v) NaCl solution to facilitate the removal of residual Carbopol; and (3) alginate structures fabricated using the proposed approach demonstrate better mechanical properties than those fabricated using the conventional 'gelation-while-printing' approach.

In vitro fermentation of alginate and its derivatives by human gut microbiota.

PubMed

Li, Miaomiao; Li, Guangsheng; Shang, Qingsen; Chen, Xiuxia; Liu, Wei; Pi, Xiong'e; Zhu, Liying; Yin, Yeshi; Yu, Guangli; Wang, Xin

2016-06-01

Alginate (Alg) has a long history as a food ingredient in East Asia. However, the human gut microbes responsible for the degradation of alginate and its derivatives have not been fully understood yet. Here, we report that alginate and the low molecular polymer derivatives of mannuronic acid oligosaccharides (MO) and guluronic acid oligosaccharides (GO) can be completely degraded and utilized at various rates by fecal microbiota obtained from six Chinese individuals. However, the derivative of propylene glycol alginate sodium sulfate (PSS) was not hydrolyzed. The bacteria having a pronounced ability to degrade Alg, MO and GO were isolated from human fecal samples and were identified as Bacteroides ovatus, Bacteroides xylanisolvens, and Bacteroides thetaiotaomicron. Alg, MO and GO can increase the production level of short chain fatty acids (SCFA), but GO generates the highest level of SCFA. Our data suggest that alginate and its derivatives could be degraded by specific bacteria in the human gut, providing the basis for the impacts of alginate and its derivates as special food additives on human health. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Correlation between adenylosuccinate lyase (ADSL) gene polymorphism and inosine monophosphate acid (IMP) content in domestic fowl and genetic relationship between red jungle fowl and domestic fowl].

PubMed

Shu, Jing-Ting; Bao, Wen-Bin; Zhang, Hong-Xia; Zhang, Xue-Yu; Ji, Cong-Liang; Chen, Guo-Hong

2007-03-01

This study investigates single nucleotide polymorphism (SNP) of the adenylosuccinate lyase(ADSL) gene in variety chicken breeds, including Recessive White chickens, Silkies chickens, Baier chickens, Tibetan chickens and two red jungle fowls. Primers for exon 2 in ADSL gene were designed based on the chicken genomic sequence and a SNP(C/T at 3484) was detected by PCR-SSCP and DNA sequencing. Three genotypes within all breeds were found and least square analysis showed that TT genotype birds had a significant higher inosine monophosphate acid (IMP) content than TC (P < 0.01) and CC (P < 0.05) genotype birds, TC genotype birds had a little higher IMP content than CC genotype birds, but the difference was not significant. We proposed this SNP site correlated with IMP content in chickens. A neighbour-joining dendrogram was constructed based on the Nei's genentic distance. The genetic relationship between Chinese red jungle fowl and Tibetan chickens was the nearest, whereas Baier chickens were more closer to Silkies chickens. The Chinese red jungle fowls were relatively closer to the domestic fowls, whereas Thailand red jungle fowls were relatively diverging to the Chinese native breeds. These results supported the theory concerning the independent origins of Chinese native fowl breeds.

Self-emulsifying excipient platform for improving technological properties of alginate-hydroxypropylcellulose pellets.

PubMed

Mannina, Paolo; Segale, Lorena; Giovannelli, Lorella; Bonda, Andrea Foglio; Pattarino, Franco

2016-02-29

In this work, alginate, alginate-pectin and alginate-hydroxypropylcellulose pellets were produced by ionotropic gelation and characterized. Ibuprofen was selected as model drug; it was suspended in the polymeric solution in crystalline form or dissolved in a self-emulsifying phase and then dispersed into the polymeric solution. The self-emulsifying excipient platform composed of Labrasol (PEG-8 caprylic/capric glycerides) and d-α-tocopherol polyethylene glycol 1000 succinate (TPGS), able to solubilize the drug was used to improve the technological and biopharmaceutical properties of the alginate pellets. The pellets had diameters between 1317 and 2026 μm and a high drug content (>51%). DSC analysis showed the amorphous state of drug in the pellets containing the self-emulsifying phase. All the systems restricted drug release in conditions simulating the gastric environment and made the drug completely available at a pH value typical for the intestine. Only alginate-HPC systems containing the drug solubilized into the self-emulsifying phase showed the ability to partially control the release of ibuprofen at neutral pH. The self-emulsifying excipient platform is a useful tool to improve technological and biopharmaceutical properties of alginate-HPC pellets. Copyright © 2015 Elsevier B.V. All rights reserved.

In vivo degradation of alginate in the presence and in the absence of resistant starch.

PubMed

Jonathan, Melliana; Souza da Silva, Carol; Bosch, Guido; Schols, Henk; Gruppen, Harry

2015-04-01

This study evaluated the intestinal degradability of alginate during 74 days intake in pigs as models for humans. Diets contained pregelatinized starch, retrograded starch, alginate, or a mix of retrograded starch and alginate. Faeces were collected on day 1, 3, 7, 14, 39 and 74. Clear trends in intestinal alginate degradation were observed. Up to day 39, the total tract digestibility of alginate was limited (0.52 ± 0.10), and was lower with the inclusion of retrograded starch in the diet (0.34 ± 0.02). More than 90% of the faecal alginate was insoluble in water, which may explain the low digestibility of the alginate. The digestibility of mannuronic acid (M) was 2-3 times higher than that of guluronic acid (G). The changes of G:M ratio and the relative amounts of alginate oligosaccharides between day 39 and 74 indicated that the microbiota needed more than 39 days to adapt to alginate. This study demonstrated that in-depth analyses of dietary fibres are valuable in understanding the fate of the dietary fibres in the large intestine as it was shown that degradation of a dietary fibre depends not only on the properties of the fibre itself, but also on the other dietary fibres present in the diet and the adaptation time. Copyright © 2014 Elsevier Ltd. All rights reserved.

Clinical and laboratory studies of the antacid and raft-forming properties of Rennie alginate suspension.

PubMed

Tytgat, G N; Simoneau, G

2006-03-15

Acid pockets at the gastro-oesophageal junction escape buffering from meals in the stomach. Combining high-dose antacid with alginate may therefore be of benefit in gastro-oesophageal reflux disease. To characterize the antacid and raft-forming properties of Rennie alginate suspension (containing high-dose antacid and alginate; Bayer Consumer Care, Bladel, the Netherlands). The in vitro acid-neutralizing capacity of Rennie algniate was compared with Gaviscon (Reckitt Benckiser, Slough, UK) by pH-recorded HCl titration. Alginate raft weight formed in vitro at different pH was used to evaluate the pH dependency of raft formation with each product. A double-blind, placebo-controlled, randomized crossover study also compared the antacid activity of Rennie alginate vs. placebo in vivo using continuous intragastric pH monitoring in 12 healthy fasting volunteers. Compared with Gaviscon, Rennie alginate had a higher acid-neutralizing capacity, greater maximum pH and longer duration of antacid activity in vitro. However, the two products produced comparable alginate rafts at each pH evaluated. In vivo, Rennie alginate provided rapid, effective and long-lasting acid neutralization, with an onset of action of <5 min, and duration of action of almost 90 min. The dual mode of action of Rennie alginate offers an effective treatment option for mild symptomatic gastro-oesophageal reflux disease particularly considering recent findings regarding 'acid pockets'.

Requirement for two or more Erwinia carotovora subsp. carotovora pectolytic gene products for maceration of potato tuber tissue by Escherichia coli.

PubMed

Roberts, D P; Berman, P M; Allen, C; Stromberg, V K; Lacy, G H; Mount, M S

1986-07-01

Several genes encoding enzymes capable of degrading plant cell wall components have been cloned from Erwinia carotovora subsp. carotovora EC14. Plasmids containing cloned EC14 DNA mediate the production of endo-pectate lyases, exo-pectate lyase, endo-polygalacturonase, and cellulase(s). Escherichia coli strains containing one of these plasmids or combinations of two plasmids were tested for their ability to macerate potato tuber slices. Only one E. coli strain, containing two plasmids that encode endo-pectate lyases, exo-pectate lyase, and endo-polygalacturonase, caused limited maceration. The pectolytic proteins associated with one of these plasmids, pDR1, have been described previously (D. P. Roberts, P. M. Berman, C. Allen, V. K. Stromberg, G. H. Lacy, and M. S. Mount, Can. J. Plant Pathol. 8:17-27, 1986) and include two secreted endo-pectate lyases. The second plasmid, pDR30, contains a 2.1-kilobase EC14 DNA insert that mediates the production of an exo-pectate lyase and an endo-polygalacturonase. These enzymes are similar in physicochemical properties to those produced by EC14. Our results suggest that the concerted activities of endo-pectate lyases with endo-polygalacturonase or exo-pectate lyase or both cause maceration.

Inhibitory effects of laminaran and alginate on production of putrefactive compounds from soy protein by intestinal microbiota in vitro and in rats.

PubMed

Nakata, Toru; Kyoui, Daisuke; Takahashi, Hajime; Kimura, Bon; Kuda, Takashi

2016-06-05

Soybean is one of the major components of the Japanese diet. In traditional Japanese cuisine, soybean-based food items are often consumed with brown algae. In this study, we examined the effect of water-soluble and fermentable polysaccharides, laminaran and sodium alginate, from brown algae, on putrefactive compound production, by human faecal microbiota in broth containing 3% (w/v) soy protein. We also investigated the effect of 2% laminaran or alginate diet on caecal putrefactive compounds in rats maintained on diets containing 20% (w/w) soy protein. The caecal microbiota was also analysed using denaturing gradient gel electrophoresis and pyrosequencing with primers targeting the bacterial 16S rRNA gene. The polysaccharides, particularly laminaran, inhibited ammonia, phenol, and indole production by human faecal microbiota. Both the algal polysaccharides lowered the caecal indole content. Laminaran was found to increase the number of Coprobacter, whereas Helicobacter was found to decrease in the presence of both laminaran and sodium alginate. Copyright © 2016 Elsevier Ltd. All rights reserved.

High surface area mesoporous activated carbon-alginate beads for efficient removal of methylene blue.

PubMed

Nasrullah, Asma; Bhat, A H; Naeem, Abdul; Isa, Mohamed Hasnain; Danish, Mohammed

2018-02-01

High surface area mesoporous activated carbon-alginate (AC-alginate) beads were successfully synthesized by entrapping activated carbon powder derived from Mangosteen fruit peel into calcium-alginate beads for methylene blue (MB) removal from aqueous solution. The structure and surface characteristics of AC-alginate beads were analyzed using Fourier transform infra-red (FTIR) spectroscopy, scanning electron microscopy (SEM) and surface area analysis (S BET ), while thermal properties were tested using thermogravimetric analysis (TGA). The effect of AC-alginate dose, pH of solution, contact time, initial concentration of MB solution and temperature on MB removal was elucidated. The results showed that the maximum adsorption capacity of 230mg/g was achieved for 100mg/L of MB solution at pH 9.5 and temperature 25°C. Furthermore, the adsorption of MB on AC-alginate beads followed well pseudo-second order equation and equilibrium adsorption data were better fitted by the Freundlich isotherm model. The findings reveal the feasibility of AC-alginate beads composite to be used as a potential and low cost adsorbent for removal of cationic dyes. Copyright © 2017 Elsevier B.V. All rights reserved.

The relationship between digital model accuracy and time-dependent deformation of alginate impressions.

PubMed

Alcan, Toros; Ceylanoğlu, Cenk; Baysal, Bekir

2009-01-01

To investigate the effects of different storage periods of alginate impressions on digital model accuracy. A total of 105 impressions were taken from a master model with three different brands of alginates and were poured into stone models in five different storage periods. In all, 21 stone models were poured and immediately were scanned, and 21 digital models were prepared. The remaining 84 impressions were poured after 1, 2, 3, and 4 days, respectively. Five linear measurements were made by three researchers on the master model, the stone models, and the digital models. Time-dependent deformation of alginate impressions at different storage periods and the accuracy of traditional stone models and digital models were evaluated separately. Both the stone models and the digital models were highly correlated with the master model. Significant deformities in the alginate impressions were noted at different storage periods of 1 to 4 days. Alginate impressions of different brands also showed significant differences between each other on the first, third, and fourth days. Digital orthodontic models are as reliable as traditional stone models and probably will become the standard for orthodontic clinical use. Storing alginate impressions in sealed plastic bags for up to 4 days caused statistically significant deformation of alginate impressions, but the magnitude of these deformations did not appear to be clinically relevant and had no adverse effect on digital modeling.

Encapsulation of Lactobacillus kefiri in alginate microbeads using a double novel aerosol technique.

PubMed

Demitri, Christian; Lamanna, Leonardo; De Benedetto, Egidio; Damiano, Fabrizio; Cappello, Maria Stella; Siculella, Luisa; Sannino, Alessandro

2017-08-01

Alginate micro beads containing Lactobacillus kefiri (the principal bacteria present in the kefir probiotic drink) were produced by a novel technique based on dual aerosols spaying of alginate based solution and CaCl 2 as cross linking agent. Carboxymethylcellulose (CMC) has been also added to the alginate in order to change the physic-chemical properties (viscosity and permeability) of the microbeads. Calcium alginate and CMC are biopolymers that can be used for developing oral drug-delivery systems. These biopolymers have been reported to show a pH-dependent swelling behaviour. Calcium alginate and CMC have also been known to possess an excellent mucoadhesive property. The loaded microbeads have been characterized in terms of morphology, chemical composition and stability in different conditions mimicking the gastric environment. In this study, we demonstrate the feasibility of a continuous fabrication of alginate microbeads in a range of 50-70μm size, encapsulating L. kefiri as active ingredient. The technique involves the use of a double aerosols of alginate based solution and CaCl 2 as crosslinking agent. Moreover, the encapsulation process was proved to be effective and not detrimental to bacteria viability. At the same time, it was verified the protective efficacy of the microcapsules against the gastric environment using both SGF pH1.2 (fasted state) and pH2.2 (feed state). Copyright © 2017 Elsevier B.V. All rights reserved.

Antimicrobial cerium ion-chitosan crosslinked alginate biopolymer films: A novel and potential wound dressing.

PubMed

Kaygusuz, Hakan; Torlak, Emrah; Akın-Evingür, Gülşen; Özen, İlhan; von Klitzing, Regine; Erim, F Bedia

2017-12-01

Wound dressings require good antiseptic properties, mechanical strength and, more trustably, natural material ingredients. Antimicrobial properties of cerium ions and chitosan are known and alginate based wound dressings are commercially available. In this study, the advantages of these materials were combined and alginate films were crosslinked with cerium(III) solution and chitosan added cerium(III) solution. Films were characterized by Fourier transform infrared spectroscopy (FTIR), light transmittance, scanning electron microscopy (SEM), swelling experiments, water vapor transmittance tests, and mechanical stretching tests. The antibacterial and physical properties of the films were compared with those of conventional calcium alginate films. Both cerium ion crosslinked and cerium ion-chitosan crosslinked alginate films gained antibacterial activity against Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria. Cerium alginate-chitosan films showed high resistance to being deformed elastically. Results show that cerium alginate-chitosan films can be flexible, ultraviolet-protecting, and antibacterial wound dressings. Copyright © 2017 Elsevier B.V. All rights reserved.

Alginate Microcapsules Incorporating Hyaluronic Acid Recreate Closer in Vivo Environment for Mesenchymal Stem Cells.

PubMed

Cañibano-Hernández, Alberto; Saenz Del Burgo, Laura; Espona-Noguera, Albert; Orive, Gorka; Hernández, Rosa M; Ciriza, Jesús; Pedraz, Jose Luis

2017-07-03

The potential clinical application of alginate cell microencapsulation has advanced enormously during the past decade. However, the 3D environment created by alginate beads does not mimic the natural extracellular matrix surrounding cells in vivo, responsible of cell survival and functionality. As one of the most frequent macromolecules present in the extracellular matrix is hyaluronic acid, we have formed hybrid beads with alginate and hyaluronic acid recreating a closer in vivo cell environment. Our results show that 1% alginate-0.25% hyaluronic acid microcapsules retain 1.5% alginate physicochemical properties. Moreover, mesenchymal stem cells encapsulated in these hybrid beads show enhanced viability therapeutic protein release and mesenchymal stem cells' potential to differentiate into chondrogenic lineage. Although future studies with additional proteins need to be done in order to approach even more the extracellular matrix features, we have shown that hyaluronic acid protects alginate encapsulated mesenchymal stem cells by providing a niche-like environment and remaining them competent as a sustainable drug delivery system.

Applications of alginate in bioseparation of proteins.

PubMed

Jain, Sulakshana; Mondal, Kalyani; Gupta, Munishwar N

2006-01-01

Alginate is a polysaccharide that is a block polymer consisting of block units of guluronic acid and mannuronic acid. It shows inherent biological affinity for a variety of enzymes such as pectinase, lipase, phospholipase D, a and ss amylases and glucoamylase. Taking advantage of its precipitation with Ca2+ and the above-mentioned property, alginate has been used for purification of these enzymes by affinity precipitation, aqueous two phase separation, macroaffinity ligand facilitated three phase partitioning, immobilized metal affinity chromatography and expanded bed affinity chromatography. Thus, this versatile marine resource has tremendous potential in bioseparation of proteins.

Nanocrystalline cellulose (NCC) reinforced alginate based biodegradable nanocomposite film.

PubMed

Huq, Tanzina; Salmieri, Stephane; Khan, Avik; Khan, Ruhul A; Le Tien, Canh; Riedl, Bernard; Fraschini, Carole; Bouchard, Jean; Uribe-Calderon, Jorge; Kamal, Musa R; Lacroix, Monique

2012-11-06

Nanocrystalline cellulose (NCC) reinforced alginate-based nanocomposite film was prepared by solution casting. The NCC content in the matrix was varied from 1 to 8% ((w/w) % dry matrix). It was found that the nanocomposite reinforced with 5 wt% NCC content exhibits the highest tensile strength which was increased by 37% compared to the control. Incorporation of NCC also significantly improved water vapor permeability (WVP) of the nanocomposite showing a 31% decrease due to 5 wt% NCC loading. Molecular interactions between alginate and NCC were supported by Fourier Transform Infrared Spectroscopy. The X-ray diffraction studies also confirmed the appearance of crystalline peaks due to the presence of NCC inside the films. Thermal stability of alginate-based nanocomposite films was improved after incorporation of NCC. Copyright © 2012 Elsevier Ltd. All rights reserved.

Investigation of cell viability and morphology in 3D bio-printed alginate constructs with tunable stiffness.

PubMed

Shi, Pujiang; Laude, Augustinus; Yeong, Wai Yee

2017-04-01

In this article, mouse fibroblast cells (L929) were seeded on 2%, 5%, and 10% alginate hydrogels, and they were also bio-printed with 2%, 5%, and 10% alginate solutions individually to form constructs. The elastic and viscous moduli of alginate solutions, their interior structure and stiffness, interactions of cells and alginate, cell viability, migration and morphology were investigated by rheometer, MTT assay, scanning electron microscope (SEM), and fluorescent microscopy. The three types of bio-printed scaffolds of distinctive stiffness were prepared, and the seeded cells showed robust viability either on the alginate hydrogel surfaces or in the 3D bio-printed constructs. Majority of the proliferated cells in the 3D bio-printed constructs weakly attached to the surrounding alginate matrix. The concentration of alginate solution and hydrogel stiffness influenced cell migration and morphology, moreover the cells formed spheroids in the bio-printed 10% alginate hydrogel construct. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1009-1018, 2017. © 2017 Wiley Periodicals, Inc.

Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site

PubMed Central

Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

2016-01-01

Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2 domains reveal that the (HhH)2 domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2 domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2 domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2 domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide. PMID:26908655

Assessment of alginate hydrogel degradation in biological tissue using viscosity-sensitive fluorescent dyes

NASA Astrophysics Data System (ADS)

Shkand, Tatiana V.; Chizh, Mykola O.; Sleta, Iryna V.; Sandomirsky, Borys P.; Tatarets, Anatoliy L.; Patsenker, Leonid D.

2016-12-01

The main goal of this study is to investigate a combination of viscosity-sensitive and viscosity-insensitive fluorescent dyes to distinguish different rheological states of hydrogel based biostructural materials and carriers in biological tissues and to assess their corresponding location areas. The research is done in the example of alginate hydrogel stained with viscosity-sensitive dyes Seta-470 and Seta-560 as well as the viscosity-insensitive dye Seta-650. These dyes absorb/emit at 469/518, 565/591 and 651/670 nm, respectively. The rheological state of the alginate, the area of the fluorescence signal and the mass of the dense alginate versus the calcium gluconate concentration utilized for alginate gelation were studied in vitro. The most pronounced change in the fluorescence signal area was found at the same concentrations of calcium gluconate (below ~1%) as the change in the alginate plaque mass. The stained alginate was also implanted in situ in rat hip and myocardium and monitored using fluorescence imaging. In summary, our data indicate that the viscosity sensitive dye in combination with the viscosity-insensitive dye allow tracking the biodegradation of the alginate hydrogel and determining the rheological state of hydrogel in biological tissue, which both should have relevance for research and clinical applications. Using this method we estimated the half-life of the dense alginate hydrogel in a rat hip to be in the order of 4 d and about 6-8 d in rat myocardium. The half-life of the dense hydrogel in the myocardium was found to be long enough to prevent aneurysm rupture of the left ventricle wall, one of the more severe complications of the early post-infarction period.

Physical, mechanical, and barrier properties of sodium alginate/gelatin emulsion based-films incorporated with canola oil

NASA Astrophysics Data System (ADS)

Syarifuddin, A.; Hasmiyani; Dirpan, A.; Mahendradatta, M.

2017-12-01

The development of mixed emulsion-based films formed by sodium alginate/gelatin incorporated with canola oil can offer particular properties such as water vapor barrier properties. The different ratios of sodium alginate/gelatin and sodium alginate/gelatin emulsion-based films incorporated with canola oil were developed and their effects on films’ physical, mechanical and barrier properties were assessed. Here we set out to examine whether canola oil addition and different ratio of sodium alginate/gelatin modified physical, mechanical, and barrier properties of films. To do so, the films were prepared by vary the ratio of sodium alginate/gelatin (2.5, 1, 0.5). Canola oil addition induced changes in moisture content, thickness, solubility, water vapor transmission rate (WVTR), percent elongation at break (p<0.05). In addition, it is apparent that varying ratio of sodium alginate to gelatin induced change the mechanical properties of films. The reduction of sodium alginate to gelatin decreased the tensile strength of both films. Improved values of WVTR, tensile strength and solubility at break were observed when the ratio of sodium alginate/gelatin emulsion film incorporated with canola oil was 2.5. Therefore, different ratio of sodium alginate/gelatin incorporated with canola oil can be used to tailor emulsion films with enhanced water vapor barrier and mechanical properties.

Fabrication of patterned calcium cross-linked alginate hydrogel films and coatings through reductive cation exchange.

PubMed

Bruchet, Marion; Melman, Artem

2015-10-20

Calcium cross-linked alginate hydrogels are widely used in targeted drug delivery, tissue engineering, wound treatment, and other biomedical applications. We developed a method for preparing homogeneous alginate hydrogels cross-linked with Ca(2+) cations using reductive cation exchange in homogeneous iron(III) cross-linked alginate hydrogels. Treatment of iron(III) cross-linked alginate hydrogels with calcium salts and sodium ascorbate results in reduction of iron(III) cations to iron(II) that are instantaneously replaced with Ca(2+) cations, producing homogeneous ionically cross-linking hydrogels. Alternatively, the cation exchange can be performed by photochemical reduction in the presence of calcium chloride using a sacrificial photoreductant. This approach allows fabrication of patterned calcium alginate hydrogels through photochemical patterning of iron(III) cross-linked alginate hydrogel followed by the photochemical reductive exchange of iron cations to calcium. Copyright © 2015 Elsevier Ltd. All rights reserved.

Halloysite nanotubes as carriers of vancomycin in alginate-based wound dressing.

PubMed

Kurczewska, Joanna; Pecyna, Paulina; Ratajczak, Magdalena; Gajęcka, Marzena; Schroeder, Grzegorz

2017-09-01

The influence of an inorganic support - halloysite nanotubes - on the release rate and biological activity of the antibiotic encapsulated in alginate-based dressings was studied. The halloysite samples were loaded with approx. 10 wt.% of the antibiotic and then encapsulated in Alginate and Gelatin/Alginate gels. The material functionalized with aliphatic amine significantly extended the release of vancomycin from alginate-based gels as compared to that achieved when silica was used. After 24 h, the released amounts of the antibiotic immobilized at silica reached 70%, while for the drug immobilized at halloysite the released amount of vancomycin reached 44% for Alginate discs. The addition of gelatin resulted in even more prolonged sustained release of the drug. The antibiotic was released from the system with a double barrier with Higuchi kinetic model and Fickian diffusion mechanism. Only the immobilized drug encapsulated in Alginate gel demonstrated very good antimicrobial activity against various bacteria. The inhibition zones were greater than those of the standard discs for the staphylococci and enterococci bacteria tested. The addition of gelatin adversely affected the biological activity of the system. The inhibition zones were smaller than those of the reference samples. A reduction in the drug dose by half had no significant effect on changing the release rate and microbiological activity. The in vivo toxicity studies of the material with immobilized drug were carried out with Acutodesmus acuminatus and Daphnia magna . The material studied had no effect on the living organisms used in the bioassays. The proposed system with a double barrier demonstrated high storage stability.

Subcellular Distribution of O-Acetylserine(thiol)lyase in Cauliflower (Brassica oleracea L.) Inflorescence.

PubMed

Rolland, N; Droux, M; Douce, R

1992-03-01

The subcellular localization of O-acetyiserine(thiol)lyase (EC 4.2.99.8) in nongreen tissue from higher plants has been studied using purified proplastids, mitochondria, and protoplasts from cauliflower (Brassica oleracea L.) buds as a source of subcellular fractions. O-Acetylserine(thiol)lyase has been detected in both organelles (proplastids and mitochondria) and a cytosolic extract obtained by protoplast fractionation. We confirmed these observations, demonstrating that a form of the enzyme different in global charge and separated from others by anion-exchange chromatography corresponded to each subcellular location. Our observations are consistent with the need for cysteine biosynthesis in each subcellular compartment where the synthesis of proteins occurs.

ATP-Citrate Lyase Controls a Glucose-to-Acetate Metabolic Switch.

PubMed

Zhao, Steven; Torres, AnnMarie; Henry, Ryan A; Trefely, Sophie; Wallace, Martina; Lee, Joyce V; Carrer, Alessandro; Sengupta, Arjun; Campbell, Sydney L; Kuo, Yin-Ming; Frey, Alexander J; Meurs, Noah; Viola, John M; Blair, Ian A; Weljie, Aalim M; Metallo, Christian M; Snyder, Nathaniel W; Andrews, Andrew J; Wellen, Kathryn E

2016-10-18

Mechanisms of metabolic flexibility enable cells to survive under stressful conditions and can thwart therapeutic responses. Acetyl-coenzyme A (CoA) plays central roles in energy production, lipid metabolism, and epigenomic modifications. Here, we show that, upon genetic deletion of Acly, the gene coding for ATP-citrate lyase (ACLY), cells remain viable and proliferate, although at an impaired rate. In the absence of ACLY, cells upregulate ACSS2 and utilize exogenous acetate to provide acetyl-CoA for de novo lipogenesis (DNL) and histone acetylation. A physiological level of acetate is sufficient for cell viability and abundant acetyl-CoA production, although histone acetylation levels remain low in ACLY-deficient cells unless supplemented with high levels of acetate. ACLY-deficient adipocytes accumulate lipid in vivo, exhibit increased acetyl-CoA and malonyl-CoA production from acetate, and display some differences in fatty acid content and synthesis. Together, these data indicate that engagement of acetate metabolism is a crucial, although partial, mechanism of compensation for ACLY deficiency. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Factors affecting the morphology of isocitrate lyase crystals

NASA Technical Reports Server (NTRS)

Demattei, Robert C.; Feigelson, Robert S.; Weber, Patricia C.

1992-01-01

Isocitrate lyase crystals have been grown by the hanging drop vapor equilibration method in both 1-g and microgravity and by vapor equilibrium in small capillaries. The crystal morphologies obtained have ranged from dendritic to 'octagonal' prisms. Theoretical evaporation models have been applied to these growth regimes. The results of these analyses along with other experimental results, indicate the factors which must be controlled to produce good growth morphologies.

Characterisation of bare and tannase-loaded calcium alginate beads by microscopic, thermogravimetric, FTIR and XRD analyses.

PubMed

Larosa, Claudio; Salerno, Marco; de Lima, Juliana Silva; Merijs Meri, Remo; da Silva, Milena Fernandes; de Carvalho, Luiz Bezerra; Converti, Attilio

2018-08-01

Incorporating enzymes into calcium alginate beads is an effective method to immobilise them and to preserve, at the same time, their catalytic activity. Sodium alginate was mixed with Aspergillus ficuum tannase in aqueous solution, and tannase-loaded calcium alginate beads were prepared using a simple droplet-based microfluidic system. Extensive experimental analysis was carried out to characterise the samples. Microscopic imaging revealed morphological differences between the surfaces of bare alginate matrix and tannase-loaded alginate beads. Thermal analysis allowed assessing the hydration contents of alginate and revealed the presence of tannase entrapped in the loaded beads, which was confirmed by vibrational spectroscopy. X-ray diffraction allowed us to conclude that alginate of tannase-loaded beads is not crystalline, which would make them suitable as carriers for possible controlled release. Moreover, they could be used in food applications to improve tea quality or clarify juices. Copyright © 2018 Elsevier B.V. All rights reserved.

Digital microfluidic three-dimensional cell culture and chemical screening platform using alginate hydrogels

PubMed Central

2015-01-01

Electro wetting-on-dielectric (EWOD) digital microfluidics (DMF) can be used to develop improved chemical screening platforms using 3-dimensional (3D) cell culture. Alginate hydrogels are one common method by which a 3D cell culture environment is created. This paper presents a study of alginate gelation on EWOD DMF and investigates designs to obtain uniform alginate hydrogels that can be repeatedly addressed by any desired liquids. A design which allows for gels to be retained in place during liquid delivery and removal without using any physical barriers or hydrophilic patterning of substrates is presented. A proof of concept screening platform is demonstrated by examining the effects of different concentrations of a test chemical on 3D cells in alginate hydrogels. In addition, the temporal effects of the various chemical concentrations on different hydrogel posts are demonstrated, thereby establishing the benefits of an EWOD DMF 3D cell culture and chemical screening platform using alginate hydrogels. PMID:25945142

Effects of alginate on frozen-thawed boar spermatozoa quality, lipid peroxidation and antioxidant enzymes activities.

PubMed

Hu, Jinghua; Geng, Guoxia; Li, Qingwang; Sun, Xiuzhu; Cao, Hualin; Liu, Yawei

2014-06-30

Although alginate was reported to play an important role as free radical scavengers in vitro and could be used as sources of natural antioxidants, there was no study about the cryoprotective effects of alginate on boar spermatozoa freezing. The objective of this research was to evaluate the effects of different concentrations of alginate added to the freezing extenders on boar spermatozoa motility, plasma membrane integrity, acrosomal integrity, mitochondrial activities, lipid peroxidation and antioxidative enzymes activities (SOD and GSH-Px) after thawing. Alginate was added to the TCG extender to yield six different final concentrations: 0, 0.2, 0.4, 0.6, 0.8, and 1.0mg/mL. The semen extender supplemented with various doses of alginate increased (P<0.05) total motility. The spermatozoa plasma membrane integrity and mitochondrial activity were improved at four different concentrations: 0.4, 0.6, 0.8, 1.0mg/mL. The addition of alginate also provided significantly positive effect on post-thaw boar spermatozoa acrosomal integrity at concentrations of 0.6, 0.8, 1.0mg/mL, compared with that of the control (P<0.05). The freezing extenders with the presence of alginate led to higher SOD and GSH-Px activities and lower MDA levels, in comparison to the control (P<0.05). In summary, alginate exhibited a dose-related response on frozen-thawed boar spermatozoa motility, functional integrity and antioxidative capacity at appropriate concentrations. Therefore alginate could be employed as an effective cryoprotectant in boar spermatozoa cryopreservation. Copyright © 2014 Elsevier B.V. All rights reserved.

Altered Fermentative Metabolism in Chlamydomonas reinhardtii Mutants Lacking Pyruvate Formate Lyase and Both Pyruvate Formate Lyase and Alcohol Dehydrogenase

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

Catalanotti, C.; Dubini, A.; Subramanian, V.

2012-02-01

Chlamydomonas reinhardtii, a unicellular green alga, often experiences hypoxic/anoxic soil conditions that activate fermentation metabolism. We isolated three Chlamydomonas mutants disrupted for the pyruvate formate lyase (PFL1) gene; the encoded PFL1 protein catalyzes a major fermentative pathway in wild-type Chlamydomonas cells. When the pfl1 mutants were subjected to dark fermentative conditions, they displayed an increased flux of pyruvate to lactate, elevated pyruvate decarboxylation, ethanol accumulation, diminished pyruvate oxidation by pyruvate ferredoxin oxidoreductase, and lowered H2 production. The pfl1-1 mutant also accumulated high intracellular levels of lactate, succinate, alanine, malate, and fumarate. To further probe the system, we generated a doublemore » mutant (pfl1-1 adh1) that is unable to synthesize both formate and ethanol. This strain, like the pfl1 mutants, secreted lactate, but it also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars and a decrease in dark, fermentative H2 production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant reroutes glycolytic carbon to lactate and glycerol. Although the metabolic adjustments observed in the mutants facilitate NADH reoxidation and sustained glycolysis under dark, anoxic conditions, the observed changes could not have been predicted given our current knowledge of the regulation of fermentation metabolism.« less

Immobilization of ammonia-oxidizing bacteria by polyvinyl alcohol and sodium alginate.

PubMed

Dong, Yuwei; Zhang, Yanqiu; Tu, Baojun

Ammonia-oxidizing bacteria were immobilized by polyvinyl alcohol (PVA) and sodium alginate. The immobilization conditions and ammonia oxidation ability of the immobilized bacteria were investigated. The following immobilization conditions were observed to be optimal: PVA, 12%; sodium alginate, 1.1%; calcium chloride, 1.0%; inoculum concentration, 1.3 immobilized balls/mL of immobilized medium; pH, 10; and temperature, 30°C. The immobilized ammonia-oxidizing bacteria exhibited strong ammonia oxidation ability even after being recycled four times. The ammonia nitrogen removal rate of the immobilized ammonia-oxidizing bacteria reached 90.30% under the optimal immobilization conditions. When compared with ammonia-oxidizing bacteria immobilized by sodium alginate alone, the bacteria immobilized by PVA and sodium alginate were superior with respect to pH resistance, the number of reuses, material cost, heat resistance, and ammonia oxidation ability. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Development of a Spirulina Extract/Alginate-Imbedded PCL Nanofibrous Cosmetic Patch.

PubMed

Byeon, Seon Yeong; Cho, Myung Kwon; Shim, Kyou Hee; Kim, Hye Jin; Song, Hyeon Gi; Shin, Hwa Sung

2017-09-28

Cosmetic patches have recently been developed as skin products for personal care owing to rapid advances in the technology of delivery of active ingredients, moisture, and adhesiveness to skin. Alginate and Spirulina are typical marine resources used in cosmetic products. This research involved the development of a Spirulina extract-impregnated alginate nanofiber cosmetic patch supported by a polycaprolactone (PCL) nanofiber cover ( Spi /Alg-PCL NF patch). In addition to the ability of alginate to affect moisture and adhesiveness to skin, the impregnation of Spirulina extract strengthened those abilities as well as its own bioactive effectiveness. All fabrication processing steps were undertaken in aqueous solution. The three components (alginate, Spirulina extract, and PCL) had no detected cytotoxicity in human keratinocyte cell-based examination. In addition, wetting the pre-dried patch on the skin resulted in the Spirulina extract being released within 30 min. The results indicate the excellence of the Spi /Alg-PCL NF patch as a skin-care cosmetic device.

Efficacy of chitosan and sodium alginate scaffolds for repair of spinal cord injury in rats

PubMed Central

Yao, Zi-ang; Chen, Feng-jia; Cui, Hong-li; Lin, Tong; Guo, Na; Wu, Hai-ge

2018-01-01

Spinal cord injury results in the loss of motor and sensory pathways and spontaneous regeneration of adult mammalian spinal cord neurons is limited. Chitosan and sodium alginate have good biocompatibility, biodegradability, and are suitable to assist the recovery of damaged tissues, such as skin, bone and nerve. Chitosan scaffolds, sodium alginate scaffolds and chitosan-sodium alginate scaffolds were separately transplanted into rats with spinal cord hemisection. Basso-Beattie-Bresnahan locomotor rating scale scores and electrophysiological results showed that chitosan scaffolds promoted recovery of locomotor capacity and nerve transduction of the experimental rats. Sixty days after surgery, chitosan scaffolds retained the original shape of the spinal cord. Compared with sodium alginate scaffolds- and chitosan-sodium alginate scaffolds-transplanted rats, more neurofilament-H-immunoreactive cells (regenerating nerve fibers) and less glial fibrillary acidic protein-immunoreactive cells (astrocytic scar tissue) were observed at the injury site of experimental rats in chitosan scaffold-transplanted rats. Due to the fast degradation rate of sodium alginate, sodium alginate scaffolds and composite material scaffolds did not have a supporting and bridging effect on the damaged tissue. Above all, compared with sodium alginate and composite material scaffolds, chitosan had better biocompatibility, could promote the regeneration of nerve fibers and prevent the formation of scar tissue, and as such, is more suitable to help the repair of spinal cord injury. PMID:29623937

Requirement for two or more Erwinia carotovora subsp. carotovora pectolytic gene products for maceration of potato tuber tissue by Escherichia coli.

PubMed Central

Roberts, D P; Berman, P M; Allen, C; Stromberg, V K; Lacy, G H; Mount, M S

1986-01-01

Several genes encoding enzymes capable of degrading plant cell wall components have been cloned from Erwinia carotovora subsp. carotovora EC14. Plasmids containing cloned EC14 DNA mediate the production of endo-pectate lyases, exo-pectate lyase, endo-polygalacturonase, and cellulase(s). Escherichia coli strains containing one of these plasmids or combinations of two plasmids were tested for their ability to macerate potato tuber slices. Only one E. coli strain, containing two plasmids that encode endo-pectate lyases, exo-pectate lyase, and endo-polygalacturonase, caused limited maceration. The pectolytic proteins associated with one of these plasmids, pDR1, have been described previously (D. P. Roberts, P. M. Berman, C. Allen, V. K. Stromberg, G. H. Lacy, and M. S. Mount, Can. J. Plant Pathol. 8:17-27, 1986) and include two secreted endo-pectate lyases. The second plasmid, pDR30, contains a 2.1-kilobase EC14 DNA insert that mediates the production of an exo-pectate lyase and an endo-polygalacturonase. These enzymes are similar in physicochemical properties to those produced by EC14. Our results suggest that the concerted activities of endo-pectate lyases with endo-polygalacturonase or exo-pectate lyase or both cause maceration. Images PMID:3013836

Generation of 2-Furfurylthiol by Carbon-Sulfur Lyase from the Baijiu Yeast Saccharomyces cerevisiae G20.

PubMed

Zha, Musu; Sun, Baoguo; Yin, Sheng; Mehmood, Arshad; Cheng, Lei; Wang, Chengtao

2018-03-07

2-Furfurylthiol is the representative aroma compound of Chinese sesame-flavored baijiu. Previous studies demonstrated that baijiu yeasts could generate 2-furfurylthiol using furfural and l-cysteine as precursors and that the Saccharomyces cerevisiae genes STR3 and CYS3 are closely related to 2-furfurylthiol biosynthesis. To confirm the mechanism of the STR3- and CYS3-gene products on 2-furfurylthiol biosynthesis, their encoded proteins were purified, and we confirmed their activities as carbon-sulfur lyases. Str3p and Cys3p were able to cleave the cysteine-furfural conjugate to release 2-furfurylthiol. Moreover, the characterization of the enzymatic properties of the purified proteins shows good thermal stabilities and wide pH tolerances, which enable their strong potential for various applications. These data provide direct evidence that yeast Str3p and Cys3p release 2-furfurylthiol in vitro, which can be applied to improve baijiu flavor.

Influence of glucagon or 5-(tetradecyloxy)-2-furoic acid on binding to mitochondria and phosphorylation of ATP-citrate lyase.

PubMed

Janski, A M; Cornell, N W

1982-02-01

To study the binding to mitochondria and the phosphorylation of ATP-citrate lyase (EC 4.1.3.8), isolated rat hepatocytes were fractionated by exposure to digitonin. After incubation of hepatocytes with the hypolipidemic agent 5-(tetradecyloxy)-2-furoic acid, which decreases the cellular CoA, the amount of bound ATP-citrate lyase was increased, but the content of acid-stable phosphate in the enzyme was diminished. Glucagon, in contrast, decreased the amount of bound enzyme but increased phosphorylation. This inverse relationship might indicate either that the bound ATP-citrate lyase is less readily phosphorylated or that the phosphorylated enzyme binds less readily to mitochondria.

Inhibition of tyrosine phenol-lyase by tyrosine homologues.

PubMed

Do, Quang; Nguyen, Giang T; Phillips, Robert S

2016-09-01

We have designed, synthesized, and evaluated tyrosine homologues and their O-methyl derivatives as potential inhibitors for tyrosine phenol lyase (TPL, E.C. 4.1.99.2). Recently, we reported that homologues of tryptophan are potent inhibitors of tryptophan indole-lyase (tryptophanase, TIL, E.C. 4.1.99.1), with K i values in the low µM range (Do et al. Arch Biochem Biophys 560:20-26, 2014). As the structure and mechanism for TPL is very similar to that of TIL, we postulated that tyrosine homologues could also be potent inhibitors of TPL. However, we have found that homotyrosine, bishomotyrosine, and their corresponding O-methyl derivatives are competitive inhibitors of TPL, which exhibit K i values in the range of 0.8-1.5 mM. Thus, these compounds are not potent inhibitors, but instead bind with affinities similar to common amino acids, such as phenylalanine or methionine. Pre-steady-state kinetic data were very similar for all compounds tested and demonstrated the formation of an equilibrating mixture of aldimine and quinonoid intermediates upon binding. Interestingly, we also observed a blue-shift for the absorbance peak of external aldimine complexes of all tyrosine homologues, suggesting possible strain at the active site due to accommodating the elongated side chains.

Safety and Efficacy of Alginate Adhesion Barrier Gel in Compromised Intestinal Anastomosis.

PubMed

Chaturvedi, Ankit A; Yauw, Simon T K; Lomme, Roger M L M; Hendriks, Thijs; van Goor, Harry

For any anti-adhesive barrier developed for abdominal surgery, the use under conditions in which anastomotic healing is compromised needs to be investigated. The current study evaluates the effect of a new ultrapure alginate gel on early healing of high-risk anastomoses in the ileum and compares this with the gold standard used in clinical practice. In 75 adult male Wistar rats, a 5 mm ileal segment was resected and continuity was restored by construction of an inverted anastomosis. Rats were divided randomly into a control group and groups receiving either alginate gel or a sodium hyaluronate carboxymethylcellulose (HA/CMC) film around the anastomosis (n = 25 each). Carprofen, given in a daily dose of 1.25 mg/kg, was used to compromise anastomotic healing. At day three, animals were killed and scored for signs of anastomotic leakage and the presence of adhesions. The incidence of adhesion formation was 95% in the HA/CMC film group, which was significantly higher than in the controls (64%, p = 0.010) and the alginate gel group (52%, p = 0.004). The adhesion score was nearly 40% lower in the alginate gel group compared with the HA/CMC film group. The incidence of ileal leakage in the HA/CMC film group (92%) was significantly higher than in the controls (68%, p = 0.016). Leakage rate did not differ between the alginate gel and control groups. There was no significant difference between groups in either incision bursting pressure or incision breaking strength. Ultrapure alginate gel does not interfere with repair of ileal anastomoses constructed under conditions in which chances of anastomotic dehiscence are high. The alginate gel performs better than the HA/CMC film.

Copper-releasing, boron-containing bioactive glass-based scaffolds coated with alginate for bone tissue engineering.

PubMed

Erol, M M; Mouriňo, V; Newby, P; Chatzistavrou, X; Roether, J A; Hupa, L; Boccaccini, Aldo R

2012-02-01

The aim of this study was to synthesize and characterize new boron-containing bioactive glass-based scaffolds coated with alginate cross-linked with copper ions. A recently developed bioactive glass powder with nominal composition (wt.%) 65 SiO2, 15 CaO, 18.4 Na2O, 0.1 MgO and 1.5 B2O3 was fabricated as porous scaffolds by the foam replica method. Scaffolds were alginate coated by dipping them in alginate solution. Scanning electron microscopy investigations indicated that the alginate effectively attached on the surface of the three-dimensional scaffolds leading to a homogeneous coating. It was confirmed that the scaffold structure remained amorphous after the sintering process and that the alginate coating improved the scaffold bioactivity and mechanical properties. Copper release studies showed that the alginate-coated scaffolds allowed controlled release of copper ions. The novel copper-releasing composite scaffolds represent promising candidates for bone regeneration. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

A new family of β-helix proteins with similarities to the polysaccharide lyases

DOE PAGES

Close, Devin W.; D'Angelo, Sara; Bradbury, Andrew R. M.

2014-09-27

Microorganisms that degrade biomass produce diverse assortments of carbohydrate-active enzymes and binding modules. Despite tremendous advances in the genomic sequencing of these organisms, many genes do not have an ascribed function owing to low sequence identity to genes that have been annotated. Consequently, biochemical and structural characterization of genes with unknown function is required to complement the rapidly growing pool of genomic sequencing data. A protein with previously unknown function (Cthe_2159) was recently isolated in a genome-wide screen using phage display to identify cellulose-binding protein domains from the biomass-degrading bacterium Clostridium thermocellum. Here, the crystal structure of Cthe_2159 is presentedmore » and it is shown that it is a unique right-handed parallel β-helix protein. Despite very low sequence identity to known β-helix or carbohydrate-active proteins, Cthe_2159 displays structural features that are very similar to those of polysaccharide lyase (PL) families 1, 3, 6 and 9. Cthe_2159 is conserved across bacteria and some archaea and is a member of the domain of unknown function family DUF4353. This suggests that Cthe_2159 is the first representative of a previously unknown family of cellulose and/or acid-sugar binding β-helix proteins that share structural similarities with PLs. More importantly, these results demonstrate how functional annotation by biochemical and structural analysis remains a critical tool in the characterization of new gene products.« less

A new family of β-helix proteins with similarities to the polysaccharide lyases

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

Close, Devin W.; D'Angelo, Sara; Bradbury, Andrew R. M.

Microorganisms that degrade biomass produce diverse assortments of carbohydrate-active enzymes and binding modules. Despite tremendous advances in the genomic sequencing of these organisms, many genes do not have an ascribed function owing to low sequence identity to genes that have been annotated. Consequently, biochemical and structural characterization of genes with unknown function is required to complement the rapidly growing pool of genomic sequencing data. A protein with previously unknown function (Cthe_2159) was recently isolated in a genome-wide screen using phage display to identify cellulose-binding protein domains from the biomass-degrading bacterium Clostridium thermocellum. Here, the crystal structure of Cthe_2159 is presentedmore » and it is shown that it is a unique right-handed parallel β-helix protein. Despite very low sequence identity to known β-helix or carbohydrate-active proteins, Cthe_2159 displays structural features that are very similar to those of polysaccharide lyase (PL) families 1, 3, 6 and 9. Cthe_2159 is conserved across bacteria and some archaea and is a member of the domain of unknown function family DUF4353. This suggests that Cthe_2159 is the first representative of a previously unknown family of cellulose and/or acid-sugar binding β-helix proteins that share structural similarities with PLs. More importantly, these results demonstrate how functional annotation by biochemical and structural analysis remains a critical tool in the characterization of new gene products.« less

Biochemical, Kinetic, and Spectroscopic Characterization of Ruegeria pomeroyi DddW—A Mononuclear Iron-Dependent DMSP Lyase

PubMed Central

Brummett, Adam E.; Schnicker, Nicholas J.; Crider, Alexander; Todd, Jonathan D.; Dey, Mishtu

2015-01-01

The osmolyte dimethylsulfoniopropionate (DMSP) is a key nutrient in marine environments and its catabolism by bacteria through enzymes known as DMSP lyases generates dimethylsulfide (DMS), a gas of importance in climate regulation, the sulfur cycle, and signaling to higher organisms. Despite the environmental significance of DMSP lyases, little is known about how they function at the mechanistic level. In this study we biochemically characterize DddW, a DMSP lyase from the model roseobacter Ruegeria pomeroyi DSS-3. DddW is a 16.9 kDa enzyme that contains a C-terminal cupin domain and liberates acrylate, a proton, and DMS from the DMSP substrate. Our studies show that as-purified DddW is a metalloenzyme, like the DddQ and DddP DMSP lyases, but contains an iron cofactor. The metal cofactor is essential for DddW DMSP lyase activity since addition of the metal chelator EDTA abolishes its enzymatic activity, as do substitution mutations of key metal-binding residues in the cupin motif (His81, His83, Glu87, and His121). Measurements of metal binding affinity and catalytic activity indicate that Fe(II) is most likely the preferred catalytic metal ion with a nanomolar binding affinity. Stoichiometry studies suggest DddW requires one Fe(II) per monomer. Electronic absorption and electron paramagnetic resonance (EPR) studies show an interaction between NO and Fe(II)-DddW, with NO binding to the EPR silent Fe(II) site giving rise to an EPR active species (g = 4.29, 3.95, 2.00). The change in the rhombicity of the EPR signal is observed in the presence of DMSP, indicating that substrate binds to the iron site without displacing bound NO. This work provides insight into the mechanism of DMSP cleavage catalyzed by DddW. PMID:25993446

Subcellular Distribution of O-Acetylserine(thiol)lyase in Cauliflower (Brassica oleracea L.) Inflorescence

PubMed Central

Rolland, Norbert; Droux, Michel; Douce, Roland

1992-01-01

The subcellular localization of O-acetyiserine(thiol)lyase (EC 4.2.99.8) in nongreen tissue from higher plants has been studied using purified proplastids, mitochondria, and protoplasts from cauliflower (Brassica oleracea L.) buds as a source of subcellular fractions. O-Acetylserine(thiol)lyase has been detected in both organelles (proplastids and mitochondria) and a cytosolic extract obtained by protoplast fractionation. We confirmed these observations, demonstrating that a form of the enzyme different in global charge and separated from others by anion-exchange chromatography corresponded to each subcellular location. Our observations are consistent with the need for cysteine biosynthesis in each subcellular compartment where the synthesis of proteins occurs. ImagesFigure 1 PMID:16668766

Fabrication of individual alginate-TCP scaffolds for bone tissue engineering by means of powder printing.

PubMed

Castilho, Miguel; Rodrigues, Jorge; Pires, Inês; Gouveia, Barbara; Pereira, Manuel; Moseke, Claus; Groll, Jürgen; Ewald, Andrea; Vorndran, Elke

2015-01-06

The development of polymer-calcium phosphate composite scaffolds with tailored architectures and properties has great potential for bone regeneration. Herein, we aimed to improve the functional performance of brittle ceramic scaffolds by developing a promising biopolymer-ceramic network. For this purpose, two strategies, namely, direct printing of a powder composition consisting of a 60:40 mixture of α/β-tricalcium phosphate (TCP) powder and alginate powder or vacuum infiltration of printed TCP scaffolds with an alginate solution, were tracked. Results of structural characterization revealed that the scaffolds printed with 2.5 wt% alginate-modified TCP powders presented a uniformly distributed and interfusing alginate TCP network. Mechanical results indicated a significant increase in strength, energy to failure and reliability of powder-modified scaffolds with an alginate content in the educts of 2.5 wt% when compared to pure TCP, as well as to TCP scaffolds containing 5 wt% or 7.5 wt% in the educts, in both dry and wet states. Culture of human osteoblast cells on these scaffolds also demonstrated a great improvement of cell proliferation and cell viability. While in the case of powder-mixed alginate TCP scaffolds, isolated alginate gels were formed between the calcium phosphate crystals, the vacuum-infiltration strategy resulted in the covering of the surface and internal pores of the TCP scaffold with a thin alginate film. Furthermore, the prediction of the scaffolds' critical fracture conditions under more complex stress states by the applied Mohr fracture criterion confirmed the potential of the powder-modified scaffolds with 2.5 wt% alginate in the educts as structural biomaterial for bone tissue engineering.

Alginate/sodium caseinate aqueous-core capsules: a pH-responsive matrix.

PubMed

Ben Messaoud, Ghazi; Sánchez-González, Laura; Jacquot, Adrien; Probst, Laurent; Desobry, Stéphane

2015-02-15

Alginate capsules have several applications. Their functionality depends considerably on their permeability, chemical and mechanical stability. Consequently, the creation of composite system by addition of further components is expected to control mechanical and release properties of alginate capsules. Alginate and alginate-sodium caseinate composite liquid-core capsules were prepared by a simple extrusion. The influence of the preparation pH and sodium caseinate concentration on capsules physico-chemical properties was investigated. Results showed that sodium caseinate influenced significantly capsules properties. As regards to the membrane mechanical stability, composite capsules prepared at pH below the isoelectric point of sodium caseinate exhibited the highest surface Young's modulus, increasing with protein content, explained by potential electrostatic interactions between sodium caseinate amino-groups and alginate carboxylic group. The kinetic of cochineal red A release changed significantly for composite capsules and showed a pH-responsive release. Sodium caseinate-dye mixture studied by absorbance and fluorescence spectroscopy confirmed complex formation at pH 2 by electrostatic interactions between sodium caseinate tryptophan residues and cochineal red sulfonate-groups. Consequently, the release mechanism was explained by membrane adsorption process. This global approach is useful to control release mechanism from macro and micro-capsules by incorporating guest molecules which can interact with the entrapped molecule under specific conditions. Copyright © 2014 Elsevier Inc. All rights reserved.

Grafting of alginates on UF/NF ceramic membranes for wastewater treatment.

PubMed

Athanasekou, C P; Romanos, G E; Kordatos, K; Kasselouri-Rigopoulou, V; Kakizis, N K; Sapalidis, A A

2010-10-15

The mechanism of heavy metal ion removal in processes involving multi-layered tubular ultrafiltration and nanofiltration (UF/NF) membranes was investigated by conducting retention experiments in both flow-through and cross-flow modes. The prospect of the regeneration of the membranes through an acidic process was also examined and discussed. The UF/NF membranes were functionalised with alginates to develop hybrid inorganic/organic materials for continuous, single pass, wastewater treatment applications. The challenge laid in the induction of additional metal adsorption and improved regeneration capacity. This was accomplished by stabilizing alginates either into the pores or on the top-separating layer of the membrane. The preservation of efficient water fluxes at moderate trans-membrane pressures introduced an additional parameter that was pursued in parallel to the membrane modification process. The deposition and stabilization of alginates was carried out via physical (filtration/cross-linking) and chemical (grafting) procedures. The materials developed by means of the filtration process exhibited a 25-60% enhancement of their Cd(2+) binding capacity, depending on the amount of the filtered alginate solution. The grafting process led to the development of alginate layers with adequate stability under acidic regeneration conditions and metal retention enhancement of 25-180%, depending on the silane involved as grafting agent and the solvent of silanisation. 2010 Elsevier B.V. All rights reserved.

Development of PVA-alginate as a matrix for enzymatic decolorization of textile dye in bioreactor system

NASA Astrophysics Data System (ADS)

Yanto, Dede Heri Yuli; Zahara, Syifa; Laksana, Raden Permana Budi; Anita, Sita Heris; Oktaviani, Maulida; Sari, Fahriya Puspita

2017-01-01

An immobilization technique using polyvinyl alcohol (PVA) crosslinked with sodium alginate as a matrix has been developed for textile dyes decolorization. Textiles use dye as an addition to the aesthetic value of the product. Dyes are generally used is a textile dye where the waste will be released directly into the waters around 2-20%. Therefore, it is important to develop an enzyme immobilization method using PVA-Alginate as a matrix. Based on the results of the study showed that the PVA-Alginate beads produced high decolorization percent compared to beads which contains only Ca-alginate alone and formula matrix is optimum at PVA 6% and alginate 1.5%. Encapsulation with boric acid at 7% showed optimum decolorization and reduction for enzyme leakage during decolorization. This study suggested that immobilization of enzymes into PVA-alginate matrix might be used as a biodecolorating agent.

Alginate/hyaluronic acid hydrogel delivery system characteristics regulate the differentiation of periodontal ligament stem cells toward chondrogenic lineage.

PubMed

Ansari, Sahar; Diniz, Ivana M; Chen, Chider; Aghaloo, Tara; Wu, Benjamin M; Shi, Songtao; Moshaverinia, Alireza

2017-09-15

Cartilage tissue regeneration often presents a challenging clinical situation. Recently, it has been shown that Periodontal Ligament Stem Cells (PDLSCs) possess high chondrogenic differentiation capacity. In this study, we developed a stem cell delivery system based on alginate/hyaluronic acid (HA) loaded with TGF-β1 ligand, encapsulating PDLSCs; and investigated the chondrogenic differentiation of encapsulated cells in alginate/HA hydrogel microspheres in vitro and in vivo. The results showed that PDLSCs, as well as human bone marrow mesenchymal stem cells (hBMMSCs), as the positive control, were stained positive for both toluidine blue and alcian blue staining, while exhibiting high levels of gene expression related to chondrogenesis (Col II, Aggrecan and Sox-9), as assessed via qPCR. The quantitative PCR analyses exhibited that the chondrogenic differentiation of encapsulated MSCs can be regulated by the modulus of elasticity of hydrogel delivery system, confirming the vital role of the microenvironment, and the presence of inductive signals for viability and differentiation of MSCs. In vivo, histological and immunofluorescence staining for chondrogenic specific protein markers confirmed ectopic cartilage-like tissue regeneration inside transplanted hydrogels. PDLSCs presented significantly greater capability for chondrogenic differentiation than hBMMSCs (P < 0.05). Altogether, our findings confirmed that alginate/HA hydrogels encapsulating PDLSCs are a promising candidate for cartilage regeneration.

Mesenchymal stem cells and alginate microcarriers for craniofacial bone tissue engineering: A review.

PubMed

Saltz, Adam; Kandalam, Umadevi

2016-05-01

Craniofacial bone is a complex structure with an intricate anatomical and physiological architecture. The defects that exist in this region therefore require a precise control of osteogenesis in their reconstruction. Unlike traditional surgical intervention, tissue engineering techniques mediate bone development with limited postoperative risk and cost. Alginate stands as the premier polymer in bone repair because of its mild ionotropic gelation and excellent biocompatibility, biodegradability, and injectability. Alginate microcarriers are candidates of choice to mediate cells and accommodate into 3-D environment. Several studies reported the use of alginate microcarriers for delivering cells, drugs, and growth factors. This review will explore the potential use of alginate microcarrier for stem cell systems and its application in craniofacial bone tissue engineering. © 2016 Wiley Periodicals, Inc.

Enzymatically cross-linked injectable alginate-g-pyrrole hydrogels for neovascularization.

PubMed

Devolder, Ross; Antoniadou, Eleni; Kong, Hyunjoon

2013-11-28

Microparticles capable of releasing protein drugs are often incorporated into injectable hydrogels to minimize their displacement at an implantation site, reduce initial drug burst, and further control drug release rates over a broader range. However, there is still a need to develop methods for releasing drug molecules over extended periods of time, in order to sustain the bioactivity of drug molecules at an implantation site. In this study, we hypothesized that a hydrogel formed through the cross-linking of pyrrole units linked to a hydrophilic polymer would release protein drugs in a more sustained manner, because of an enhanced association between cross-linked pyrrole groups and the drug molecules. To examine this hypothesis, we prepared hydrogels of alginate substituted with pyrrole groups, alginate-g-pyrrole, through a horse-radish peroxidase (HRP)-activated cross-linking of the pyrrole groups. The hydrogels were encapsulated with poly(lactic-co-glycolic acid) (PLGA) microparticles loaded with vascular endothelial growth factor (VEGF). The resulting hydrogel system released VEGF in a more sustained manner than Ca(2+) alginate or Ca(2+) alginate-g-pyrrole gel systems. Finally, implantations of the VEGF-releasing HRP-activated alginate-g-pyrrole hydrogel system on chicken chorioallantoic membranes resulted in the formation of blood vessels in higher densities and with larger diameters, compared to other control conditions. Overall, the drug releasing system developed in this study will be broadly useful for regulating release rates of a wide array of protein drugs, and further enhance the quality of protein drug-based therapies. © 2013 Elsevier B.V. All rights reserved.

Performance evaluation of bipolar and tripolar excitations during nozzle-jetting-based alginate microsphere fabrication

NASA Astrophysics Data System (ADS)

Herran, C. Leigh; Huang, Yong; Chai, Wenxuan

2012-08-01

Microspheres, small spherical (polymeric) particles with or without second phase materials embedded or encapsulated, are important for many biomedical applications such as drug delivery and organ printing. Scale-up fabrication with the ability to precisely control the microsphere size and morphology has always been of great manufacturing interest. The objective of this work is to experimentally study the performance differences of bipolar and tripolar excitation waveforms in using drop-on-demand (DOD)-based single nozzle jetting for alginate microsphere fabrication. The fabrication performance has been evaluated based on the formability of alginate microspheres as a function of materials properties (sodium alginate and calcium chloride concentrations) and operating conditions. The operating conditions for each excitation include voltage rise/fall times, dwell times and excitation voltage amplitudes. Overall, the bipolar excitation is more robust in making spherical, monodispersed alginate microspheres as good microspheres for its wide working range of material properties and operating conditions, especially during the fabrication of highly viscous materials such as the 2% sodium alginate solution. For both bipolar and tripolar excitations, the sodium alginate concentration and the voltage dwell times should be carefully selected to achieve good microsphere formability.

The effect of ionotropic gelation residence time on alginate cross-linking and properties.

PubMed

Patel, Mitulkumar A; AbouGhaly, Mohamed H H; Schryer-Praga, Jacqueline V; Chadwick, Keith

2017-01-02

The ability to engineer biocompatible polymers with controllable properties is highly desirable. One such approach is to cross-link carbohydrate polymers using ionotropic gelation (IG). Previous studies have investigated the effect of curing time on alginate cross-linking. Herein, we discuss a novel study detailing the effect of IG residence time (IGRT) on the cross-linking of alginate with calcium ions (Ca 2+ ) along with water migration (syneresis) and their subsequent impact on the pharmaceutical properties of alginate particles. IGRT was shown to have a significant effect on particle size, porosity, density, mechanical strength and swelling of calcium alginate particles as well as drug release mechanism. Furthermore, we describe a novel application of electron dispersive spectroscopy (EDS), in conjunction with Fourier Transform- infra red (FT-IR) spectroscopy, to analyze and monitor the changes in Ca 2+ concentration during cross-linking. A simple procedure to determine the concentration and distribution of the surface and internal Ca 2+ involved in alginate cross-linking was successfully developed. Copyright © 2016 Elsevier Ltd. All rights reserved.

Tough Al-alginate/poly(N-isopropylacrylamide) hydrogel with tunable LCST for soft robotics.

PubMed

Zheng, Wen Jiang; An, Ning; Yang, Jian Hai; Zhou, Jinxiong; Chen, Yong Mei

2015-01-28

Tough Al-alginate/poly(N-isopropylacrylamide) (PNIPAM) hydrogel has been synthesized by introducing an interpenetrating network with hybrid physically cross-linked alginate and chemically cross-linked PNIPAM. Varying the concentration of AlCl3 regulates the mechanical properties of the tough hydrogel and tunes its lower critical solution temperature (LCST) as well. The tough Al-alginate/PNIPAM exhibits 6.3 ± 0.3 MPa of compressive stress and 9.95 of uniaxial stretch. Tunability of LCST is also achieved in a wide range within 22.5-32 °C. A bending beam actuator and a four-arm gripper made of bilayer (Na-alginate/PNIPAM)/(Al-alginate/PNIPAM) hydrogel as prototype of all-hydrogel soft robotics are demonstrated. A finite element (FE) simulation model is developed to simulate the deformation of the soft robotics. The FE simulation not only reproduces the deformation process of performed experiments but also predicts more complicated devices that can be explored in the future. This work broadens the application of temperature-responsive PNIPAM-based hydrogels.

As(III) and As(V) removal from the aqueous phase via adsorption onto acid mine drainage sludge (AMDS) alginate beads and goethite alginate beads.

PubMed

Lee, Hongkyun; Kim, Dohyeong; Kim, Jongsik; Ji, Min-Kyu; Han, Young-Soo; Park, Young-Tae; Yun, Hyun-Shik; Choi, Jaeyoung

2015-07-15

Acid mine drainage sludge (AMDS) is a solid waste generated following the neutralization of acid mine drainage (AMD). This material entrapped in calcium alginate was investigated for the sorption of As(III) and As(V). Three different adsorbent materials were prepared: AMDS alginate beads (AABs), goethite alginate beads (GABs), and pure alginate beads. The effects of pH and the adsorption kinetics were investigated, and the adsorption isotherms were also evaluated. The optimum pH range using the AABs was determined to be within 2-10 for As(III) and 2-9 for As(V). Adsorption equilibrium data were evaluated using the Langmuir isotherm model, and the maximum adsorption capacity qmax was 18.25 and 4.97 mg g(-1) for As(III) on AAB and GAB, respectively, and 21.79 and 10.92 mg g(-1) for As(V) on AAB and GAB, respectively. The adsorption of As(III) and As(V) was observed to follow pseudo-second order kinetics. The As K-edge X-ray absorption near-edge structure (XANES) revealed that the adsorbed As(III) on the AABs was oxidized to As(V) via manganese oxide in the AMDS. Copyright © 2015 Elsevier B.V. All rights reserved.

An additive manufacturing-based PCL-alginate-chondrocyte bioprinted scaffold for cartilage tissue engineering.

PubMed

Kundu, Joydip; Shim, Jin-Hyung; Jang, Jinah; Kim, Sung-Won; Cho, Dong-Woo

2015-11-01

Regenerative medicine is targeted to improve, restore or replace damaged tissues or organs using a combination of cells, materials and growth factors. Both tissue engineering and developmental biology currently deal with the process of tissue self-assembly and extracellular matrix (ECM) deposition. In this investigation, additive manufacturing (AM) with a multihead deposition system (MHDS) was used to fabricate three-dimensional (3D) cell-printed scaffolds using layer-by-layer (LBL) deposition of polycaprolactone (PCL) and chondrocyte cell-encapsulated alginate hydrogel. Appropriate cell dispensing conditions and optimum alginate concentrations for maintaining cell viability were determined. In vitro cell-based biochemical assays were performed to determine glycosaminoglycans (GAGs), DNA and total collagen contents from different PCL-alginate gel constructs. PCL-alginate gels containing transforming growth factor-β (TGFβ) showed higher ECM formation. The 3D cell-printed scaffolds of PCL-alginate gel were implanted in the dorsal subcutaneous spaces of female nude mice. Histochemical [Alcian blue and haematoxylin and eosin (H&E) staining] and immunohistochemical (type II collagen) analyses of the retrieved implants after 4 weeks revealed enhanced cartilage tissue and type II collagen fibril formation in the PCL-alginate gel (+TGFβ) hybrid scaffold. In conclusion, we present an innovative cell-printed scaffold for cartilage regeneration fabricated by an advanced bioprinting technology. Copyright © 2013 John Wiley & Sons, Ltd.

Immobilization of naringinase in PVA-alginate matrix using an innovative technique.

PubMed

Nunes, Mário A P; Vila-Real, Hélder; Fernandes, Pedro C B; Ribeiro, Maria H L

2010-04-01

A synthetic polymer, polyvinyl alcohol (PVA), a cheap and nontoxic synthetic polymer to organism, has been ascribed for biocatalyst immobilization. In this work PVA-alginate beads were developed with thermal, mechanical, and chemical stability to high temperatures (<80 degrees C). The combination of alginate and bead treatment with sodium sulfate not only prevented agglomeration but produced beads of high gel strength and conferred enzyme protection from inactivation by boric acid. Naringinase from Penicillium decumbens was immobilized in PVA (10%)-alginate beads with three different sizes (1-3 mm), at three different alginate concentrations (0.2-1.0%), and these features were investigated in terms of swelling ratio within the beads, enzyme activity, and immobilization yield during hydrolysis of naringin. The pH and temperature optimum were 4.0 and 70 degrees C for the PVA-alginate-immobilized naringinase. The highest naringinase activity yield in PVA (10%)-alginate (1%) beads of 2 mm was 80%, at pH 4.0 and 70 degrees C. The Michaelis constant (K(Mapp)) and the maximum reaction velocity (V(maxapp)) were evaluated for both free (K(Mapp) = 0.233 mM; V(maxapp) = 0.13 mM min(-1)) and immobilized naringinase (K(Mapp) = 0.349 mM; V(maxapp) = 0.08 mM min(-1)). The residual activity of the immobilized enzyme was followed in eight consecutive batch runs with a retention activity of 70%. After 6 weeks, upon storage in acetate buffer pH 4 at 4 degrees C, the immobilized biocatalyst retained 90% of the initial activity. These promising results are illustrative of the potential of this immobilization strategy for the system evaluated and suggest that its application may be effectively performed for the entrapment of other biocatalysts.

Synthesis of Thiolated Alginate and Evaluation of Mucoadhesiveness, Cytotoxicity and Release Retardant Properties

PubMed Central

Jindal, A. B.; Wasnik, M. N.; Nair, Hema A.

2010-01-01

Modification of polymers by covalent attachment of thiol bearing pendant groups is reported to impart many beneficial properties to them. Hence in the present study, sodium alginate–cysteine conjugate was synthesized by carbodiimide mediated coupling under varying reaction conditions and the derivatives characterized for thiol content. The thiolated alginate species synthesized had bound thiol content ranging from 247.8±11.03–324.54±10.107 ΅mol/g of polymer depending on the reaction conditions. Matrix tablets based on sodium alginate-cysteine conjugate and native sodium alginate containing tramadol hydrochloride as a model drug were prepared and mucoadhesive strength and in vitro drug release from the tablets were compared. Tablets containing 75 mg sodium alginate-cysteine conjugate could sustain release of 10 mg of model drug for 3 h, whereas 90% of the drug was released within 1 h from corresponding tablets prepared using native sodium alginate. An approximately 2-fold increase in the minimal detachment force of the tablets from an artificial mucin film was observed for sodium alginate–cysteine conjugate as compared to native sodium alginate. In vitro cytotoxicity studies in L-929 mouse fibroblast cells studied using an MTT assay revealed that at low concentrations of polymer, sodium alginate–cysteine conjugate was less toxic to L-929 mouse fibroblast cell line when compared to native sodium alginate. Hence, thiolation is found to be a simple route to improving polymer performance. The combination of improved controlled drug release and mucoadhesive properties coupled with the low toxicity of these new excipients builds up immense scope for the use of thiolated polymers in mucoadhesive drug delivery systems. PMID:21969750

Spectroscopic studies on the active site of hydroperoxide lyase; the influence of detergents on its conformation.

PubMed

Noordermeer, M A; Veldink, G A; Vliegenthart, J F

2001-02-02

Expression of high quantities of alfalfa hydroperoxide lyase in Escherichia coli made it possible to study its active site and structure in more detail. Circular dichroism (CD) spectra showed that hydroperoxide lyase consists for about 75% of alpha-helices. Electron paramagnetic resonance (EPR) spectra confirmed its classification as a cytochrome P450 enzyme. The positive influence of detergents on the enzyme activity is paralleled by a spin state transition of the heme Fe(III) from low to high spin. EPR and CD spectra showed that detergents induce a subtle conformational change, which might result in improved substrate binding. Because hydroperoxide lyase is thought to be a membrane bound protein and detergents mimic a membrane environment, the more active, high spin form likely represents the in vivo conformation. Furthermore, the spin state appeared to be temperature-dependent, with the low spin state favored at low temperature. Point mutants of the highly conserved cysteine in domain D indicated that this residue might be involved in heme binding.

Microbial β-etherases and glutathione lyases for lignin valorisation in biorefineries: current state and future perspectives.

PubMed

Husarcíková, Jana; Voß, Hauke; Domínguez de María, Pablo; Schallmey, Anett

2018-05-04

Lignin is the major aromatic biopolymer in nature, and it is considered a valuable feedstock for the future supply of aromatics. Hence, its valorisation in biorefineries is of high importance, and various chemical and enzymatic approaches for lignin depolymerisation have been reported. Among the enzymes known to act on lignin, β-etherases offer the possibility for a selective cleavage of the β-O-4 aryl ether bonds present in lignin. These enzymes, together with glutathione lyases, catalyse a reductive, glutathione-dependent ether bond cleavage displaying high stereospecificity. β-Etherases and glutathione lyases both belong to the superfamily of glutathione transferases, and several structures have been solved recently. Additionally, different approaches for their application in lignin valorisation have been reported in the last years. This review gives an overview on the current knowledge on β-etherases and glutathione lyases, their biochemical and structural features, and critically discusses their potential for application in biorefineries.

Quantitative determination of alginic acid in pharmaceutical formulations using capillary electrophoresis.

PubMed

Moore, Douglas E; Miao, William G; Benikos, Con

2004-01-27

A capillary electrophoresis (CE) method has been developed and validated for the quantitative determination of alginic acid, which is used as a rafting agent in complex antacid formulations. The method involves a preliminary separation of the alginic acid from the formulation by washing the sample matrix with methanol, diluted HCl and water. This is followed by electrophoresis within a fused silica capillary using borate/boric acid buffer as the electrolyte, and the quantification is performed by a UV detector monitoring at 200 nm, where the intrinsic absorption of alginic acid is measured. An assay precision of better than 3% was achieved in intra- and interday determinations. No interference was found from the matrix of the antacid formulations.

Chitosan/alginate complexes for vaginal delivery of chlorhexidine digluconate.

PubMed

Abruzzo, A; Bigucci, F; Cerchiara, T; Saladini, B; Gallucci, M C; Cruciani, F; Vitali, B; Luppi, B

2013-01-16

Chitosan/alginate complexes were prepared at different polycation/polyanion molar ratios and freeze-dried vaginal inserts were obtained for chlorhexidine digluconate local delivery in genital infections. Complex yield, FT-IR spectra, and TGA thermograms were studied to confirm the interaction between the two polyions. The influence of different complexes on physical handling, morphology, and drug distribution in the samples were evaluated by friability test, scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDS), respectively. In vitro water-uptake, mucoadhesion and release tests were performed as well as microbiological tests toward pathogenic vaginal microorganisms. The results showed that the selection of suitable chitosan/alginate molar ratio and drug loading allowed modulate insert ability to hydrate, adhere to the mucosa, and release chlorhexidine digluconate. The insert containing an excess of alginate was found to be the best performing formulation and showed good antimicrobial activity toward the pathogens Candida albicans and Escherichia coli. Copyright © 2012 Elsevier Ltd. All rights reserved.

Methylene blue adsorption on graphene oxide/calcium alginate composites.

PubMed

Li, Yanhui; Du, Qiuju; Liu, Tonghao; Sun, Jiankun; Wang, Yonghao; Wu, Shaoling; Wang, Zonghua; Xia, Yanzhi; Xia, Linhua

2013-06-05

Graphene oxide has been used as an adsorbent in wastewater treatment. However, the dispersibility in aqueous solution and the biotoxicity to human cells of graphene oxide limits its practical application in environmental protection. In this research, a novel environmental friendly adsorbent, calcium alginate immobilized graphene oxide composites was prepared. The effects of pH, contact time, temperature and dosage on the adsorption properties of methylene blue onto calcium alginate immobilized graphene oxide composites were investigated. The equilibrium adsorption data were described by the Langmuir and Freundlich isotherms. The maximum adsorption capacity obtained from Langmuir isotherm equation was 181.81 mg/g. The pseudo-first order, pseudo-second order, and intraparticle diffusion equation were used to evaluate the kinetic data. Thermodynamic analysis of equilibriums indicated that the adsorption reaction of methylene blue onto calcium alginate immobilized graphene oxide composites was exothermic and spontaneous in nature. Copyright © 2013 Elsevier Ltd. All rights reserved.

Alginate based nanocomposite for microencapsulation of probiotic: Effect of cellulose nanocrystal (CNC) and lecithin.

PubMed

Huq, Tanzina; Fraschini, Carole; Khan, Avik; Riedl, Bernard; Bouchard, Jean; Lacroix, Monique

2017-07-15

Probiotic (Lactobacillus rhamnosus ATCC 9595) was encapsulated in alginate-CNC-lecithin microbeads to produce nutraceutical microcapsules. Addition of CNC and lecithin in alginate microbeads (ACL-1) improved the viability of L. rhamnosus during gastric passage and storage. The compression strength of the freeze-dried ACL-1 microbeads improved 40% compared to alginate microbeads alone. Swelling studies revealed that addition of CNC and lecithin in alginate microbeads decreased (around 47%) the gastric fluid absorption but increased the dissolution time by 20min compared to alginate microbeads (A-0). During transition through the gastric passage, the viability of L. rhamnosus in dried ACL-1 microbeads was increased 37% as compared to A-0 based beads. At 25 and 4°C storage conditions, the viability of L. rhamnosus encapsulated in ACL-1 microbeads decreased by 1.23 and 1.08 log respectively, whereas the encapsulation with A-0 microbeads exhibited a 3.17 and 1.93 log reduction respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

Innovative plasticized alginate obtained by thermo-mechanical mixing: Effect of different biobased polyols systems.

PubMed

Gao, Chengcheng; Pollet, Eric; Avérous, Luc

2017-02-10

Plasticized alginate films with different biobased polyols (glycerol and sorbitol) and their mixtures were successfully prepared by thermo-mechanical mixing instead of the usual casting-evaporation procedure. The microstructure and properties of the different plasticized alginate formulations were investigated by SEM, FTIR, XRD, DMTA and uniaxial tensile tests. SEM and XRD results showed that native alginate particles were largely destructured with the plasticizers (polyols and water), under a thermo-mechanical input. With increasing amount of plasticizers, the samples showed enhanced homogeneity while their thermal and mechanical properties decreased. Compared to sorbitol, glycerol resulted in alginate films with a higher flexibility due to its better plasticization efficiency resulting from its smaller size and higher hydrophilic character. Glycerol and sorbitol mixtures seemed to be an optimum to obtain the best properties. This work showed that thermo-mechanical mixing is a promising method to produce, at large scale, plasticized alginate-based films with improved properties. Copyright © 2016 Elsevier Ltd. All rights reserved.

Enzymatically Cross-linked Alginic-Hyaluronic acid Composite Hydrogels As Cell Delivery Vehicles

PubMed Central

Ganesh, Nitya; Hanna, Craig; Nair, Shantikumar V.; Nair, Lakshmi S.

2013-01-01

An injectable composite gel was developed from alginic and hyaluronic acid. The ezymatically cross-linked injectable gels were prepared via the oxidative coupling of tyramine modified sodium algiante and sodium hyaluronate in the presence of horse radish peroxidase (HRP) and hydrogen peroxide (H2O2). The composite gels were prepared by mixing equal parts of the two tryaminated polymer solutions in 10U HRP and treating with 1.0% H2O2. The properties of the alginate gels were significanly affected by the addition of hyaluronic acid. The percentage water absorption and storage modulus of the composite gels were found to be lower than the alginate gels. The alginate and composite gels showed lower protein release compared to hyaluronate gels in the absence of hyaluronidase. Even hyaluronate gels showed only approximately 10% protein release after 14 days incubation in phosphate buffer solution. ATDC-5 cells encapsulated in the injectable gels showed high cell viability. The composite gels showed the presence of enlarged spherical cells with significantly higher metabolic activity compared to cells in hyaluronic and alginic acid gels. The results suggest the potential of the composite approach to develop covalently cross-linked hydrogels with tuneable physical, mechanical, and biological properties. PMID:23357799

Antifibrotic effect of dexamethasone/alginate-coated silicone sheet in the abraded middle ear mucosa.

PubMed

Jang, Chul Ho; Ahn, Seung Hyun; Kim, Geun Hyung

2016-12-01

Silicone sheet is a material which is commonly used in middle ear surgery to prevent the formation of adhesions between the tympanic membrane and the medial bony wall of the middle ear cavity. However, silicone sheet can induce a tight and hard fibrous capsule in the region of the stapes, and this is particularly common in cases of eustachian tube dysfunction. As a result of the fibrous encapsulation around the silicone sheet, postoperative aeration of the stapes can be interrupted causing poor hearing gain. In this study, we performed an in vitro and in vivo evaluation of the antifibrotic effects of a dexamethasone and alginate (Dx/alginate) coating on silicone sheet. The Dx/alginate-coated silicone sheets were fabricated using a plasma-treatment and coating method. The Dx/alginate-coated silicone sheets effectively limited in vitro fibroblast attachment and proliferation due to the controlled release of Dx, which can be modified by manipulation of the alginate coating. For the in-vivo evaluation, guinea pigs (albino, male, weighing 250g) were divided into two groups, with the control group (n=5) implanted with silicone sheet and the test group (n=5) receiving Dx/alginate-coated silicone sheet. Animals were sacrificed 3 weeks after implantation, and histological analysis was performed using hematoxylin and eosin (H&E) and immunohistochemical staining techniques. Dx/alginate-coated silicone sheets showed marked inhibition of fibrosis in both the in vitro and in vivo studies. Silicone sheet that incorporates a Dx/alginate coating can release Dx and inhibit fibrosis in the middle ear. This material could be utilized in middle ear surgery as a means of preserving proper aeration and hearing gain following ossiculoplasty. Copyright © 2016 Elsevier B.V. All rights reserved.

Encapsulation of Autoinducer Sensing Reporter Bacteria in Reinforced Alginate-Based Microbeads.

PubMed

Li, Ping; Müller, Mareike; Chang, Matthew Wook; Frettlöh, Martin; Schönherr, Holger

2017-07-12

Quorum sensing, in which bacteria communities use signaling molecules for inter- and intracellular communication, has been intensively studied in recent decades. In order to fabricate highly sensitive easy-to-handle point of care biosensors that detect quorum sensing molecules, we have developed, as is reported here, reporter bacteria loaded alginate-methacrylate (alginate-MA) hydrogel beads. The alginate-MA beads, which were obtained by electrostatic extrusion, were reinforced by photo-cross-linking to increase stability and thereby to reduce bacteria leaching. In these beads the genetically engineered fluorescent reporter bacterium Escherichia coli pTetR-LasR-pLuxR-GFP (E. coli pLuxR-GFP) was encapsulated, which responds to the autoinducer N-(3-oxododecanoyl)homoserine lactone secreted by Pseudomonas aeruginosa. After encapsulation in alginate-MA hydrogel beads with diameters in the range of 100-300 μm that were produced by an electrostatic extrusion method and rapid photo-cross-linking, the E. coli pLuxR-GFP were found to possess a high degree of viability and sensing activity. The encapsulated bacteria could proliferate inside the hydrogel beads, when exposed to bacteria culture medium. In media containing the autoinducer N-(3-oxododecanoyl)homoserine lactone, the encapsulated reporter bacteria responded with a strong fluorescence signal due to an increased green fluorescent protein (GFP) expression. A prototype dipstick type sensor developed here underlines the potential of encapsulation of viable and functional reporter bacteria inside reinforced alginate-methacrylate hydrogel beads for whole cell sensors for bacteria detection.

Alginate nanoparticles protect ferrous from oxidation: Potential iron delivery system.

PubMed

Katuwavila, Nuwanthi P; Perera, A D L C; Dahanayake, Damayanthi; Karunaratne, V; Amaratunga, Gehan A J; Karunaratne, D Nedra

2016-11-20

A novel, efficient delivery system for iron (Fe 2+ ) was developed using the alginate biopolymer. Iron loaded alginate nanoparticles were synthesized by a controlled ionic gelation method and was characterized with respect to particle size, zeta potential, morphology and encapsulation efficiency. Successful loading was confirmed with Fourier Transform Infrared spectroscopy and Thermogravimetric Analysis. Electron energy loss spectroscopy study corroborated the loading of ferrous into the alginate nanoparticles. Iron encapsulation (70%) was optimized at 0.06% Fe (w/v) leading to the formation of iron loaded alginate nanoparticles with a size range of 15-30nm and with a negative zeta potential (-38mV). The in vitro release studies showed a prolonged release profile for 96h. Release of iron was around 65-70% at pH of 6 and 7.4 whereas it was less than 20% at pH 2.The initial burst release upto 8h followed zero order kinetics at all three pH values. All the release profiles beyond 8h best fitted the Korsmeyer-Peppas model of diffusion. Non Fickian diffusion was observed at pH 6 and 7.4 while at pH 2 Fickian diffusion was observed. Copyright © 2016 Elsevier B.V. All rights reserved.

Copper and nitrophenol pollutants removal by Na-montmorillonite/alginate microcapsules.

PubMed

Ely, Abdellahi; Baudu, Michel; Basly, Jean-Philippe; Kankou, Mohamed Ould Sid'Ahmed Ould

2009-11-15

The use of renewable bioresources allows the development of low cost adsorbents that are versatile. In the present paper, the affinity and the removal capacity of montmorillonite/alginate microcapsules for a hydrophobic organic pollutant (4-nitrophenol) and an inorganic pollutant (copper) were evaluated. The physicochemical processes through sorption and kinetic experiments under different ratios of montmorillonite vs. alginate and initial contaminant concentrations were investigated. The total weight loss and diameter decrease during the drying process were 90-96% and 64%, respectively. A significant decrease in beads diameter, related to water elimination, has been observed during the first 24h. Structural modifications that occur during the drying process were evaluated using thermal analysis. From correlation coefficients, the second-order equation depicts properly the adsorption of copper by the microbeads adsorption capacity increases to saturation with time; 3 and 6h were needed to reach equilibrium on wet and dry mixed microcapsules. The pseudo-second order model properly depicts the adsorption process of 4-NP onto Na-mont and (Na-mont/SA) mixed microcapsules but failed to reproduce the data observed for the alginate beads. Isotherms data were fitted with good correlation using the Langmuir model; alginate and montmorillonite adsorption capacities (q(m)/wet beads) agree with those obtained by various studies.

Molecular Characterization of a Recombinant Zea mays Phenylalanine Ammonia-Lyase (ZmPAL2) and Its Application in trans-Cinnamic Acid Production from L-Phenylalanine.

PubMed

Zang, Ying; Jiang, Ting; Cong, Ying; Zheng, Zhaojuan; Ouyang, Jia

2015-06-01

Phenylalanine ammonia-lyase (PAL) is one of the most extensively studied enzymes with its crucial role in secondary phenylpropanoid metabolism of plants. Recently, its demand has been increased for aromatic chemical production, but its applications in trans-cinnamic acid production were not much explored. In the present study, a putative PAL gene from Zea mays designated as ZmPAL2 was expressed and characterized in Escherichia coli BL21 (DE3). The recombinant ZmPAL2 exhibited a high PAL activity (7.14 U/mg) and a weak tyrosine ammonia-lyase activity. The optimal temperature of ZmPAL2 was 55 °C, and the thermal stability results showed that about 50 % of enzyme activity remained after a treatment at 60 °C for 6 h. The recombinant ZmPAL2 is a good candidate for the production of trans-cinnamic acid. The vitro conversion indicated that the recombinant ZmPAL2 could effectively catalyze the L-phenylalanine to trans-cinnamic acid, and the trans-cinnamic acid concentration can reach up to 5 g/l.

Strategies to overcome pH-dependent solubility of weakly basic drugs by using different types of alginates.

PubMed

Gutsche, S; Krause, M; Kranz, H

2008-12-01

Weakly basic drugs demonstrate higher solubility at lower pH, thus often leading to faster drug release at lower pH. The objective of this study was to achieve pH-independent release of weakly basic drugs from extended release formulations based on the naturally occurring polymer sodium alginate. Three approaches to overcome the pH-dependent solubility of the weakly basic model drug verapamil hydrochloride were investigated. First, matrix tablets were prepared by direct compression of drug substance with different types of sodium alginate only. Second, pH-modifiers were added to the drug/alginate matrix systems. Third, press-coated tablets consisting of an inner pH-modifier tablet core and an outer drug/sodium alginate coat were prepared. pH-Independent drug release was achieved from matrix tablets consisting of selected alginates and drug substance only. Alginates are better soluble at higher pH. Therefore, they are able to compensate the poor solubility of weakly basic drugs at higher pH as the matrix of the tablets dissolves faster. This approach was successful when using alginates that demonstrated fast hydration and erosion at higher pH. The approach failed for alginates with less-pronounced erosion at higher pH. The addition of fumaric acid to drug/alginate-based matrix systems decreased the microenvironmental pH within the tablets thus increasing the solubility of the weakly basic drug at higher pH. Therefore, pH-independent drug release was achieved irrespective of the type of alginate used. Drug release from press-coated tablets did not provide any further advantages as compound release remained pH-dependent.

Improvement of stability of oil-in-water emulsions containing caseinate-coated droplets by addition of sodium alginate.

PubMed

Pallandre, S; Decker, E A; McClements, D J

2007-11-01

The potential of sodium alginate for improving the stability of emulsions containing caseinate-coated droplets was investigated. One wt% corn oil-in-water emulsions containing anionic caseinate-coated droplets (0.15 wt% sodium caseinate) and anionic sodium alginate (0 to 1 wt%) were prepared at pH 7. The pH of these emulsions was then adjusted to 3.5, so that the anionic alginate molecules adsorbed to the cationic caseinate-coated droplets. Extensive droplet aggregation occurred when there was insufficient alginate to completely saturate the droplet surfaces due to bridging flocculation, and when the nonadsorbed alginate concentration was high enough to induce depletion flocculation. Emulsions with relatively small particle sizes could be formed over a range of alginate concentrations (0.1 to 0.4 wt%). The influence of pHs (3 to 7) and sodium chloride (0 to 500 mM) on the properties of primary (0 wt% alginate) and secondary (0.15 wt% alginate) emulsions was studied. Alginate adsorbed to the droplet surfaces at pHs 3, 4, and 5, but not at pHs 6 and 7, due to electrostatic attraction between anionic groups on the alginate and cationic groups on the adsorbed caseinate. Secondary emulsions had better stability than primary emulsions at pH values near caseinate's isoelectric point (pHs 4 and 5). In addition, secondary emulsions were stable up to higher ionic strengths (< 300 mM) than primary emulsions (<50 mM). The controlled electrostatic deposition method utilized in this study could be used to extend the range of application of dairy protein emulsifiers in the food industry.

Montmorillonite-Alginate Composites as a Drug delivery System: Intercalation and In vitro Release of Diclofenac sodium

PubMed Central

Kevadiya, B. D.; Patel, H. A.; Joshi, G. V.; Abdi, S. H. R.; Bajaj, H. C.

2010-01-01

Diclofenac sodium and alginate was intercalated into montmorillonite to form uniform sized beads by gelation method. The structure and surface morphology of the synthesized composite beads were characterized by powdered X-ray diffraction, Fourier transform infrared spectroscopy, thermo gravimetric analysis and scanning electron microscopy. Diclofenac release kinetics of the composite in simulated intestinal fluid medium (pH 7.4) and effect of montmorillonite content on the in vitro release of diclofenac from diclofenac-montmorillonite-alginate composites bead was investigated by UV/Vis spectrophotometer. Diclofenac encapsulation efficiency in the montmorillonite-alginate composites bead increases with an increase in the montmorillonite content. The control release of diclofenac from diclofenac-montmorillonite-alginate composites beads was observed to be better as compared to diclofenac-alginate beads. PMID:21969745

Temporal regulation and forespore-specific expression of the spore photoproduct lyase gene by sigma-G RNA polymerase during Bacillus subtilis sporulation.

PubMed Central

Pedraza-Reyes, M; Gutiérrez-Corona, F; Nicholson, W L

1994-01-01

Bacterial spores are highly resistant to killing by UV radiation and exhibit unique DNA photochemistry. UV irradiation of spore DNA results in formation of spore photoproduct (SP), the thymine dimer 5-thyminyl-5,6-dihydrothymine. Repair of SP occurs during germination of Bacillus subtilis spores by two distinct routes, either by the general nucleotide excision repair (uvr) pathway or by a novel SP-specific monomerization reaction mediated by the enzyme SP lyase, which is encoded by the spl gene. Repair of SP occurs early in spore germination and is independent of de novo protein synthesis, suggesting that the SP repair enzymes are synthesized during sporulation and are packaged in the dormant spore. To test this hypothesis, the expression of a translational spl-lacZ fusion integrated at the spl locus was monitored during B. subtilis growth and sporulation. beta-Galactosidase expression from the spl-lacZ fusion was silent during vegetative growth and was not DNA damage inducible, but it was activated at morphological stage III of sporulation specifically in the forespore compartment, coincident with activation of expression of the stage III marker enzyme glucose dehydrogenase. Expression of the spl-lacZ fusion was shown to be dependent upon the sporulation-specific RNA polymerase containing the sigma-G factor (E sigma G), as spl-lacZ expression was abolished in a mutant harboring a deletion in the sigG gene and restored by expression of the sigG gene in trans. Primer extension analysis of spl mRNA revealed a major extension product initiating upstream from a small open reading frame of unknown function which precedes spl, and it revealed two other shorter minor extension products. All three extension products were present in higher quantities during sporulation and after sigG induction. The three putative transcripts are all preceded by sequences which share homology with the consensus sigma-G factor-type promoter sequence, but in vitro transcription by purified

Hybrid 3D printing and electrodeposition approach for controllable 3D alginate hydrogel formation.

PubMed

Shang, Wanfeng; Liu, Yanting; Wan, Wenfeng; Hu, Chengzhi; Liu, Zeyang; Wong, Chin To; Fukuda, Toshio; Shen, Yajing

2017-06-07

Calcium alginate hydrogels are widely used as biocompatible materials in a substantial number of biomedical applications. This paper reports on a hybrid 3D printing and electrodeposition approach for forming 3D calcium alginate hydrogels in a controllable manner. Firstly, a specific 3D hydrogel printing system is developed by integrating a customized ejection syringe with a conventional 3D printer. Then, a mixed solution of sodium alginate and CaCO 3 nanoparticles is filled into the syringe and can be continuously ejected out of the syringe nozzle onto a conductive substrate. When applying a DC voltage (∼5 V) between the substrate (anode) and the nozzle (cathode), the Ca 2+ released from the CaCO 3 particles can crosslink the alginate to form calcium alginate hydrogel on the substrate. To elucidate the gel formation mechanism and better control the gel growth, we can further establish and verify a gel growth model by considering several key parameters, i.e., applied voltage and deposition time. The experimental results indicate that the alginate hydrogel of various 3D structures can be formed by controlling the movement of the 3D printer. A cell viability test is conducted and shows that the encapsulated cells in the gel can maintain a high survival rate (∼99% right after gel formation). This research establishes a reliable method for the controllable formation of 3D calcium alginate hydrogel, exhibiting great potential for use in basic biology and applied biomedical engineering.

Effects on intestinal microbiota and immune genes of Solea senegalensis after suspension of the administration of Shewanella putrefaciens Pdp11.

PubMed

Vidal, Sara; Tapia-Paniagua, Silvana Teresa; Moriñigo, Jesús Miguel; Lobo, Carmen; García de la Banda, Inés; Balebona, María Del Carmen; Moriñigo, Miguel Ángel

2016-11-01

The interaction host-intestinal microbiota is essential for the immunological homeostasis of the host. Probiotics, prebiotics and synbiotics are promising tools for the manipulation of the intestinal microbiota towards beneficial effects to the host. The objective of this study was to evaluate the modulation effect on the intestinal microbiota and the transcription of genes involved in the immune response in head kidney of Solea senegalensis after administration of diet supplemented with the prebiotic alginate and the probiotic Shewanella putrefaciens Pdp11 CECT 7627 (SpPdp11). The results showed higher adaptability to dietary changes in the intestinal microbiota of fish fed diet with alginate and SpPdp11 together compared to those fish that received an alginate-supplemented diet. The alginate-supplemented diet produced up-regulation of genes encoding proteins involved in immunological responses, such as complement, lysozyme G and transferrin, and oxidative stress, such as NADPH oxidase and glutation peroxidase. On the other hand, the administration of alginate combined with SpPdp11 resulted in a significant increase of the transcription of genes encoding for glutation peroxidase and HSP70, indicating a potential protective effect of SpPdp11 against oxidative stress. In addition, these effects were maintained after the suspension of the probiotic treatment. The relationship between the modulation of the intestinal microbiota and the expression of genes with protective effect against the oxidative stress was demonstrated by the Principal Components Analysis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cloned Erwinia chrysanthemi out genes enable Escherichia coli to selectively secrete a diverse family of heterologous proteins to its milieu.

PubMed Central

He, S Y; Lindeberg, M; Chatterjee, A K; Collmer, A

1991-01-01

The out genes of the enterobacterial plant pathogen Erwinia chrysanthemi are responsible for the efficient extracellular secretion of multiple plant cell wall-degrading enzymes, including four isozymes of pectate lyase, exo-poly-alpha-D-galacturonosidase, pectin methylesterase, and cellulase. Out- mutants of Er. chrysanthemi are unable to export any of these proteins beyond the periplasm and are severely reduced in virulence. We have cloned out genes from Er. chrysanthemi in the stable, low-copy-number cosmid pCPP19 by complementing several transposon-induced mutations. The cloned out genes were clustered in a 12-kilobase chromosomal DNA region, complemented all existing out mutations in Er. chrysanthemi EC16, and enabled Escherichia coli strains to efficiently secrete the extracellular pectic enzymes produced from cloned Er. chrysanthemi genes, while retaining the periplasmic marker protein beta-lactamase. DNA sequencing of a 2.4-kilobase EcoRI fragment within the out cluster revealed four genes arranged colinearly and sharing substantial similarity with the Klebsiella pneumoniae genes pulH, pulI, pulJ, and pulK, which are necessary for pullulanase secretion. However, K. pneumoniae cells harboring the cloned Er. chrysanthemi pelE gene were unable to secrete the Erwinia pectate lyase. Furthermore, the Er. chrysanthemi Out system was unable to secrete an extracellular pectate lyase encoded by a gene from a closely related plant pathogen. Erwinia carotovora ssp. carotovora. The results suggest that these enterobacteria secrete polysaccharidases by a conserved mechanism whose protein-recognition capacities have diverged. Images PMID:1992458

Cloned Erwinia chrysanthemi out genes enable Escherichia coli to selectively secrete a diverse family of heterologous proteins to its milieu.

PubMed

He, S Y; Lindeberg, M; Chatterjee, A K; Collmer, A

1991-02-01

The out genes of the enterobacterial plant pathogen Erwinia chrysanthemi are responsible for the efficient extracellular secretion of multiple plant cell wall-degrading enzymes, including four isozymes of pectate lyase, exo-poly-alpha-D-galacturonosidase, pectin methylesterase, and cellulase. Out- mutants of Er. chrysanthemi are unable to export any of these proteins beyond the periplasm and are severely reduced in virulence. We have cloned out genes from Er. chrysanthemi in the stable, low-copy-number cosmid pCPP19 by complementing several transposon-induced mutations. The cloned out genes were clustered in a 12-kilobase chromosomal DNA region, complemented all existing out mutations in Er. chrysanthemi EC16, and enabled Escherichia coli strains to efficiently secrete the extracellular pectic enzymes produced from cloned Er. chrysanthemi genes, while retaining the periplasmic marker protein beta-lactamase. DNA sequencing of a 2.4-kilobase EcoRI fragment within the out cluster revealed four genes arranged colinearly and sharing substantial similarity with the Klebsiella pneumoniae genes pulH, pulI, pulJ, and pulK, which are necessary for pullulanase secretion. However, K. pneumoniae cells harboring the cloned Er. chrysanthemi pelE gene were unable to secrete the Erwinia pectate lyase. Furthermore, the Er. chrysanthemi Out system was unable to secrete an extracellular pectate lyase encoded by a gene from a closely related plant pathogen. Erwinia carotovora ssp. carotovora. The results suggest that these enterobacteria secrete polysaccharidases by a conserved mechanism whose protein-recognition capacities have diverged.

Generation of Stable Co-Cultures of Vascular Cells in a Honeycomb Alginate Scaffold

PubMed Central

Yamamoto, Masaya; James, Daylon; Li, Hui; Butler, Jason; Rafii, Shahin

2010-01-01

Scaffold-guided vascular tissue engineering has been investigated as a means to generate functional and transplantable vascular tissue grafts that increase the efficacy of cell-based therapeutic strategies in regenerative medicine. In this study, we employed confocal microscopy and three-dimensional reconstruction to assess the engraftment and growth potential of vascular cells within an alginate scaffold with aligned pores. We fabricated honeycomb alginate scaffolds with aligned pores, whose surface was immobilized with fibronectin and subsequently coated with matrigel. Endothelial cells were seeded into aligned pore scaffolds in the presence and absence of human smooth muscle cells. We showed that endothelial cells seeded into alginate scaffolds attach on the surface of aligned pores in vitro, giving rise to stable co-cultures of vascular cells. Moreover, the three-dimensional alginate depots containing the cells were exposed to laminar flow in order to recapitulate physiological shear stress found in the vasculature in vivo. After the flow exposure, the scaffold remained intact and some cells remained adherent to the scaffold and aligned in the flow direction. These studies demonstrate that alginate scaffolds provide a suitable matrix for establishing durable angiogenic modules that may ultimately enhance organ revascularization. PMID:19705957

Kefiran-alginate gel microspheres for oral delivery of ciprofloxacin.

PubMed

Blandón, Lina M; Islan, German A; Castro, Guillermo R; Noseda, Miguel D; Thomaz-Soccol, Vanete; Soccol, Carlos R

2016-09-01

Ciprofloxacin is a broad-spectrum antibiotic associated with gastric and intestinal side effects after extended oral administration. Alginate is a biopolymer commonly employed in gel synthesis by ionotropic gelation, but unstable in the presence of biological metal-chelating compounds and/or under dried conditions. Kefiran is a microbial biopolymer able to form gels with the advantage of displaying antimicrobial activity. In the present study, kefiran-alginate gel microspheres were developed to encapsulate ciprofloxacin for antimicrobial controlled release and enhanced bactericidal effect against common pathogens. Scanning electron microscopy (SEM) analysis of the hybrid gel microspheres showed a spherical structure with a smoother surface compared to alginate gel matrices. In vitro release of ciprofloxacin from kefiran-alginate microspheres was less than 3.0% and 5.0% at pH 1.2 (stomach), and 5.0% and 25.0% at pH 7.4 (intestine) in 3 and 21h, respectively. Fourier transform infrared spectroscopy (FTIR) of ciprofloxacin-kefiran showed the displacement of typical bands of ciprofloxacin and kefiran, suggesting a cooperative interaction by hydrogen bridges between both molecules. Additionally, the thermal analysis of ciprofloxacin-kefiran showed a protective effect of the biopolymer against ciprofloxacin degradation at high temperatures. Finally, antimicrobial assays of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhymurium, and Staphylococcus aureus demonstrated the synergic effect between ciprofloxacin and kefiran against the tested microorganisms. Copyright © 2016 Elsevier B.V. All rights reserved.

Electrospray-assisted encapsulation of caffeine in alginate microhydrogels.

PubMed

Nikoo, Alireza Mehregan; Kadkhodaee, Rassoul; Ghorani, Behrouz; Razzaq, Hussam; Tucker, Nick

2018-05-02

One of the major challenges with microencapsulation and delivery of low molecular weight bioactive compounds is their diffusional loss during storage and process conditions as well as under gastric conditions. In an attempt to slow down the release rate of core material, electrospray fabricated calcium alginate microhydrogels were coated with low molecular weight and high molecular weight chitosans. Caffeine as a hydrophilic model compound was used due to its several advantages on human behavior especially increasing consciousness. Mathematical modeling of the caffeine release by fitting the data with Korsmeyer-Peppas model showed that Fick's diffusion law could be the prevalent mechanism of the release. Electrostatic interaction between alginate and chitosan (particularly in the presence of 1% low molecular weight chitosan) provided an effective barrier against caffeine release and significantly reduced swelling of particles compared to control samples. The results of this study demonstrated that calcium alginate microhydrogels coated by chitosan could be used for encapsulation of low molecular compounds. However, more complementary research must be done in this field. In addition, electrospray, by producing monodisperse particles, would be as an alternative method for fabrication of microparticles based on natural polymers. Copyright © 2018. Published by Elsevier B.V.

Cystathionine γ-lyase deficiency mediates neurodegeneration in Huntington’s disease

PubMed Central

Paul, Bindu D.; Sbodio, Juan I.; Xu, Risheng; Vandiver, M. Scott; Cha, Jiyoung Y.; Snowman, Adele M.; Snyder, Solomon H.

2015-01-01

Huntington’s disease is an autosomal dominant disease associated with a mutation in the gene encoding huntingtin (Htt) leading to expanded polyglutamine repeats of mutant Htt (mHtt) that elicit oxidative stress, neurotoxicity, and motor and behavioural changes1. Huntington’s disease is characterized by highly selective and profound damage to the corpus striatum, which regulates motor function. Striatal selectivity of Huntington’s disease may reflect the striatally selective small G protein Rhes binding to mHtt and enhancing its neurotoxicity2. Specific molecular mechanisms by which mHtt elicits neurodegeneration have been hard to determine. Here we show a major depletion of cystathionine γ-lyase (CSE), the biosynthetic enzyme for cysteine, in Huntington’s disease tissues, which may mediate Huntington’s disease pathophysiology. The defect occurs at the transcriptional level and seems to reflect influences of mHtt on specificity protein 1, a transcriptional activator for CSE. Consistent with the notion of loss of CSE as a pathogenic mechanism, supplementation with cysteine reverses abnormalities in cultures of Huntington’s disease tissues and in intact mouse models of Huntington’s disease, suggesting therapeutic potential. PMID:24670645

Identification of compounds inhibiting the C-S lyase activity of a cell extract from a Staphylococcus sp. isolated from human skin.

PubMed

Egert, M; Höhne, H-M; Weber, T; Simmering, R; Banowski, B; Breves, R

2013-12-01

The C-S lyase activity of bacteria in the human armpit releases highly malodorous, volatile sulfur compounds from nonvolatile precursor molecules. Such compounds significantly contribute to human body odour. Hence, C-S lyase represents an attractive target for anti-body-odour cosmetic products. Here, aiming at a final use in an ethanol-based deodorant formulation, 267 compounds and compound mixtures were screened for their ability to inhibit the C-S lyase activity of a Stapyhlococcus sp. crude extract. Staphylococcus sp. Isolate 128, closely related to Staphylococcus hominis, was chosen as the test bacterium, as it showed a reproducibly high specific C-S lyase activity on three different culturing media. Using a photometric assay and benzylcysteine as substrate, six rather complex, plant-derived compound mixtures and five well defined chemical compounds or compound mixtures were identified as inhibitors, leading to an inhibition of ≥70% at concentrations of ≤0·5% in the assay. The inhibition data have demonstrated that compounds with two vicinal hydroxyl groups or one hydroxyl and one keto group bound to an aryl residue are characteristic for the inhibition. The substances identified as C-S lyase inhibitors have the potential to improve the performance of anti-body-odour cosmetic products, for example, ethanol-based deodorants. Bacterial C-S lyase represents one of the key enzymes involved in human body odour formation. The aim of this study was to identify compounds inhibiting the C-S lyase activity of a Staphylococcus sp. isolate from the human skin. The compounds identified as the best inhibitors are characterized by the following features: two vicinal hydroxyl groups or one hydroxyl and one keto group bound to an aryl residue. They might be used to improve the performance of cosmetic products aiming to prevent the formation of microbially caused human body odour, for example, ethanol-based deodorants. © 2013 The Society for Applied Microbiology.

Methionine biosynthesis in higher plants. II. Purification and characterization of cystathionine beta-lyase from spinach chloroplasts.

PubMed

Droux, M; Ravanel, S; Douce, R

1995-01-10

Cystathionine beta-lyase, the second enzyme of the transsulfuration pathway leading to homocysteine synthesis was purified over 16,000-fold from spinach (Spinacia oleracea L.) leaf chloroplasts (soluble fraction). Enzyme activity was followed along the purification scheme by either a colorimetric method for the determination of cysteine or by fluorescence detection of the bimane derivative of L-homocysteine after reverse-phase HPLC. Cystathionine beta-lyase has a molecular mass of 170,000 +/- 5000 Da and consists of four identical subunits of 44,000 Da. The enzyme exhibits an absorption spectrum in the visible range with a maximum at 418 nm due to pyridoxal 5'-phosphate. The chloroplastic enzyme catalyzes alpha,beta-cleavage of the thioether L-cystathionine and the dithioacetal L-djenkolate with apparent Km values of 0.15 and 0.34 mM, respectively, and apparent Vm values corresponding to a specific activity of 13 Units mg-1. However, no activity was detected toward the disulfide L-cysteine. With either L-cystathionine and L-djenkolate as substrate, maximal activity was obtained between pH 8.3 and pH 9.0. Besides the chloroplastic enzyme form, anion exchange chromatography of a total spinach leaf extract allowed the detection of a second pool of cystathionine beta-lyase activity that is associated with the cytosolic compartment and eluted at a lower salt concentration than the chloroplastic isoform. Kinetics of inactivation of cystathionine beta-lyase by the L-alpha-(2-aminoethoxyvinyl) glycine (AVG), an analogue of L-cystathionine, are consistent with the existence of an intermediate reversible enzyme inhibitor complex (apparent inhibition constant Kappi of 110 microM) preceding the irreversible formation of a final inactivated state of the enzyme (kd = 4.8 x 10(-3) s-1). Pyridoxal 5'-phosphate free in solution binds AVG with an apparent dissociation constant Kapp in the order of 350 microM. The comparison between the Kapp (free pyridoxal 5'-phosphate) and Kappi

A comparative study on the raft chemical properties of various alginate antacid raft-forming products.

PubMed

Dettmar, Peter W; Gil-Gonzalez, Diana; Fisher, Jeanine; Flint, Lucy; Rainforth, Daniel; Moreno-Herrera, Antonio; Potts, Mark

2018-01-01

Research to measure the chemical characterization of alginate rafts for good raft performance and ascertain how formulation can affect chemical parameters. A selection of alginate formulations was investigated all claiming to be proficient raft formers with significance between products established and ranked. Procedures were selected which demonstrated the chemical characterization allowing rafts to effectively impede the reflux into the esophagus or in severe cases to be refluxed preferentially into the esophagus and exert a demulcent effect, with focus of current research on methods which complement previous studies centered on physical properties. The alginate content was analyzed by a newly developed HPLC method. Methods were used to determine the neutralization profile and the acid neutralization within the raft determined along with how raft structure affects neutralization. Alginate content of Gaviscon Double Action (GDA) within the raft was significantly superior (p < .0001) to all competitor products. The two products with the highest raft acid neutralization capacity were GDA and Rennie Duo, the latter product not being a raft former. Raft structure was key and GDA had the right level of porosity to allow for longer duration of neutralization. Alginate formulations require three chemical reactions to take place simultaneously: transformation to alginic acid, sodium carbonate reacting to form carbon dioxide, calcium releasing free calcium ions to bind with alginic acid providing strength to raft formation. GDA was significantly superior (p <.0001) to all other comparators.

Core-shell alginate-ghatti gum modified montmorillonite composite matrices for stomach-specific flurbiprofen delivery.

PubMed

Bera, Hriday; Ippagunta, Sohitha Reddy; Kumar, Sanoj; Vangala, Pavani

2017-07-01

Novel alginate-arabic gum (AG) gel membrane coated alginate-ghatti gum (GG) modified montmorillonite (MMT) composite matrices were developed for intragastric flurbiprofen (FLU) delivery by combining floating and mucoadhesion mechanisms. The clay-biopolymer composite matrices containing FLU as core were accomplished by ionic-gelation technique. Effects of polymer-blend (alginate:GG) ratios and crosslinker (CaCl 2 ) concentrations on drug entrapment efficiency (DEE, %) and cumulative drug release after 8h (Q 8h , %) were studied to optimize the core matrices by a 3 2 factorial design. The optimized matrices (F-O) demonstrated DEE of 91.69±1.43% and Q 8h of 74.96±1.56% with minimum errors in prediction. The alginate-AG gel membrane enveloped optimized matrices (F-O, coated) exhibited superior buoyancy, better ex vivo mucoadhesion and slower drug release rate. The drug release profile of FLU-loaded uncoated and coated optimized matrices was best fitted in Korsmeyer-Peppas model with anomalous diffusion and case-II transport driven mechanism, respectively. The uncoated and coated matrices containing FLU were also characterized for drug-excipients compatibility, drug crystallinity, thermal behaviour and surface morphology. Thus, the newly developed alginate-AG gel membrane coated alginate-GG modified MMT composite matrices are appropriate for intragastric delivery of FLU over an extended period of time with improved therapeutic benefits. Copyright © 2017 Elsevier B.V. All rights reserved.

Synergistic effect of divalent cations in improving technological properties of cross-linked alginate beads.

PubMed

Cerciello, Andrea; Del Gaudio, Pasquale; Granata, Veronica; Sala, Marina; Aquino, Rita P; Russo, Paola

2017-08-01

Gelling solution parameters are some of the most important variables in ionotropic gelation and consequently influence the technological characteristics of the product. To date, only a few studies have focused on the simultaneous use of multiple cations as gelling agents. With the aim to deeply explore this possibility, in this research we investigated the effect of two divalent cations (Ca 2+ and Zn 2+ ) on alginate beads formation and properties. Alginate beads containing prednisolone (P) as model drug were prepared by prilling technique. The main critical variables of the ionotropic gelation process i.e. composition of the aqueous feed solutions (sodium alginate and prednisolone concentration) and cross-linking conditions (Ca 2+ , Zn 2+ or Ca 2+ +Zn 2+ ), were studied. The obtained beads were characterized and their in vitro release performances were assessed in conditions simulating the gastrointestinal environment. Results evidenced a synergistic effect of the two cations, affecting positively both the encapsulation efficiency and the ability of the alginate polymeric matrix to control the drug release. A Ca 2+ /Zn 2+ ratio of 4:1, in fact, exploited the Ca 2+ ability of establish quicker electrostatic interactions with guluronic groups of alginate and the Zn 2+ ability to establish covalent-like bonds with carboxylate groups of both guluronic and mannuronic moieties of alginate. Copyright © 2017 Elsevier B.V. All rights reserved.

Fabrication of monodispersive nanoscale alginate-chitosan core-shell particulate systems for controlled release studies

NASA Astrophysics Data System (ADS)

Körpe, Didem Aksoy; Malekghasemi, Soheil; Aydın, Uğur; Duman, Memed

2014-12-01

Biopolymers such as chitosan and alginate are widely used for controlled drug delivery systems. The present work aimed to develop a new protocol for preparation of monodisperse alginate-coated chitosan nanoparticles at nanoscale. Modifications of preparation protocol contain changing the pH of polymer solutions and adding extra centrifugation steps into the procedure. While chitosan nanoparticles were synthesized by ionic gelation method, they were coated with alginate by electrostatic interaction. The size, morphology, charge, and structural characterization of prepared core-shell nanoparticulated system were performed by AFM, Zeta sizer, and FTIR. BSA and DOX were loaded as test biomolecules to core and shell part of the nanoparticle, respectively. Release profiles of BSA and DOX were determined by spectrophotometry. The sizes of both chitosan and alginate-coated chitosan nanoparticles which were prepared by modified protocol were measured to be 50 ± 10 and 60 ± 3 nm, respectively. After loading BSA and DOX, the average size of the particles increased to 80 ± 7 nm. Moreover, while the zeta potential of chitosan nanoparticles was positive value, the value was inverted to negative after alginate coating. Release profile measurements of BSA and DOX were determined during 57 and 2 days, respectively. Our results demonstrated that monodisperse alginate-coated nanoparticles were synthesized and loaded successfully using our modified protocol.

Development of silver nanoparticles loaded chitosan-alginate constructs with biomedical potentialities.

PubMed

Bilal, Muhammad; Rasheed, Tahir; Iqbal, Hafiz M N; Li, Chuanlong; Hu, Hongbo; Zhang, Xuehong

2017-12-01

Herein, a facile biosynthesis of silver nanoparticles (AgNPs) and AgNPs-loaded chitosan-alginate constructs with biomedical potentialities is reported. The UV-vis spectroscopic profile confirmed the synthesis of AgNPs using methanolic leaves extract of Euphorbia helioscopia. The newly developed AgNPs were characterized using various analytical and imaging techniques including UV-vis and FT-IR spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), atomic force microscopy (AFM), and transmission electron microscopy (TEM). The optimally yielded AgNPs at 24h reaction period were loaded onto various chitosan-alginate constructs. A maximum of 95% loading efficiency (LE) was recorded with a chitosan: alginate ratio at 2:1, followed by 81% at 2:2 ratios. The anti-bacterial activities of AgNPs and AgNPs loaded chitosan-alginate constructs were tested against six bacterial strains i.e. Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Morganella morganii and Haemophilus influenza. A significant reduction in the log values was recorded for all test constructs, in comparison to the initial bacterial count (control value, i.e., 1.5×10 8 CFU/mL). The cytotoxicity profile revealed complete biocompatibility against normal cell line i.e. L929. Almost all constructs showed considerable cytotoxicity up to certain extant against human epithelial cells (HeLa) cancer cells. In summary, the highest antibacterial activities along with anti-cancer behavior both suggest the biomedical potentialities of newly engineered AgNPs and AgNPs-loaded chitosan-alginate constructs. Copyright © 2017 Elsevier B.V. All rights reserved.

Chondrocyte response to cyclic hydrostatic pressure in alginate versus pellet culture.

PubMed

Elder, Steven H; Sanders, Shawn W; McCulley, William R; Marr, Misti L; Shim, Joon W; Hasty, Karen A

2006-04-01

Cells are often cultured at high density (e.g., confluent monolayer and as pellets) to promote chondrogenic differentiation and to maintain the chondrocyte phenotype. They are also frequently suspended in hydrogels such as agarose or alginate for the same purposes. These culture techniques differ markedly with respect to frequency of direct contact between cells and overall intercellular spacing. Because these factors may significantly affect mechanotransduction, the purpose of this study was to determine if the response of articular chondrocytes to cyclic hydrostatic pressure would depend on the culture condition. Primary articular chondrocytes from young and mature pigs were cultured either as pellets or suspended in alginate beads. Both groups were exposed to dynamic hydrostatic pressure (4 MPa, 1 Hz, 5400 cycles per day) for 7 days. Cell proliferation was unaffected by pressure, but pressurized chondrocytes in pellet culture had significantly greater sGAG content and incorporated [3H]proline at a higher rate than nonpressurized controls. Electron microscopy revealed a fibrous extracellular matrix (ECM) surrounding pellets, but not cells in alginate. In addition, expression of Connexin 43 (Cx43) mRNA was slightly lower in alginate than in pellet cultures and was not significantly altered by loading. Thus, metabolic response of chondrocytes to dynamic hydrostatic pressure was affected by culture technique; chondrocytes cultured as pellets exhibited the classical anabolic response to dynamic hydrostatic pressure, but those in alginate did not. Although cell-ECM interaction could be important, the differential response is not likely attributable to differential expression of Cx43 mRNA. Copyright 2006 Orthopaedic Research Society

Work of adhesion between mucin macromolecule and calcium-alginate gels on molecular level.

PubMed

Popeski-Dimovski, Riste

2015-06-05

The bioadhesion of biopolymers to mucus layers is of great interest for the development of drug delivery systems. Herein we use AFM force measurements to evaluate the interaction on molecular level between a mucin macromolecule attached to an AFM tip and a calcium-alginate gel layer. The total work of adhesion is measured from the AFM force curves depending on different parameters: time of contact, G/M ratio of the alginate, and crosslink ratio of the gel. The total work of adhesion is found to be in the range of 1×10(-19) to 6×10(-18)J. The results show that the work of adhesion increases with the time of contact but it is independent from the molecular mass of the alginate, the G/M ratio of the alginate and crosslink ratio of the gel. Copyright © 2015 Elsevier Ltd. All rights reserved.

Detecting De-gelation through Tissue Using Magnetically Modulated Optical Nanoprobes (MagMOONs)

PubMed Central

Nguyen, KhanhVan T.; Anker, Jeffrey N.

2014-01-01

Alginate gels are widely used for drug delivery and implanted devices. The rate at which these gels break down is important for controlling drug release. Since the de-gelation may be different in vivo, monitoring this process in situ is essential. However, it is challenging to monitor the gel through tissue due to optical scattering and tissue autofluorescence. Herein we describe a method to detect through tissue the chemically-induced changes in viscosity and de-gelation process of alginate gels using magnetically modulated optical nanoprobes (MagMOONs). The MagMOONs are fluorescent magnetic microspheres coated with a thin layer of opaque metal on one hemisphere. The metal layer prevents excitation and emission light from passing through one side of the MagMOONs, which creates orientation-dependent fluorescence intensity. The magnetic particles also align in an external magnetic field and give blinking signals when they rotate to follow an external modulated magnetic field. The blinking signals from these MagMOONs are distinguished from background autofluorescence and can be tracked on a single particle level in the absence of tissue, or for an ensemble average of particles blinking through tissue. When these MagMOONs are dispersed in calcium alginate gel, they become sensors for detecting gel degradation upon addition of either ammonium ion or alginate lyase. Our results show MagMOONs start blinking approximately 10 minutes after 2 mg/mL alginate lyase addition and this blinking is clearly detected even through up to 4 mm chicken breast. This approach can potentially be employed to detect bacterial biofilm formation on medical implants by sensing specific proteases that either activate a related function or regulate biofilm formation. It can also be applied to other biosensors and drug delivery systems based on enzyme-catalyzed breakdown of gel components. PMID:26273129

Detecting De-gelation through Tissue Using Magnetically Modulated Optical Nanoprobes (MagMOONs).

PubMed

Nguyen, KhanhVan T; Anker, Jeffrey N

2014-12-15

Alginate gels are widely used for drug delivery and implanted devices. The rate at which these gels break down is important for controlling drug release. Since the de-gelation may be different in vivo , monitoring this process in situ is essential. However, it is challenging to monitor the gel through tissue due to optical scattering and tissue autofluorescence. Herein we describe a method to detect through tissue the chemically-induced changes in viscosity and de-gelation process of alginate gels using magnetically modulated optical nanoprobes (MagMOONs). The MagMOONs are fluorescent magnetic microspheres coated with a thin layer of opaque metal on one hemisphere. The metal layer prevents excitation and emission light from passing through one side of the MagMOONs, which creates orientation-dependent fluorescence intensity. The magnetic particles also align in an external magnetic field and give blinking signals when they rotate to follow an external modulated magnetic field. The blinking signals from these MagMOONs are distinguished from background autofluorescence and can be tracked on a single particle level in the absence of tissue, or for an ensemble average of particles blinking through tissue. When these MagMOONs are dispersed in calcium alginate gel, they become sensors for detecting gel degradation upon addition of either ammonium ion or alginate lyase. Our results show MagMOONs start blinking approximately 10 minutes after 2 mg/mL alginate lyase addition and this blinking is clearly detected even through up to 4 mm chicken breast. This approach can potentially be employed to detect bacterial biofilm formation on medical implants by sensing specific proteases that either activate a related function or regulate biofilm formation. It can also be applied to other biosensors and drug delivery systems based on enzyme-catalyzed breakdown of gel components.

Sodium alginate hydrogel-based bioprinting using a novel multinozzle bioprinting system.

PubMed

Song, Seung-Joon; Choi, Jaesoon; Park, Yong-Doo; Hong, Soyoung; Lee, Jung Joo; Ahn, Chi Bum; Choi, Hyuk; Sun, Kyung

2011-11-01

Bioprinting is a technology for constructing bioartificial tissue or organs of complex three-dimensional (3-D) structure with high-precision spatial shape forming ability in larger scale than conventional tissue engineering methods and simultaneous multiple components composition ability. It utilizes computer-controlled 3-D printer mechanism or solid free-form fabrication technologies. In this study, sodium alginate hydrogel that can be utilized for large-dimension tissue fabrication with its fast gelation property was studied regarding material-specific printing technique and printing parameters using a multinozzle bioprinting system developed by the authors. A sodium alginate solution was prepared with a concentration of 1% (wt/vol), and 1% CaCl(2) solution was used as cross-linker for the gelation. The two materials were loaded in each of two nozzles in the multinozzle bioprinting system that has a total of four nozzles of which the injection speed can be independently controlled. A 3-D alginate structure was fabricated through layer-by-layer printing. Each layer was formed through two phases of printing, the first phase with the sodium alginate solution and the second phase with the calcium chloride solution, in identical printing pattern and speed condition. The target patterns were lattice shaped with 2-mm spacing and two different line widths. The nozzle moving speed was 6.67 mm/s, and the injection head speed was 10 µm/s. For the two different line widths, two injection needles with inner diameters of 260 and 410 µm were used. The number of layers accumulated was five in this experiment. By varying the nozzle moving speed and the injection speed, various pattern widths could be achieved. The feasibility of sodium alginate hydrogel free-form formation by alternate printing of alginate solution and sodium chloride solution was confirmed in the developed multinozzle bioprinting system. © 2011, Copyright the Authors. Artificial Organs © 2011, International

Free-standing polyelectrolyte membranes made of chitosan and alginate

PubMed Central

Caridade, Sofia G.; Monge, Claire; Gilde, Flora; Boudou, Thomas; Mano, João F.; Picart, Catherine

2014-01-01

Free-standing films have increasing applications in the biomedical field as drug delivery systems, for wound healing and tissue engineering. Here, we prepared free-standing membranes by the layer-by-layer assembly of chitosan and alginate, two widely used biomaterials. Our aim was to produce thick membrane, to study the permeation of model drugs and the adhesion of muscle cells. We first defined the optimal growth conditions in terms of pH and alginate concentration. The membranes could be easily detached from polystyrene or polypropylene substrate without any post-processing step. They dry thickness was varied over a large range from 4 to 35 μm. A two-fold swelling was observed by confocal microscopy when they were immersed in PBS. In addition, we quantified the permeation of model drugs (fluorescent dextrans) through the free standing membrane, which depended on the dextran molecular weight. Finally, we showed that myoblast cells exhibited a preferential adhesion on the alginate-ending membrane as compared to the chitosan-ending membrane or to the substrate side. PMID:23590116

Preparation of highly stable zeolite-alginate foam composite for strontium(90Sr) removal from seawater and evaluation of Sr adsorption performance.

PubMed

Hong, Hye-Jin; Kim, Byoung-Gyu; Ryu, Jungho; Park, In-Su; Chung, Kang-Sup; Lee, Sang Moon; Lee, Jin-Bae; Jeong, Hyeon Su; Kim, Hyunchul; Ryu, Taegong

2018-01-01

Alginate bead is a promising strontium (Sr) adsorbent in seawater, but highly concentrated Na ions caused over-swelling and damaged the hydrogel bead. To improve the mechanical stability of alginate bead, flexible foam-type zeolite-alginate composite was synthesized and Sr adsorption performance was evaluated in seawater; 1-10% zeolite immobilized alginate foams were prepared by freeze-dry technique. Immobilization of zeolite into alginate foam converted macro-pores to meso-pores which lead to more compact structure. It resulted in less swollen composite in seawater medium and exhibited highly improved mechanical stability compared with alginate bead. Besides, Sr adsorption efficiency and selectivity were enhanced by immobilization of zeolite in alginate foam due to the increase of Sr binding sites (zeolite). In particular, Sr selectivity against Na was highly improved. The 10% zeolite-alginate foam exhibited a higher log K d of 3.3, while the pure alginate foam exhibited 2.7 in the presence of 0.1 M Na. Finally, in the real seawater, the 10% zeolite-alginate foam exhibited 1.5 times higher Sr adsorption efficiency than the pure alginate foam. This result reveals that zeolite-alginate foam composite is appropriate material for Sr removal in seawater due to its swelling resistance as well as improved Sr adsorption performance in complex media. Copyright © 2017 Elsevier Ltd. All rights reserved.

Inhibition of cystathionine-gamma-lyase leads to loss of glutathione and aggravation of mitochondrial dysfunction mediated by excitatory amino acid in the CNS.

PubMed

Diwakar, Latha; Ravindranath, Vijayalakshmi

2007-01-01

Oxidative stress has been implicated in the pathogenesis and progression of neurodegenerative disorders and antioxidants potentially have a major role in neuroprotection. Optimum levels of glutathione (gamma-glutamylcysteinyl glycine), an endogenous thiol antioxidant are required for the maintenance of the redox status of cells. Cystathionine gamma-lyase is the rate-limiting enzyme for the synthesis of cysteine from methionine and availability of cysteine is a critical factor in glutathione synthesis. In the present study, we have examined the role of cystathionine gamma-lyase in maintaining the redox homeostasis in brain, particularly with reference to mitochondrial function since the complex I of the electron transport chain is sensitive to redox perturbation. Inhibition of cystathionine gamma-lyase by l-propargylglycine caused loss of glutathione and decrease in complex I activity in the brain although the enzyme activity in mouse brain was 1% of the corresponding hepatic activity. We then examined the effect of this inhibition on the neurotoxicity mediated by the excitatory amino acid, l-beta-oxalyl amino-l-alanine, which is the causative factor of a type of motor neuron disease, neurolathyrism. l-beta-Oxalyl amino-l-alanine toxicity was exacerbated by l-propargylglycine measured as loss of complex I activity indicating the importance of cystathionine gamma-lyase in maintaining glutathione levels and in turn the mitochondrial function during excitotoxicity. Oxidative stress generated by l-beta-oxalyl amino-l-alanine itself inhibited cystathionine gamma-lyase, which could be prevented by prior treatment with thiol antioxidant. Thus, cystathionine gamma-lyase itself is susceptible to inactivation by oxidative stress and this can potentially exacerbate oxidant-induced damage. Cystathionine gamma-lyase is present in neuronal cells in human brain and its activity is several-fold higher compared to mouse brain. It could potentially play an important role in

An acidic pectin lyase from Aspergillus niger with favourable efficiency in fruit juice clarification.

PubMed

Xu, S X; Qin, X; Liu, B; Zhang, D Q; Zhang, W; Wu, K; Zhang, Y H

2015-02-01

The pectin lyase gene pnl-zj5a from Aspergillus niger ZJ5 was identified and expressed in Pichia pastoris. PNL-ZJ5A was purified by ultrafiltration, anion exchange and gel chromatography. The Km and Vmax values determined using citrus pectin were 0.66 mg ml(-1) and 32.6 μmol min(-1) mg(-1) , respectively. PNL-ZJ5A exhibited optimal activity at 43°C and retained activity over 25-50°C. PNL-ZJ5A was optimally active at pH 5 and effective in apple juice clarification. Compared with controls, PNL-ZJ5A increased the fruit juice yield significantly. Furthermore, PNL-ZJ5A reduced the viscosity of apple juice by 38.8% and increased its transmittance by 86.3%. PNL-ZJ5A combined with a commercial pectin esterase resulted in higher juice volume. © 2014 The Society for Applied Microbiology.

Generation and Phenotypic Characterization of Aspergillus nidulans Methylisocitrate Lyase Deletion Mutants: Methylisocitrate Inhibits Growth and Conidiation

PubMed Central

Brock, Matthias

2005-01-01

Propionate is a very abundant carbon source in soil, and many microorganisms are able to use this as the sole carbon source. Nevertheless, propionate not only serves as a carbon source for filamentous fungi but also acts as a preservative when added to glucose containing media. To solve this contradiction between carbon source and preservative effect, propionate metabolism of Aspergillus nidulans was studied and revealed the methylcitrate cycle as the responsible pathway. Methylisocitrate lyase is one of the key enzymes of that cycle. It catalyzes the cleavage of methylisocitrate into succinate and pyruvate and completes the α-oxidation of propionate. Previously, methylisocitrate lyase was shown to be highly specific for the substrate (2R,3S)-2-methylisocitrate. Here, the identification of the genomic sequence of the corresponding gene and the generation of deletion mutants is reported. Deletion mutants did not grow on propionate as sole carbon and energy source and were severely inhibited during growth on alternative carbon sources, when propionate was present. The strongest inhibitory effect was observed, when glycerol was the main carbon source, followed by glucose and acetate. In addition, asexual conidiation was strongly impaired in the presence of propionate. These effects might be caused by competitive inhibition of the NADP-dependent isocitrate dehydrogenase, because the Ki of (2R,3S)-2-methylisocitrate, the product of the methylcitrate cycle, on NADP-dependent isocitrate dehydrogenase was determined as 1.55 μM. Other isomers had no effect on enzymatic activity. Therefore, methylisocitrate was identified as a potential toxic compound for cellular metabolism. PMID:16151139

Systematic study of alginate-based microcapsules by micropipette aspiration and confocal fluorescence microscopy.

PubMed

Kleinberger, Rachelle M; Burke, Nicholas A D; Dalnoki-Veress, Kari; Stöver, Harald D H

2013-10-01

Micropipette aspiration and confocal fluorescence microscopy were used to study the structure and mechanical properties of calcium alginate hydrogel beads (A beads), as well as A beads that were additionally coated with poly-L-lysine (P) and sodium alginate (A) to form, respectively, AP and APA hydrogels. A beads were found to continue curing for up to 500 h during storage in saline, due to residual calcium chloride carried over from the gelling bath. In subsequent saline washes, micropipette aspiration proved to be a sensitive indicator of gel weakening and calcium loss. Aspiration tests were used to compare capsule stiffness before and after citrate extraction of calcium. They showed that the initial gel strength is largely due to the calcium alginate gel cores, while the long term strength is solely due to the poly-L-lysine-alginate polyelectrolyte complex (PEC) shells. Confocal fluorescence microscopy showed that calcium chloride exposure after PLL deposition led to PLL redistribution into the hydrogel bead, resulting in thicker but more diffuse and weaker PEC shells. Adding a final alginate coating to form APA capsules did not significantly change the PEC membrane thickness and stiffness, but did speed the loss of calcium from the bead core. © 2013.

Synthesis and characterization of guar-alginate hybrid bead templated mercury sorbing titania spheres.

PubMed

Singh, Vandana; Preeti; Singh, Angela; Singh, Devendra; Singh, Yadveer; Pandey, Arvind Kumar

2015-01-01

Present communication reports on the synthesis and characterization of Hg(II) sorbing millimeter sized porous titania spheres (TSP). The synthesis utilizes guar gum-alginate hybrid beads as sacrificial template to polymerize titanium(IV) isopropoxide. The hybrid beads are crafted by pouring guar-alginate mixed solution to calcium bath. The mechanical strength of the beads depended on guar to alginate ratio in the mixed solution. The equal weight ratio of the two polysaccharides is appropriate for adequate mechanical strength beads. The unique performance of the templating beads is attributed to the synergistic interaction between guar gum and sodium alginate. FTIR, BET, SEM, TEM, XRD, TGA, and DTG analyses have been used for the characterization of the optimum performance TSP (TSPAG2). TSPAG2 is a mesoporous material that has higher surface area and narrower pore size distribution than pure alginate derived titania spheres (TSPA). TEM study demonstrated that TSPAG2 spheres are constituted of aggregated TiO2 nanoparticles of ∼ 10 nm size. TSPAG2 is able to capture >95% Hg(II) from synthetic Hg(II) solution in 10h at pH 5 as opposed to only 68% removal by TSPA. Copyright © 2014 Elsevier B.V. All rights reserved.

Design and performance of a sericin-alginate interpenetrating network hydrogel for cell and drug delivery.

PubMed

Zhang, Yeshun; Liu, Jia; Huang, Lei; Wang, Zheng; Wang, Lin

2015-07-24

Although alginate hydrogels have been extensively studied for tissue engineering applications, their utilization is limited by poor mechanical strength, rapid drug release, and a lack of cell adhesive ability. Aiming to improve these properties, we employ the interpenetrating hydrogel design rationale. Using alginate and sericin (a natural protein with many unique properties and a major component of silkworm silk), we develop an interpenetrating polymer network (IPN) hydrogel comprising interwoven sericin and alginate double networks. By adjusting the sericin-to-alginate ratios, IPNs' mechanical strength can be adjusted to meet stiffness requirements for various tissue repairs. The IPNs with high sericin content show increased stability during degradation, avoiding pure alginate's early collapse. These IPNs have high swelling ratios, benefiting various applications such as drug delivery. The IPNs sustain controlled drug release with the adjustable rates. Furthermore, these IPNs are adhesive to cells, supporting cell proliferation, long-term survival and migration. Notably, the IPNs inherit sericin's photoluminescent property, enabling bioimaging in vivo. Together, our study indicates that the sericin-alginate IPN hydrogels may serve as a versatile platform for delivering cells and drugs, and suggests that sericin may be a building block broadly applicable for generating IPN networks with other biomaterials for diverse tissue engineering applications.

Effect of lecithin and starch on alginate-encapsulated probiotic bacteria.

PubMed

Donthidi, A R; Tester, R F; Aidoo, K E

2010-01-01

The effect of lecithin and starch on viability of alginate encapsulated probiotics was determined at different temperatures. Probiotic organisms (1% v/v>10Log CFU ml(-1)) were encapsulated using alginate (2% w/v), gelatinized starches (2% w/v) and lecithin (0-4% w/v) and stored in sealed containers at 4, 23 and 37 degrees C (to simulate shelf storage conditions). Incorporation of lecithin improved the entrapment efficiency (p < 0.05) and the viability of encapsulated bacteria (p = 0.02). Encapsulated Lactobacillus, Bifidobacterium species and Lactococcus lactis in lecithin containing freeze-dried beads had good survival stability (above 6Log CFU ml(-1)) at 23 degrees C for 12 weeks. The bacteria in the beads showed 6Log survival by the end of 2 weeks at 37 degrees C. Encapsulated L. casei in the alginate beads containing lecithin were also more stable in the yoghurt than the beads without lecithin. SEM analysis of the beads showed an irregular surface for the beads without lecithin.

Alginate based polyurethanes: A review of recent advances and perspective.

PubMed

Zia, Khalid Mahmood; Zia, Fatima; Zuber, Mohammad; Rehman, Saima; Ahmad, Mirza Nadeem

2015-08-01

The trend of using biopolymers in combination with synthetic polymers was increasing rapidly from last two or three decades. Polysaccharide based biopolymers especially starch, cellulose, chitin, chitosan, alginate, etc. found extensive applications for different industrial uses, as they are biocompatible, biodegradable, bio-renewable resources and chiefly environment friendly. Segment block copolymer character of polyurethanes that endows them a broad range of versatility in terms of tailoring their properties was employed in conjunction with various natural polymers resulted in modified biomaterials. Alginate is biodegradable, biocompatible, bioactive, less toxic and low cost anionic polysaccharide, as a part of structural component of bacteria and brown algae (sea weed) is quite abundant in nature. It is used in combination with polyurethanes to form elastomers, nano-composites, hydrogels, etc. that especially revolutionized the food and biomedical industries. The review summarized the development in alginate based polyurethanes with their potential applications. Copyright © 2015 Elsevier B.V. All rights reserved.

Probing the Active Center of Benzaldehyde Lyase with Substitutions and the Pseudosubstrate Analogue Benzoylphosphonic Acid Methyl Ester

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

Brandt, Gabriel S.; Nemeria, Natalia; Chakraborty, Sumit

2008-07-28

Benzaldehyde lyase (BAL) catalyzes the reversible cleavage of (R)-benzoin to benzaldehyde utilizing thiamin diphosphate and Mg{sup 2+} as cofactors. The enzyme is important for the chemoenzymatic synthesis of a wide range of compounds via its carboligation reaction mechanism. In addition to its principal functions, BAL can slowly decarboxylate aromatic amino acids such as benzoylformic acid. It is also intriguing mechanistically due to the paucity of acid-base residues at the active center that can participate in proton transfer steps thought to be necessary for these types of reactions. Here methyl benzoylphosphonate, an excellent electrostatic analogue of benzoylformic acid, is used tomore » probe the mechanism of benzaldehyde lyase. The structure of benzaldehyde lyase in its covalent complex with methyl benzoylphosphonate was determined to 2.49 {angstrom} (Protein Data Bank entry 3D7K) and represents the first structure of this enzyme with a compound bound in the active site. No large structural reorganization was detected compared to the complex of the enzyme with thiamin diphosphate. The configuration of the predecarboxylation thiamin-bound intermediate was clarified by the structure. Both spectroscopic and X-ray structural studies are consistent with inhibition resulting from the binding of MBP to the thiamin diphosphate in the active centers. We also delineated the role of His29 (the sole potential acid-base catalyst in the active site other than the highly conserved Glu50) and Trp163 in cofactor activation and catalysis by benzaldehyde lyase.« less

Intragastric Gelation of Heated Soy Protein Isolate-Alginate Mixtures and Its Effect on Sucrose Release.

PubMed

Huang, Zhaozhi; Gruen, Ingolf; Vardhanabhuti, Bongkosh

2018-06-15

The goal of our study was to investigate the effect of alginate on in vitro gastric digestion and sucrose release of soy protein isolate (SPI) in model beverages. Model beverages containing 5% w/w SPI, 0% to 0.20% w/w alginate, and 10% w/w sucrose were prepared by heating the mixtures at 85 °C for 30 min at pH 6.0 or 7.0. Characterizations of beverages included determination of zeta potential, particle size and rheological properties. Digestion patterns and sucrose release profiles were determined during 2 hr in vitro gastric digestion using SDS-PAGE and HPLC analysis, respectively. Increasing alginate concentration led to increased negative surface charge, particle size, as well as viscosity and pseudoplastic behavior; however, no phase separation was observed. SPI beverages formed intragastric gel during in vitro gastric digestion when the formulations contained alginate or at pH 6.0 without alginate. Formation of the intragastric gel led to delayed protein digestion and release of sucrose. Higher resistance to digestion and a slower sucrose release rate were exhibited at increased alginate concentration, and to a lesser extent, at pH 6.0. This suggests that electrostatic interaction between SPI and alginate that occurred when the beverages were under gastric condition could be responsible for the intragastric gelation. These results could potentially lead to the formulation of SPI beverages with functionality to lower postprandial glycemic response. The results could be used to design beverages or semi solid food products with altered digestion properties and lowered or slower glucose release. © 2018 Institute of Food Technologists®.

Transcriptional regulation of Saccharomyces cerevisiaeCYS3 encoding cystathionine γ-lyase

PubMed Central

Hiraishi, Hiroyuki; Miyake, Tsuyoshi

2008-01-01

In studying the regulation of GSH11, the structural gene of the high-affinity glutathione transporter (GSH-P1) in Saccharomyces cerevisiae, a cis-acting cysteine responsive element, CCGCCACAC (CCG motif), was detected. Like GSH-P1, the cystathionine γ-lyase encoded by CYS3 is induced by sulfur starvation and repressed by addition of cysteine to the growth medium. We detected a CCG motif (−311 to −303) and a CGC motif (CGCCACAC; −193 to −186), which is one base shorter than the CCG motif, in the 5′-upstream region of CYS3. One copy of the centromere determining element 1, CDE1 (TCACGTGA; −217 to −210), being responsible for regulation of the sulfate assimilation pathway genes, was also detected. We tested the roles of these three elements in the regulation of CYS3. Using a lacZ-reporter assay system, we found that the CCG/CGC motif is required for activation of CYS3, as well as for its repression by cysteine. In contrast, the CDE1 motif was responsible for only activation of CYS3. We also found that two transcription factors, Met4 and VDE, are responsible for activation of CYS3 through the CCG/CGC and CDE1 motifs. These observations suggest a dual regulation of CYS3 by factors that interact with the CDE1 motif and the CCG/CGC motifs. PMID:18317767

21 CFR 184.1610 - Potassium alginate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Potassium alginate. 184.1610 Section 184.1610 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) DIRECT FOOD SUBSTANCES AFFIRMED AS GENERALLY RECOGNIZED AS SAFE Listing of...

21 CFR 582.7187 - Calcium alginate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Calcium alginate. 582.7187 Section 582.7187 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Stabilizers § 582.7187...

21 CFR 582.7610 - Potassium alginate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Potassium alginate. 582.7610 Section 582.7610 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Stabilizers § 582.7610...

21 CFR 582.7610 - Potassium alginate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Potassium alginate. 582.7610 Section 582.7610 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Stabilizers § 582.7610...

21 CFR 582.7133 - Ammonium alginate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Ammonium alginate. 582.7133 Section 582.7133 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Stabilizers § 582.7133...

21 CFR 184.1610 - Potassium alginate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Potassium alginate. 184.1610 Section 184.1610 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) DIRECT FOOD SUBSTANCES AFFIRMED AS GENERALLY RECOGNIZED AS SAFE Listing of...

21 CFR 582.7187 - Calcium alginate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Calcium alginate. 582.7187 Section 582.7187 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Stabilizers § 582.7187...

21 CFR 582.7133 - Ammonium alginate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Ammonium alginate. 582.7133 Section 582.7133 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Stabilizers § 582.7133...

Genome Sequence of Microbulbifer mangrovi DD-13T Reveals Its Versatility to Degrade Multiple Polysaccharides.

PubMed

Imran, Md; Pant, Poonam; Shanbhag, Yogini P; Sawant, Samir V; Ghadi, Sanjeev C

2017-02-01

Microbulbifer mangrovi strain DD-13 T is a novel-type species isolated from the mangroves of Goa, India. The draft genome sequence of strain DD-13 comprised 4,528,106 bp with G+C content of 57.15%. Out of 3479 open reading frames, functions for 3488 protein coding sequences were predicted on the basis of similarity with the cluster of orthologous groups. In addition to protein coding sequences, 34 tRNA genes and 3 rRNA genes were detected. Analysis of nucleotide sequence of predicted gene using a Carbohydrate-Active Enzymes (CAZymes) Analysis Toolkit indicates that strain DD-13 encodes a large set of CAZymes including 255 glycoside hydrolases, 76 carbohydrate esterases, 17 polysaccharide lyases, and 113 carbohydrate-binding modules (CBMs). Many genes from strain DD-13 were annotated as carbohydrases specific for degradation of agar, alginate, carrageenan, chitin, xylan, pullulan, cellulose, starch, β-glucan, pectin, etc. Some of polysaccharide-degrading genes were highly modular and were appended at least with one CBM indicating the versatility of strain DD-13 to degrade complex polysaccharides. The cell growth of strain DD-13 was validated using pure polysaccharides such as agarose or alginate as carbon source as well as by using red and brown seaweed powder as substrate. The homologous carbohydrase produced by strain DD-13 during growth degraded the polysaccharide, ensuring the production of metabolizable reducing sugars. Additionally, several other polysaccharides such as carrageenan, xylan, pullulan, pectin, starch, and carboxymethyl cellulose were also corroborated as growth substrate for strain DD-13 and were associated with concomitant production of homologous carbohydrase.

Silk sericin-alginate-chitosan microcapsules: hepatocytes encapsulation for enhanced cellular functions.

PubMed

Nayak, Sunita; Dey, Sanchareeka; Kundu, Subhas C

2014-04-01

The encapsulation based technology permits long-term delivery of desired therapeutic products in local regions of body without the need of immunosuppressant drugs. In this study microcapsules composed of sericin and alginate micro bead as inner core and with an outer chitosan shell are prepared. This work is proposed for live cell encapsulation for potential therapeutic applications. The sericin protein is obtained from cocoons of non-mulberry silkworm Antheraea mylitta. The sericin-alginate micro beads are prepared via ionotropic gelation under high applied voltage. The beads further coated with chitosan and crosslinked with genipin. The microcapsules developed are nearly spherical in shape with smooth surface morphology. Alamar blue assay and confocal microscopy indicate high cell viability and uniform encapsulated cell distribution within the sericin-alginate-chitosan microcapsules indicating that the microcapsules maintain favourable microenvironment for the cells. The functional analysis of encapsulated cells demonstrates that the glucose consumption, urea secretion rate and intracellular albumin content increased in the microcapsules. The study suggests that the developed sericin-alginate-chitosan microcapsule contributes towards the development of cell encapsulation model. It also offers to generate enriched population of metabolically and functionally active cells for the future therapeutics especially for hepatocytes transplantation in acute liver failure. Copyright © 2014 Elsevier B.V. All rights reserved.

Electrosynthesis of magnetoresponsive microrobot for targeted drug delivery using calcium alginate.

PubMed

Chengzhi Hu; Riederer, Katharina; Klemmer, Michael; Pane, Salvador; Nelson, Bradley J

2016-08-01

Targeted drug delivery systems deliver drugs precisely to a specific targeted site inside the body, and can also release the drugs with controlled kinetics to prolong the efficacy of single dose administration. The advantageous properties of hydrogels make them attractive for use in the area of drug delivery. Calcium alginate is a pH sensitive hydrogel stable in acidic media and soluble in basic media. This enables the hydrogel to absorb and release aqueous solutions at certain ranges of pH values. By absorbing an aqueous solution containing a drug, an active drug release can be triggered at a specified range of pH value. In this paper, we combined calcium alginate with cobalt nickel (CoNi) in a cylindrical hybrid micro robot by electrodeposition. The designed microrobot can be wirelessly actuated with an external magnetic manipulation system and, hence, targeted to a specific location in the human body. At this specific location, characterized by its pH range, the absorbed drug will be released. Here, the fabrication steps of the specified microrobot are characterized, namely the production of a template on a silicon chip and the subsequent template-assisted electrodeposition of CoNi and alginate. Additionally, the dynamics of drug release of calcium alginate is studied.

Oesophageal bioadhesion of sodium alginate suspensions: particle swelling and mucosal retention.

PubMed

Richardson, J Craig; Dettmar, Peter W; Hampson, Frank C; Melia, Colin D

2004-09-01

This paper describes a prospective bioadhesive liquid dosage form designed to specifically adhere to the oesophageal mucosa. It contains a swelling polymer, sodium alginate, suspended in a water-miscible vehicle and is activated by dilution with saliva to form an adherent layer of polymer on the mucosal surface. The swelling of alginate particles and the bioadhesion of 40% (w/w) sodium alginate suspensions were investigated in a range of vehicles: glycerol, propylene glycol, PEG 200 and PEG 400. Swelling of particles as a function of vehicle dilution with artificial saliva was quantified microscopically using 1,9-dimethyl methylene blue (DMMB) as a visualising agent. The minimum vehicle dilution to initiate swelling varied between vehicles: glycerol required 30% (w/w) dilution whereas PEG 400 required nearly 60% (w/w). Swelling commenced when the Hildebrand solubility parameter of the diluted vehicle was raised to 37 MPa(1/2). The bioadhesive properties of suspensions were examined by quantifying the amount of sodium alginate retained on oesophageal mucosa after washing in artificial saliva. Suspensions exhibited considerable mucoretention and strong correlations were obtained between mucosal retention, the minimum dilution to initiate swelling, and the vehicle Hildebrand solubility parameter. These relationships may allow predictive design of suspensions with specific mucoretentive properties, through judicious choice of vehicle characteristics.

A smart bioconjugate of alginate and pectinase with unusual biological activity toward chitosan.

PubMed

Sardar, Meryam; Roy, Ipsita; Gupta, Munishwar N

2003-01-01

The commercial preparation of pectinase (Pectinex Ultra SP-L) was conjugated to alginate by noncovalent interactions by employing 1% alginate during the conjugation protocol. The optimum "immobilization efficiency" was 0.76. The pH optimum and the thermal stability of the enzyme remained unchanged upon conjugation with alginate. The soluble bioconjugate showed a 3-fold increase in V(max)/K(m) as compared to the free enzyme when the smart biocatalyst was used for chitosan hydrolysis. Time course hydrolysis of chitosan thus showed higher conversion of chitosan into reducing oligosaccharides/sugars. The smart bioconjugate could be reused five times without any detectable loss of chitosanase activity.

Structure-based engineering of a pectate lyase with improved specific activity for ramie degumming.

PubMed

Zhou, Zhanping; Liu, Yang; Chang, Zhenying; Wang, Huilin; Leier, André; Marquez-Lago, Tatiana T; Ma, Yanhe; Li, Jian; Song, Jiangning

2017-04-01

Biotechnological applications of microbial pectate lyases (Pels) in plant fiber processing are promising, eco-friendly substitutes for conventional chemical degumming processes. However, to potentiate the enzymes' use for industrial applications, resolving the molecular structure to elucidate catalytic mechanisms becomes necessary. In this manuscript, we report the high resolution (1.45 Å) crystal structure of pectate lyase (pelN) from Paenibacillus sp. 0602 in apo form. Through sequence alignment and structural superposition with other members of the polysaccharide lyase (PL) family 1 (PL1), we determined that pelN shares the characteristic right-handed β-helix and is structurally similar to other members of the PL1 family, while exhibiting key differences in terms of catalytic and substrate binding residues. Then, based on information from structure alignments with other PLs, we engineered a novel pelN. Our rational design yielded a pelN mutant with a temperature for enzymatic activity optimally shifted from 67.5 to 60 °C. Most importantly, this pelN mutant displayed both higher specific activity and ramie fiber degumming ability when compared with the wild-type enzyme. Altogether, our rational design method shows great potential for industrial applications. Moreover, we expect the reported high-resolution crystal structure to provide a solid foundation for future rational, structure-based engineering of genetically enhanced pelNs.

21 CFR 184.1011 - Alginic acid.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Alginic acid. 184.1011 Section 184.1011 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) DIRECT FOOD SUBSTANCES AFFIRMED AS GENERALLY RECOGNIZED AS SAFE Listing of Specific...

21 CFR 184.1011 - Alginic acid.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Alginic acid. 184.1011 Section 184.1011 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) DIRECT FOOD SUBSTANCES AFFIRMED AS GENERALLY RECOGNIZED AS SAFE Listing of Specific...

Effects of alginate hydrogel cross-linking density on mechanical and biological behaviors for tissue engineering.

PubMed

Jang, Jinah; Seol, Young-Joon; Kim, Hyeon Ji; Kundu, Joydip; Kim, Sung Won; Cho, Dong-Woo

2014-09-01

An effective cross-linking of alginate gel was made through reaction with calcium carbonate (CaCO3). We used human chondrocytes as a model cell to study the effects of cross-linking density. Three different pore size ranges of cross-linked alginate hydrogels were fabricated. The morphological, mechanical, and rheological properties of various alginate hydrogels were characterized and responses of biosynthesis of cells encapsulated in each gel to the variation in cross-linking density were investigated. Desired outer shape of structure was maintained when the alginate solution was cross-linked with the applied method. The properties of alginate hydrogel could be tailored through applying various concentrations of CaCO3. The rate of synthesized GAGs and collagens was significantly higher in human chondrocytes encapsulated in the smaller pore structure than that in the larger pore structure. The expression of chondrogenic markers, including collagen type II and aggrecan, was enhanced in the smaller pore structure. It was found that proper structural morphology is a critical factor to enhance the performance and tissue regeneration. Copyright © 2014 Elsevier Ltd. All rights reserved.

Synthesis and evaluation of functional alginate hydrogels based on click chemistry for drug delivery applications.

PubMed

García-Astrain, Clara; Avérous, Luc

2018-06-15

Environment-sensitive alginate-based hydrogels for drug delivery applications are receiving increasing attention. However, most work in this field involves traditional cross-linking strategies which led to hydrogels with poor long-term stability. Herein, a series of chemically cross-linked alginate hydrogels was synthesized via click chemistry using Diels-Alder reaction by reacting furan-modified alginate and bifunctional cross-linkers. Alginate was successfully functionalized with furfurylamine. Then, 3D architectures were synthesized with water-soluble bismaleimides. Different substitution degrees were achieved in order to study the effect of alginate modification and the cross-linking extent over the behaviour of the hydrogels. The ensuing hydrogels were analysed in terms of microstructure, swelling, structure modification and rheological behaviour. The materials response to external stimuli such as pH was also investigated, revealing a pulsatile behaviour in a large pH range (1-13) and a clear pH-dependent swelling. Finally, vanillin release studies were conducted to demonstrate the potential of these biobased materials for drug delivery applications. Copyright © 2018 Elsevier Ltd. All rights reserved.

A simple approach for morphology tailoring of alginate particles by manipulation ionic nature of polyurethanes.

PubMed

Daemi, Hamed; Barikani, Mehdi; Barmar, Mohammad

2014-05-01

A number of different ionic aqueous polyurethane dispersions (PUDs) were synthesized based on NCO-terminated prepolymers. Two different anionic and cationic polyurethane samples were synthesized using dimethylol propionic acid and N-methyldiethanolamine emulsifiers, respectively. Then, proper amounts of PUDs and sodium alginate were mixed to obtain a number of aqueous polyurethane dispersions-sodium alginate (PUD/SA) elastomers. The chemical structure, thermal, morphological, thermo-mechanical and mechanical properties, and hydrophilicity content of the prepared samples were studied by FTIR, EDX, DSC, TGA, SEM, DMTA, tensile testing and contact angle techniques. The cationic polyurethanes and their blends with sodium alginate showed excellent miscibility and highly stretchable properties, while the samples containing anionic polyurethanes and alginate illustrated a poor compatibility and no significant miscibility. The morphology of alginate particles shifted from nanoparticles to microparticles by changing the nature of PUDs from cationic to anionic types. The final cationic elastomers not only showed better mechanical properties but also were formulated easier than anionic samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Effect of storage time of extended-pour and conventional alginate impressions on dimensional accuracy of casts.

PubMed

Rohanian, Ahmad; Ommati Shabestari, Ghasem; Zeighami, Somayeh; Samadi, Mohammad Javad; Shamshiri, Ahmad Reza

2014-11-01

Some manufacturers claim to have produced new irreversible hydro-colloids that are able to maintain their dimensional stability during storage. The present study evaluated the effect of storage time on dimensional stability of three alginates: Hydrogum 5, Tropicalgin and Alginoplast. In this experimental in-vitro trial, a total of 90 alginate impressions were made from a Dentoform model using Hydrogum 5, Tropicalgin and Alginoplast alginates. The impressions were stored in a sealed plastic bag without a damp paper towel for 0, 24, 48, 72 and 120 hours and then poured with type III dental stone. Cross-arch (facial of 6 to facial of 6 on the opposite side) and antero-posterior (distal of right first molar to the ipsilateral central incisor) measurements were made with a digital caliper on the casts. Data were analyzed by two-way and one-way ANOVA and Tukey's post-hoc test (P<0.05). Alginate type and the pouring time significantly affected the dimensional stability of alginate impressions (both Ps<0.001). Pouring of Hydrogum 5 impressions can be delayed for up to 120 hours without significant dimensional changes. Alginoplast impressions may be poured after 72 hours, but Tropicalgin should be poured immediately and the storage time should not be more than 24 hours. Immediate pouring of alginate impressions provides the highest accuracy in reproducing the teeth and adjacent tissues; however, this study demonstrated that pouring may be delayed for up to five days using extended-pour (Hydrogum 5) alginates.

Production of tannase by the immobilized cells of Bacillus licheniformis KBR6 in Ca-alginate beads.

PubMed

Mohapatra, P K D; Mondal, K C; Pati, B R

2007-06-01

The present study was aimed at finding the optimal conditions for immobilization of Bacillus licheniformis KBR6 cells in calcium-alginate (Ca-alginate) beads and determining the operational stability during the production of tannin-acyl-hydrolase (tannase) under semicontinous cultivation. The active cells of B. licheniformis KBR6 were immobilized in Ca-alginate and used for the production of tannase. The influence of alginate concentration (5, 10, 20 and 30 g l(-1)) and initial cell loading on enzyme production were studied. The production of tannase increased significantly with increasing alginate concentration and reached a maximum enzyme yield of 0.56 +/- 0.03 U ml(-1) at 20 g l(-1). This was about 1.70-fold higher than that obtained by free cells. The immobilized cells produced tannase consistently over 13 repeated cycles and reached a maximum level at the third cycle. Scanning electron microscope study indicated that the cells in Ca-alginate beads remain in normal shape. The Ca-alginate entrapment is a promising immobilization method of B. licheniformis KBR6 for repeated tannase production. Tannase production by immobilized cells is superior to that of free cells because it leads to higher volumetric activities within the same period of fermentation. This is the first report of tannase production from immobilized bacterial cells. The bacterium under study can produce higher amounts of tannase with respect to other fungal strains within a short cultivation period.

Identification of ATP Citrate Lyase as a Positive Regulator of Glycolytic Function in Glioblastomas

PubMed Central

Beckner, Marie E.; Fellows-Mayle, Wendy; Zhang, Zhe; Agostino, Naomi R.; Kant, Jeffrey A.; Day, Billy W.; Pollack, Ian F.

2009-01-01

Glioblastomas, the most malignant type of glioma, are more glycolytic than normal brain tissue. Robust migration of glioblastoma cells has been previously demonstrated under glycolytic conditions and their pseudopodia contain increased glycolytic and decreased mitochondrial enzymes. Glycolysis is suppressed by metabolic acids, including citric acid which is excluded from mitochondria during hypoxia. We postulated that glioma cells maintain glycolysis by regulating metabolic acids, especially in their pseudopodia. The enzyme that breaks down cytosolic citric acid is ATP citrate lyase (ACLY). Our identification of increased ACLY in pseudopodia of U87 glioblastoma cells on 1D gels and immunoblots prompted investigation of ACLY gene expression in gliomas for survival data and correlation with expression of ENO1, that encodes enolase 1. Queries of the NIH’s REMBRANDT brain tumor database based on Affymetrix data indicated that decreased survival correlated with increased gene expression of ACLY in gliomas. Queries of gliomas and glioblastomas found an association of upregulated ACLY and ENO1 expression by chi square for all probe sets (reporters) combined and correlation for numbers of probe sets indicating shared upregulation of these genes. Real-time quantitative PCR confirmed correlation between ACLY and ENO1 in 21 glioblastomas (p < 0.001). Inhibition of ACLY with hydroxycitrate suppressed (p < 0.05) in vitro glioblastoma cell migration, clonogenicity and brain invasion under glycolytic conditions and enhanced the suppressive effects of a Met inhibitor on cell migration. In summary, gene expression data, proteomics and functional assays support ACLY as a positive regulator of glycolysis in glioblastomas. PMID:19795461

Mechanistic studies of a novel C-S lyase in ergothioneine biosynthesis: the involvement of a sulfenic acid intermediate

PubMed Central

Song, Heng; Hu, Wen; Naowarojna, Nathchar; Her, Ampon Sae; Wang, Shu; Desai, Rushil; Qin, Li; Chen, Xiaoping; Liu, Pinghua

2015-01-01

Ergothioneine is a histidine thio-derivative isolated in 1909. In ergothioneine biosynthesis, the combination of a mononuclear non-heme iron enzyme catalyzed oxidative C-S bond formation reaction and a PLP-mediated C-S lyase (EgtE) reaction results in a net sulfur transfer from cysteine to histidine side-chain. This demonstrates a new sulfur transfer strategy in the biosynthesis of sulfur-containing natural products. Due to difficulties associated with the overexpression of Mycobacterium smegmatis EgtE protein, the proposed EgtE functionality remained to be verified biochemically. In this study, we have successfully overexpressed and purified M. smegmatis EgtE enzyme and evaluated its activities under different in vitro conditions: C-S lyase reaction using either thioether or sulfoxide as a substrate in the presence or absence of reductants. Results from our biochemical characterizations support the assignment of sulfoxide 4 as the native EgtE substrate and the involvement of a sulfenic acid intermediate in the ergothioneine C-S lyase reaction. PMID:26149121

Encapsulating Non-Human Primate Multipotent Stromal Cells in Alginate via High Voltage for Cell-Based Therapies and Cryopreservation

PubMed Central

Gryshkov, Oleksandr; Pogozhykh, Denys; Hofmann, Nicola; Pogozhykh, Olena; Mueller, Thomas; Glasmacher, Birgit

2014-01-01

Alginate cell-based therapy requires further development focused on clinical application. To assess engraftment, risk of mutations and therapeutic benefit studies should be performed in an appropriate non-human primate model, such as the common marmoset (Callithrix jacchus). In this work we encapsulated amnion derived multipotent stromal cells (MSCs) from Callithrix jacchus in defined size alginate beads using a high voltage technique. Our results indicate that i) alginate-cell mixing procedure and cell concentration do not affect the diameter of alginate beads, ii) encapsulation of high cell numbers (up to 10×106 cells/ml) can be performed in alginate beads utilizing high voltage and iii) high voltage (15–30 kV) does not alter the viability, proliferation and differentiation capacity of MSCs post-encapsulation compared with alginate encapsulated cells produced by the traditional air-flow method. The consistent results were obtained over the period of 7 days of encapsulated MSCs culture and after cryopreservation utilizing a slow cooling procedure (1 K/min). The results of this work show that high voltage encapsulation can further be maximized to develop cell-based therapies with alginate beads in a non-human primate model towards human application. PMID:25259731

Alginate as immobilization matrix and stabilizing agent in a two-phase liquid system: application in lipase-catalysed reactions.

PubMed

Hertzberg, S; Kvittingen, L; Anthonsen, T; Skjåk-Braek, G

1992-01-01

Alginate was evaluated as an immobilization matrix for enzyme-catalyzed reactions in organic solvents. In contrast to most hydrogels, calcium alginate was found to be stable in a range of organic solvents and to retain the enzyme inside the gel matrix. In hydrophobic solvents, the alginate gel (greater than 95% water) thus provided a stable, two-phase liquid system. The lipase from Candida cylindracea, after immobilization in alginate beads, catalysed esterification and transesterification in n-hexane under both batch and continuous-flow conditions. The operational stability of the lipase was markedly enhanced by alginate entrapment. In the esterification of butanoic acid with n-butanol, better results were obtained in the typical hydrophilic calcium alginate beads than in less hydrophilic matrices. The effects of substrate concentration, matrix area, and polarity of the substrate alcohols and of the organic solvent on the esterification activity were examined. The transesterification of octyl 2-bromopropanoate with ethanol was less efficient than that of ethyl 2-bromopropanoate with octanol. By using the hydrophilic alginate gel as an immobilization matrix in combination with a mobile hydrophobic phase, a two-phase liquid system was achieved with definite advantages for a continuous, enzyme-catalysed process.

Three-dimensional electrospun polycaprolactone (PCL)/alginate hybrid composite scaffolds.

PubMed

Kim, Min Seong; Kim, GeunHyung

2014-12-19

Micro/nanofibrous scaffolds have been used widely in biomedical applications because the micro/nano-scale fibres resemble natural extracellular matrix and the high surface-to-volume ratio encourages cellular activities (attachment and proliferation). However, poor mechanical properties, low controllability of various shapes and difficulties in obtaining controllable pore structure have been obstacles to their use in hard-tissue regeneration. To overcome these shortcomings, we suggest a new composite system, which uses a combination method of wet electrospinning, rapid prototyping and a physical punching process. Using the process, we obtained polycaprolactone (PCL)/alginate composite scaffolds, consisting of electrospun PCL/alginate fibres and micro-sized PCL struts, with mean pore sizes of 821 ± 55 μm. To show the feasibility of the scaffolds for hard-tissue regeneration, the scaffolds were assessed not only for physical properties, including hydrophilicity, water absorption, and tensile and compressive strength, but also in vitro cellular responses (cell viability and proliferation) and osteogenic differentiation (alkaline phosphatase (ALP) activity, and mineralisation) by culturing with pre-osteoblasts (MC3T3-E1 cells). With the reinforcing micro-sized PCL struts, the elastic modulus of the PCL/alginate scaffold was significantly improved versus a pure PCL scaffold. Additionally, due to the alginate component in the fibrous scaffold, they showed significantly enhanced hydrophilic behaviour, water absorption (∼8-fold) and significant biological activities (∼1.6-fold for cell viability at 7 days, ∼2.3-fold for ALP activity at 14 days and ∼6.4-fold for calcium mineralisation at 14 days) compared with those of a pure PCL fibrous scaffold. Copyright © 2014 Elsevier Ltd. All rights reserved.

Use of Magnetic Nanoparticles to Monitor Alginate-Encapsulated βTC-tet Cells

PubMed Central

Constantinidis, Ioannis; Grant, Samuel C.; Simpson, Nicholas E.; Oca-Cossio, Jose A.; Sweeney, Carol A.; Mao, Hui; Blackband, Stephen J.; Sambanis, Athanassios

2008-01-01

Non-invasive monitoring of tissue-engineered constructs is an important component in optimizing construct design and assessing therapeutic efficacy. In recent years, cellular and molecular imaging initiatives have spurred the use of iron oxide based contrast agents in the field of NMR imaging. Although their use in medical research has been widespread, their application in tissue engineering has been limited. In this study, the utility of Monocrystalline Iron Oxide Nanoparticles (MION) as an NMR contrast agent was evaluated for βTC-tet cells encapsulated within alginate/poly-L-lysine/alginate (APA) microbeads. The constructs were labeled with MION in two different ways: (a) MION-labeled βTC-tet cells were encapsulated in APA beads (i.e., intracellular compartment); and (b) MION particles were suspended in the alginate solution prior to encapsulation so that the alginate matrix was labeled with MION instead of the cells (i.e., extracellular compartment). The data show that although the location of cells can be identified within APA beads, cell growth or rearrangement within these constructs cannot be effectively monitored, regardless of the location of MION compartmentalization. The advantages and disadvantages of these techniques and their potential use in tissue engineering are discussed. PMID:19165877

Preparation and characterization of hydroxyapatite/sodium alginate biocomposites for bone implant application

NASA Astrophysics Data System (ADS)

Kanasan, Nanthini; Adzila, Sharifah; Suid, Mohd Syafiq; Gurubaran, P.

2016-07-01

In biomedical fields, synthetic scaffolds are being improved by using the ceramics, polymers and composites materials to avoid the limitations of allograft. Ceramic-polymer composites are appearing to be the most successful bone graft substitute in human body. The natural bones itself are well-known as composite of collagen and hydroxyapatite. In this research, precipitation method was used to synthesis hydroxyapatite (HA)/sodium alginate (SA) in various parameters. This paper describes the hydroxyapatite/sodium alginate biocomposite which suitable for use in bone defects or regeneration of bone through the characterizations which include FTIR, FESEM, EDS and DTA. In FTIR, the characteristi peaks of PO4-3 and OH- groups which corresponding to hydroxyapatite are existed in the mixing powders. The needle-size particle of hydroxyapatite/ alginate (HA/SA) are observed in FESEM in the range of 15.8nm-38.2nm.EDS confirmed the existence of HA/SA composition in the mixing powders. There is an endothermic peak which corresponds to the dehydration and the loss of physically adsorbed water molecules of the hydroxyapatite (HA)/sodium alginate (SA) powder which are described in DTA.

Oesophageal bioadhesion of sodium alginate suspensions 2. Suspension behaviour on oesophageal mucosa.

PubMed

Richardson, J Craig; Dettmar, Peter W; Hampson, Frank C; Melia, Colin D

2005-01-01

Sodium alginate suspensions in a range of water miscible vehicles were investigated as novel bioadhesive liquids for targeting the oesophageal mucosa. Such a dosage form might be utilised to coat the oesophageal surface and provide a protective barrier against gastric reflux, or to deliver therapeutic agents site-specifically. Alginate suspensions swelled and formed an adherent viscous layer on contact with the mucosa. The swelling kinetics of alginate particles on the oesophageal surface was examined with respect to vehicle composition and related to the extent, duration and location of bioadhesion within the oesophagus. Mucosal retention was evaluated in two in vitro models utilising tissue immersion and a peristaltic tube. By varying the vehicle composition it was possible to modulate the rate of swelling of alginate particles on the mucosa and the mucosal retention of suspensions. Suspensions containing predominantly glycerol exhibited superior retention and were preferentially retained within the lower oesophagus. The propensity of these suspensions to rapidly swell on the mucosa and establish adhesive/cohesive bonds may explain their enhanced retention. The potential to control, through vehicle composition, the extent, duration and location of oesophageal retention could provide a useful tool for site targeting of viscous polymers to the oesophagus.

Streptomycin-loaded PLGA-alginate nanoparticles: preparation, characterization, and assessment

NASA Astrophysics Data System (ADS)

Asadi, Asadollah

2014-04-01

The aim of this study was to formulate and characterize streptomycin-loaded PLGA-alginate nanoparticles for their potential therapeutic use in Salmonella subsp. enterica ATCC 14028 infections. The streptomycin nanoparticle was prepared by solvent diffusion method, and the other properties such as size, zeta potential, loading efficacy, release kinetics, and antimicrobial strength were evaluated. The survey shows that nanoparticles may serve as a carrier of streptomycin and may provide localized antibacterial activity in the treatment of Salmonellosis. Electron microscopy showed spherical particles with indentations. The average size of the nanoparticles was 90 nm. At pH 7.2, the release kinetics of streptomycin from the nanoparticles was successfully illustrated as an initial burst defined by a first order equation that after this stage, it has a drastic tendency to obtain steady state. Nevertheless, nanoparticles showed loading efficacy nearly about 70-75 %. In addition, the tendency of concentration of streptomycin released from nanoparticles to reach antibacterial activity was similar to that of free streptomycin against PLGA-alginate, but it had threefold more antimicrobial strength in comparison with free streptomycin. This work shows the potential use of streptomycin-loaded PLGA-alginate nanoparticles and its capability.

Fabrication of cationic chitin nanofiber/alginate composite materials.

PubMed

Sato, Koki; Tanaka, Kohei; Takata, Yusei; Yamamoto, Kazuya; Kadokawa, Jun-Ichi

2016-10-01

We have already found that an amidinated chitin, which was prepared by the reaction of a partially deacetylated chitin with N,N-dimethylacetamide dimethyl acetal, was converted into an amidinium chitin bicarbonate with nanofiber morphology by CO2 gas bubbling and ultrasonic treatments in water. In this study, we performed the fabrication of composite materials of such cationic chitin nanofibers with an anionic polysaccharide, sodium alginate, by ion exchange. When the amidinium chitin bicarbonate nanofiber aqueous dispersion was added to an aqueous solution of sodium alginate, the composite material was agglomerated, which was isolated by centrifugation, filtration, and lyophilization, to form a manipulatable sheet. The morphology of the resulting sheet at nano-scale was evaluated by SEM measurement. Copyright © 2016 Elsevier B.V. All rights reserved.

Methylcitrate cycle defines the bactericidal essentiality of isocitrate lyase for survival of Mycobacterium tuberculosis on fatty acids

PubMed Central

Eoh, Hyungjin; Rhee, Kyu Y.

2014-01-01

Few mutations attenuate Mycobacterium tuberculosis (Mtb) more profoundly than deletion of its isocitrate lyases (ICLs). However, the basis for this attenuation remains incompletely defined. Mtb’s ICLs are catalytically bifunctional isocitrate and methylisocitrate lyases required for growth on even and odd chain fatty acids. Here, we report that Mtb’s ICLs are essential for survival on both acetate and propionate because of its methylisocitrate lyase (MCL) activity. Lack of MCL activity converts Mtb’s methylcitrate cycle into a “dead end” pathway that sequesters tricarboxylic acid (TCA) cycle intermediates into methylcitrate cycle intermediates, depletes gluconeogenic precursors, and results in defects of membrane potential and intrabacterial pH. Activation of an alternative vitamin B12-dependent pathway of propionate metabolism led to selective corrections of TCA cycle activity, membrane potential, and intrabacterial pH that specifically restored survival, but not growth, of ICL-deficient Mtb metabolizing acetate or propionate. These results thus resolve the biochemical basis of essentiality for Mtb’s ICLs and survival on fatty acids. PMID:24639517

Profiling of Volatile Compounds and Associated Gene Expression and Enzyme Activity during Fruit Development in Two Cucumber Cultivars

PubMed Central

Chen, Shuxia; Zhang, Ranran; Hao, Lining; Chen, Weifeng; Cheng, Siqiong

2015-01-01

Changes in volatile content, as well as associated gene expression and enzyme activity in developing cucumber fruits were investigated in two Cucumis sativus L. lines (No. 26 and No. 14) that differ significantly in fruit flavor. Total volatile, six-carbon (C6) aldehyde, linolenic and linoleic acid content were higher during the early stages, whereas the nine-carbon (C9) aldehyde content was higher during the latter stages in both lines. Expression of C. sativus hydroperoxide lyase (CsHPL) mirrored 13-hydroperoxide lyase (13-HPL) enzyme activity in variety No. 26, whereas CsHPL expression was correlated with 9-hydroperoxide lyase (9-HPL) enzyme activity in cultivar No. 14. 13-HPL activity decreased significantly, while LOX (lipoxygenase) and 9-HPL activity increased along with fruit ripening in both lines, which accounted for the higher C6 and C9 aldehyde content at 0-6 day post anthesis (dpa) and 9-12 dpa, respectively. Volatile compounds from fruits at five developmental stages were analyzed by principal component analysis (PCA), and heatmaps of volatile content, gene expression and enzyme activity were constructed. PMID:25799542

Structure of Methylobacterium extorquens malyl-CoA lyase: CoA-substrate binding correlates with domain shift

DOE PAGES

Gonzalez, Javier M.; Marti-Arbona, Ricardo; Chen, Julian C. -H.; ...

2017-01-27

Malyl-CoA lyase (MCL) is an Mg 2+-dependent enzyme that catalyzes the reversible cleavage of (2 S)-4-malyl-CoA to yield acetyl-CoA and glyoxylate. MCL enzymes, which are found in a variety of bacteria, are members of the citrate lyase-like family and are involved in the assimilation of one- and two-carbon compounds. Here, the 1.56 Å resolution X-ray crystal structure of MCL from Methylobacterium extorquens AM1 with bound Mg 2+is presented. Structural alignment with the closely related Rhodobacter sphaeroides malyl-CoA lyase complexed with Mg 2+, oxalate and CoA allows a detailed analysis of the domain motion of the enzyme caused by substrate binding.more » Alignment of the structures shows that a simple hinge motion centered on the conserved residues Phe268 and Thr269 moves the C-terminal domain by about 30° relative to the rest of the molecule. Furthermore, this domain motion positions a conserved aspartate residue located in the C-terminal domain in the active site of the adjacent monomer, which may serve as a general acid/base in the catalytic mechanism.« less

Structure of Methylobacterium extorquens malyl-CoA lyase: CoA-substrate binding correlates with domain shift

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

Gonzalez, Javier M.; Marti-Arbona, Ricardo; Chen, Julian C. -H.

Malyl-CoA lyase (MCL) is an Mg 2+-dependent enzyme that catalyzes the reversible cleavage of (2 S)-4-malyl-CoA to yield acetyl-CoA and glyoxylate. MCL enzymes, which are found in a variety of bacteria, are members of the citrate lyase-like family and are involved in the assimilation of one- and two-carbon compounds. Here, the 1.56 Å resolution X-ray crystal structure of MCL from Methylobacterium extorquens AM1 with bound Mg 2+is presented. Structural alignment with the closely related Rhodobacter sphaeroides malyl-CoA lyase complexed with Mg 2+, oxalate and CoA allows a detailed analysis of the domain motion of the enzyme caused by substrate binding.more » Alignment of the structures shows that a simple hinge motion centered on the conserved residues Phe268 and Thr269 moves the C-terminal domain by about 30° relative to the rest of the molecule. Furthermore, this domain motion positions a conserved aspartate residue located in the C-terminal domain in the active site of the adjacent monomer, which may serve as a general acid/base in the catalytic mechanism.« less

Physiological roles of pyruvate ferredoxin oxidoreductase and pyruvate formate-lyase in Thermoanaerobacterium saccharolyticum JW/SL-YS485

DOE PAGES

Zhou, Jilai; Olson, Daniel G.; Lanahan, Anthony A.; ...

2015-09-15

We report that Thermoanaerobacter saccharolyticum is a thermophilic microorganism that has been engineered to produce ethanol at high titer (30–70 g/L) and greater than 90 % theoretical yield. However, few genes involved in pyruvate to ethanol production pathway have been unambiguously identified. In T. saccharolyticum, the products of six putative pfor gene clusters and one pfl gene may be responsible for the conversion of pyruvate to acetyl-CoA. To gain insights into the physiological roles of PFOR and PFL, we studied the effect of deletions of several genes thought to encode these activities. We found that that pyruvate ferredoxin oxidoreductase enzymemore » (PFOR) is encoded by the pforA gene and plays a key role in pyruvate dissimilation. We further demonstrated that pyruvate formate-lyase activity (PFL) is encoded by the pfl gene. Although the pfl gene is normally expressed at low levels, it is crucial for biosynthesis in T. saccharolyticum. In pforA deletion strains, pfl expression increased and was able to partially compensate for the loss of PFOR activity. Deletion of both pforA and pfl resulted in a strain that required acetate and formate for growth and produced lactate as the primary fermentation product, achieving 88 % theoretical lactate yield. PFOR encoded by Tsac_0046 and PFL encoded by Tsac_0628 are only two routes for converting pyruvate to acetyl-CoA in T. saccharolyticum. The physiological role of PFOR is pyruvate dissimilation, whereas that of PFL is supplying C1 units for biosynthesis.« less

The study of the structural properties of very low viscosity sodium alginate by small-angle neutron scattering

NASA Astrophysics Data System (ADS)

Badita, C. R.; Aranghel, D.; Radulescu, A.; Anitas, E. M.

2016-03-01

Sodium alginate is a linear polymer extract from brown algae and it is used in the biomedical, food, cosmetics and pharmaceutical industries as solution property modifiers and gelling agents. But despite the extensive studies of the alginate gelation process, still some fundamental questions remain unresolved. The fractal behavior of very low viscosity sodium alginate solutions and their influence on the critical gelation of alginate induced by Ca2+ ions were investigated using Small-Angle Neutron Scattering (SANS) measurements. SANS data are interpreted using both standard linear plots and the Beaucage model. The scattering intensity is dependent by alginate concentration and Ca2+ concentration. From a critical concentration of 1.0 % w/w our polymer swelled forming spherical structures with rough surfaces. Also the addition of the salt induces the collapse and the appearance of the aggregation and clusters formation.

A computational modeling approach for the characterization of mechanical properties of 3D alginate tissue scaffolds.

PubMed

Nair, K; Yan, K C; Sun, W

2008-01-01

Scaffold guided tissue engineering is an innovative approach wherein cells are seeded onto biocompatible and biodegradable materials to form 3-dimensional (3D) constructs that, when implanted in the body facilitate the regeneration of tissue. Tissue scaffolds act as artificial extracellular matrix providing the environment conducive for tissue growth. Characterization of scaffold properties is necessary to understand better the underlying processes involved in controlling cell behavior and formation of functional tissue. We report a computational modeling approach to characterize mechanical properties of 3D gellike biomaterial, specifically, 3D alginate scaffold encapsulated with cells. Alginate inherent nonlinearity and variations arising from minute changes in its concentration and viscosity make experimental evaluation of its mechanical properties a challenging and time consuming task. We developed an in silico model to determine the stress-strain relationship of alginate based scaffolds from experimental data. In particular, we compared the Ogden hyperelastic model to other hyperelastic material models and determined that this model was the most suitable to characterize the nonlinear behavior of alginate. We further propose a mathematical model that represents the alginate material constants in Ogden model as a function of concentrations and viscosity. This study demonstrates the model capability to predict mechanical properties of 3D alginate scaffolds.

Culture of C3A cells in alginate beads for fluidized bed bioartificial liver.

PubMed

Kinasiewicz, A; Gautier, A; Lewinska, D; Bukowski, J; Legallais, C; Weryński, A

2007-11-01

Extracorporeal bioartificial liver has been designed to sustain the detoxification and synthetic function of the failed liver in patients suffering from acute liver failure until the time of liver allotransplantation or regeneration of their own. A fluidized bed, bioartificial liver improves the mass transfer velocity between the medium and the hepatocytes. Detoxification functions of the liver could be replaced by completely artificial systems, but the synthetic functions of hepatocytes may be obtained only by metabolically active cells. The aim of our study was to investigate the influence of C3A cell culture in alginate beads on synthetic function in a fluidized bed, bioartificial liver. Cells in alginate beads were prepared using an electrostatic droplet generator of our own design using low-viscosity alginate. Beads were cultured for 24 hours then 7 days in static conditions and then 24 hours of fluidization in the bioreactor to assess albumin production. We observed significantly increased albumin production by C3A cells entrapped in alginate beads during static culture. Fluidization increased albumin production compared with static culture. Fluidization performed after 7 days of static culture resulted in a significant increase in albumin synthesis. In conclusion, static culture of alginate beads hosting hepatic cells facilitates restoration of cell function.

Chitosan and alginate types of bio-membrane in fuel cell application: An overview

NASA Astrophysics Data System (ADS)

Shaari, N.; Kamarudin, S. K.

2015-09-01

The major problems of polymer electrolyte membrane fuel cell technology that need to be highlighted are fuel crossovers (e.g., methanol or hydrogen leaking across fuel cell membranes), CO poisoning, low durability, and high cost. Chitosan and alginate-based biopolymer membranes have recently been used to solve these problems with promising results. Current research in biopolymer membrane materials and systems has focused on the following: 1) the development of novel and efficient biopolymer materials; and 2) increasing the processing capacity of membrane operations. Consequently, chitosan and alginate-based biopolymers seek to enhance fuel cell performance by improving proton conductivity, membrane durability, and reducing fuel crossover and electro-osmotic drag. There are four groups of chitosan-based membranes (categorized according to their reaction and preparation): self-cross-linked and salt-complexed chitosans, chitosan-based polymer blends, chitosan/inorganic filler composites, and chitosan/polymer composites. There are only three alginate-based membranes that have been synthesized for fuel cell application. This work aims to review the state-of-the-art in the growth of chitosan and alginate-based biopolymer membranes for fuel cell applications.

Oxygen uptake rate in alginate producer (algU+) and nonproducer (algU-) strains of Azotobacter vinelandii under nitrogen-fixation conditions.

PubMed

Castillo, T; López, I; Flores, C; Segura, D; García, A; Galindo, E; Peña, C

2018-07-01

The sigma E (AlgU) in Azotobacter vinelandii has been shown to control the expression of cydR gene, a repressor of genes of the alternative respiratory chain, and alginate has been considered a barrier for oxygen diffusion. Therefore, the aim of the present study was to compare the respiratory activity of an alginate nonproducing strain, lacking the sigma factor E (algU-), and polymer-producing strains (algU+) of A. vinelandii under diazotrophic conditions at different aeration conditions. Our results reveal that under diazotrophic and high aeration conditions, A. vinelandii strain OP (algU-) had a specific oxygen consumption rate higher (30 and 54%) than those observed in the OP algU+-complemented strain, named OPAlgU+, and the ATCC 9046 respectively. However, the specific growth rate and biomass yields (based on oxygen and sucrose) were lower for OP cultivations as compared to the algU+ strains. These differences were partially explained by an increase in 1·5-fold of cydA relative expression in the OP strain, as compared to that obtained in the isogenic OPAlgU+ strain. Overall, our results confirm the important role of algU gene on the regulation of respiratory metabolism under diazotrophic growth when A. vinelandii is exposed to high aeration. This study highlights the role of AlgU to control respiration of A. vinelandii when exposed to diazotrophy. © 2018 The Society for Applied Microbiology.

Probing the active center of benzaldehyde lyase with substitutions and the pseudo-substrate analog benzoylphosphonic acid methyl ester

PubMed Central

Brandt, Gabriel S.; Nemeria, Natalia; Chakraborty, Sumit; McLeish, Michael J.; Yep, Alejandra; Kenyon, George L.; Petsko, Gregory A.; Jordan, Frank; Ringe, Dagmar

2009-01-01

Benzaldehyde lyase (BAL) catalyzes the reversible cleavage of (R)-benzoin to benzaldehyde utilizing thiamin diphosphate and Mg2+ as cofactors. The enzyme is important for the chemoenzymatic synthesis of a wide range of compounds via its carboligation reaction mechanism. In addition to its principal functions, BAL can slowly decarboxylate aromatic amino acids such as benzoylformic acid. It is also intriguing mechanistically due to the paucity of acid-base residues at the active center that can participate in proton transfer steps thought to be necessary for these type of reactions. Here methyl benzoylphosphonate, an excellent electrostatic analog of benzoylformic acid, is used to probe the mechanism of benzaldehyde lyase. The structure of benzaldehyde lyase in its covalent complex with methyl benzoylphosphonate was determined to 2.49 Å (PDB ID: 3D7K) and represents the first structure of this enzyme with a compound bound in the active site. No large structural reorganization was detected compared to the complex of the enzyme with thiamin diphosphate. The configuration of the predecarboxylation thiamin-bound intermediate was clarified by the structure. Both spectroscopic and X-ray structural studies are consistent with inhibition resulting from the binding of MBP to the thiamin diphosphate in the active centers. We also delineated the role of His29 (the sole potential acid-base catalyst in the active site other than the highly conserved Glu50) and Trp163 in cofactor activation and catalysis by benzaldehyde lyase. PMID:18570438

Sulindac loaded alginate beads for a mucoprotective and controlled drug release.

PubMed

Yegin, Betül Arica; Moulari, Brice; Durlu-Kandilci, N Tugba; Korkusuz, Petek; Pellequer, Yann; Lamprecht, Alf

2007-06-01

Ionotropic gelation was used to entrap sulindac into calcium alginate beads as a potential drug carrier for the oral delivery of this anti-inflammatory drug. Beads were investigated in vitro for a possible sustained drug release and their use in vivo as a gastroprotective system for sulindac. Process parameters such as the polymer concentration, polymer/drug ratio, and different needle diameter were analysed for their influences on the bead properties. Size augmented with increasing needle diameter (0.9 mm needle: 1.28 to 1.44 mm; 0.45 mm needle: 1.04 to 1.07 mm) due to changes in droplet size as well as droplet viscosity. Yields varied between 87% and 98% while sulindac encapsulation efficiencies of about 88% and 94% were slightly increasing with higher alginate concentrations. Drug release profiles exhibited a complete release for all formulations within 4 hours with a faster release for smaller beads. Sulindac loaded alginate beads led to a significant reduction of macroscopic histological damage in the stomach and duodenum in mice. Similarly, microscopic analyses of the mucosal damage demonstrated a significant mucoprotective effect of all bead formulation compared to the free drug. The present alginate formulations exhibit promising properties of a controlled release form for sulindac; meanwhile they provide a distinct tissue protection in the stomach and duodenum.

Effect of Storage Time of Extended-Pour and Conventional Alginate Impressions on Dimensional Accuracy of Casts

PubMed Central

Rohanian, Ahmad; Ommati Shabestari, Ghasem; Zeighami, Somayeh; Samadi, Mohammad Javad; Shamshiri, Ahmad Reza

2014-01-01

Objectives: Some manufacturers claim to have produced new irreversible hydro-colloids that are able to maintain their dimensional stability during storage. The present study evaluated the effect of storage time on dimensional stability of three alginates: Hydrogum 5, Tropicalgin and Alginoplast. Materials and Methods: In this experimental in-vitro trial, a total of 90 alginate impressions were made from a Dentoform model using Hydrogum 5, Tropicalgin and Alginoplast alginates. The impressions were stored in a sealed plastic bag without a damp paper towel for 0, 24, 48, 72 and 120 hours and then poured with type III dental stone. Cross-arch (facial of 6 to facial of 6 on the opposite side) and antero-posterior (distal of right first molar to the ipsilateral central incisor) measurements were made with a digital caliper on the casts. Data were analyzed by two-way and one-way ANOVA and Tukey’s post-hoc test (P<0.05). Results: Alginate type and the pouring time significantly affected the dimensional stability of alginate impressions (both Ps<0.001). Pouring of Hydrogum 5 impressions can be delayed for up to 120 hours without significant dimensional changes. Alginoplast impressions may be poured after 72 hours, but Tropicalgin should be poured immediately and the storage time should not be more than 24 hours. Conclusion: Immediate pouring of alginate impressions provides the highest accuracy in reproducing the teeth and adjacent tissues; however, this study demonstrated that pouring may be delayed for up to five days using extended-pour (Hydrogum 5) alginates. PMID:25628695

Evaluation of digital dental models obtained from dental cone-beam computed tomography scan of alginate impressions

PubMed Central

Jiang, Tingting; Lee, Sang-Mi; Hou, Yanan; Chang, Xin

2016-01-01

Objective To investigate the dimensional accuracy of digital dental models obtained from the dental cone-beam computed tomography (CBCT) scan of alginate impressions according to the time elapse when the impressions are stored under ambient conditions. Methods Alginate impressions were obtained from 20 adults using 3 different alginate materials, 2 traditional alginate materials (Alginoplast and Cavex Impressional) and 1 extended-pour alginate material (Cavex ColorChange). The impressions were stored under ambient conditions, and scanned by CBCT immediately after the impressions were taken, and then at 1 hour intervals for 6 hours. After reconstructing three-dimensional digital dental models, the models were measured and the data were analyzed to determine dimensional changes according to the elapsed time. The changes within the measurement error were regarded as clinically acceptable in this study. Results All measurements showed a decreasing tendency with an increase in the elapsed time after the impressions. Although the extended-pour alginate exhibited a less decreasing tendency than the other 2 materials, there were no statistically significant differences between the materials. Changes above the measurement error occurred between the time points of 3 and 4 hours after the impressions. Conclusions The results of this study indicate that digital dental models can be obtained simply from a CBCT scan of alginate impressions without sending them to a remote laboratory. However, when the impressions are not stored under special conditions, they should be scanned immediately, or at least within 2 to 3 hours after the impressions are taken. PMID:27226958

Modification of pH Conferring Virucidal Activity on Dental Alginates

PubMed Central

Nallamuthu, Navina; Braden, Michael; Oxford, John; Williams, David; Patel, Mangala

2015-01-01

To formulate an alginate dental impression material with virucidal properties, experimental alginate dental impression materials were developed and the formulations adjusted in order to study the effect on pH profiles during setting. Commercially available materials served as a comparison. Eight experimental materials were tested for antiviral activity against Herpes Simplex Virus type 1 (HSV-1). Changing the amount of magnesium oxide (MgO) used in the experimental formulations had a marked effect on pH. Increasing MgO concentration corresponded with increased pH values. All experimental materials brought about viral log reductions ranging between 0.5 and 4.0 over a period of 4 h. The material with the lowest pH was the most effective. The current work highlights the very important role of MgO in controlling pH profiles. This knowledge has been applied to the formulation of experimental alginates; where materials with pH values of approximately 4.2–4.4 are able to achieve a significant log reduction when assayed against HSV-1. PMID:28788042

Production and characterization of the healthy brown rice milk with sodium alginate addition from brown algae Sargassum binderi as emulsifier

NASA Astrophysics Data System (ADS)

Latifah, R. N.; Warganegara, F. M.

2018-03-01

Brown rice milk, a plant milk, is potential to cure beriberi disease. Alginate was used as an emulsifier and increased the nutrition of brown rice milk. Alginate from Sargassum binderi was extracted by nonacidic treatment. The yield of alginate was 6.25 %. The moisture and water content of alginate were 18.27 % and 12.2 %, respectively. The density and viscosity of 0.1 % alginates in aqueous solution were 1.007 g·mL-1 and 7.651 × 10-3 kg·m-1s-1, respectively. The characteristics peaks of alginate appeared at 3,477; 1,633 and 1,419 cm-1, corresponding to hydroxyl (OH), carbonyl (C=O) and carboxyl (COOH), respectively. The best composition of alginate addition in brown rice milk was 0.2 %. The viscosity of the 0.2 % alginate in brown rice milk was 1.206 × 10-2 kg·m-1s-1 and showed a texture of small particle with closer spaces between the particles. The addition of alginate in brown rice milk also inhibited the process of sedimentation of milk which showed in the first order. The nutrition composition of the best brown rice milk was 40.21 mg·mL-1 carbohydrate; 1.3 mg·mL-1 protein; 0.158 mg·mL-1 fat and 13.1 mg·mL-1 total dietary fiber.

The replicative DNA polymerase of herpes simplex virus 1 exhibits apurinic/apyrimidinic and 5′-deoxyribose phosphate lyase activities

PubMed Central

Bogani, Federica; Boehmer, Paul E.

2008-01-01

Base excision repair (BER) is essential for maintaining genome stability both to counter the accumulation of unusual bases and to protect from base loss in the DNA. Herpes simplex virus 1 (HSV-1) is a large dsDNA virus that encodes its own DNA replication machinery, including enzymes involved in nucleotide metabolism. We report on a replicative family B and a herpesvirus-encoded DNA Pol that possesses DNA lyase activity. We have discovered that the catalytic subunit of the HSV-1 DNA polymerase (Pol) (UL30) exhibits apurinic/apyrimidinic (AP) and 5′-deoxyribose phosphate (dRP) lyase activities. These activities are integral to BER and lead to DNA cleavage on the 3′ side of abasic sites and 5′-dRP residues that remain after cleavage by 5′-AP endonuclease. The UL30-catalyzed reaction occurs independently of divalent cation and proceeds via a Schiff base intermediate, indicating that it occurs via a lyase mechanism. Partial proteolysis of the Schiff base shows that the DNA lyase activity resides in the Pol domain of UL30. These observations together with the presence of a virus-encoded uracil DNA glycosylase indicates that HSV-1 has the capacity to perform critical steps in BER. These findings have implications on the role of BER in viral genome maintenance during lytic replication and reactivation from latency. PMID:18695225

The Structure of RdDddP from Roseobacter denitrificans Reveals That DMSP Lyases in the DddP-Family Are Metalloenzymes

PubMed Central

Hehemann, Jan-Hendrik; Law, Adrienne; Redecke, Lars; Boraston, Alisdair B.

2014-01-01

Marine microbes degrade dimethylsulfoniopropionate (DMSP), which is produced in large quantities by marine algae and plants, with DMSP lyases into acrylate and the gas dimethyl sulfide (DMS). Approximately 10% of the DMS vents from the sea into the atmosphere and this emission returns sulfur, which arrives in the sea through rivers and runoff, back to terrestrial systems via clouds and rain. Despite their key role in this sulfur cycle DMSP lyases are poorly understood at the molecular level. Here we report the first X-ray crystal structure of the putative DMSP lyase RdDddP from Roseobacter denitrificans, which belongs to the abundant DddP family. This structure, determined to 2.15 Å resolution, shows that RdDddP is a homodimeric metalloprotein with a binuclear center of two metal ions located 2.7 Å apart in the active site of the enzyme. Consistent with the crystallographic data, inductively coupled plasma mass spectrometry (ICP-MS) and total reflection X-ray fluorescence (TRXF) revealed the bound metal species to be primarily iron. A 3D structure guided analysis of environmental DddP lyase sequences elucidated the critical residues for metal binding are invariant, suggesting all proteins in the DddP family are metalloenzymes. PMID:25054772

The effect of histidine ammonia-lyase on some murine tumours.

PubMed

Jack, G W; Wiblin, C N; McMahon, P C

1983-01-01

The histidine ammonia-lyase from bacterial strain CAMR 5315 was partially purified to assess its effect on the growth of murine tumours. This strain was selected as the source after an extensive screening programme for histidine ammonia-lyases. The enzyme was partially purified by ammonium sulphate fractionation, chromatography on DEAE-cellulose and Sephadex G-150. The enzyme reduced circulating L-histidine levels in Wistar rats and in mice persisted with a half-life of 6-7 h. Neither LDH virus nor chemical modification with ethylacetimidate increased the half-life as observed with L-asparaginase and L-glutaminase. The enzyme was tested in mice against Ehrlich carcinoma, L5178Y lymphoblastic leukaemia, Mc/S sarcoma, B16 melanoma, P8157 mastocytoma, P1798 lymphosarcoma and the Gardner 6C3HED lymphosarcoma. The only tumours to show sensitivity to the enzyme were the Mc/S sarcoma against which a 65% increase in life span was observed at the highest enzyme dose, 1000 U/kg on alternate days over 14 days and the Ehrlich ascites carcinoma where cures were obtained at 250 U/kg on alternate days over 14 days, but only at inocula levels of 10(5) and 10(3) cells/animal respectively.

Light-addressable electrodeposition of cell-encapsulated alginate hydrogels for a cellular microarray using a digital micromirror device

PubMed Central

Huang, Shih-Hao; Hsueh, Hui-Jung; Jiang, Yeu-Long

2011-01-01

This paper describes a light-addressable electrolytic system used to perform an electrodeposition of calcium alginate hydrogels using a digital micromirror device (DMD). In this system, a patterned light illumination is projected onto a photoconductive substrate serving as a photo-anode to electrolytically produce protons, which can lead to a decreased pH gradient. The low pH generated at the anode can locally release calcium ions from insoluble calcium carbonate (CaCO3) to cause gelation of calcium alginate through sol-gel transition. By controlling the illumination pattern on the DMD, a light-addressable electrodeposition of calcium alginate hydrogels with different shapes and sizes, as well as multiplexed micropatterning was performed. The effects of the concentration of the alginate and CaCO3 solutions on the dimensional resolution of alginate hydrogel formation were experimentally examined. A 3 × 3 array of cell-encapsulated alginate hydrogels was also successfully demonstrated through light-addressable electrodeposition. Our proposed method provides a programmable method for the spatiotemporally controllable assembly of cell populations into cellular microarrays and could have a wide range of biological applications in cell-based biosensing, toxicology, and drug discovery. PMID:22685500

Light-addressable electrodeposition of cell-encapsulated alginate hydrogels for a cellular microarray using a digital micromirror device.

PubMed

Huang, Shih-Hao; Hsueh, Hui-Jung; Jiang, Yeu-Long

2011-09-01

This paper describes a light-addressable electrolytic system used to perform an electrodeposition of calcium alginate hydrogels using a digital micromirror device (DMD). In this system, a patterned light illumination is projected onto a photoconductive substrate serving as a photo-anode to electrolytically produce protons, which can lead to a decreased pH gradient. The low pH generated at the anode can locally release calcium ions from insoluble calcium carbonate (CaCO(3)) to cause gelation of calcium alginate through sol-gel transition. By controlling the illumination pattern on the DMD, a light-addressable electrodeposition of calcium alginate hydrogels with different shapes and sizes, as well as multiplexed micropatterning was performed. The effects of the concentration of the alginate and CaCO(3) solutions on the dimensional resolution of alginate hydrogel formation were experimentally examined. A 3 × 3 array of cell-encapsulated alginate hydrogels was also successfully demonstrated through light-addressable electrodeposition. Our proposed method provides a programmable method for the spatiotemporally controllable assembly of cell populations into cellular microarrays and could have a wide range of biological applications in cell-based biosensing, toxicology, and drug discovery.

Degradation of trichloroethylene (TCE) by nanoscale zero-valent iron (nZVI) immobilized in alginate bead.

PubMed

Kim, Hojeong; Hong, Hye-Jin; Jung, Juri; Kim, Seong-Hye; Yang, Ji-Won

2010-04-15

Nowadays, many researchers have studied the environmental application of the nanoscale zero-valent iron (nZVI) and several field applications for the groundwater remediation have been reported. Still, there are many concerns on the fate and transport of the nZVI and the corresponding risks. To avoid such concerns, it was investigated to immobilize nZVI in a support and then it was applied to degrade trichloroethylene (TCE). The nZVI and palladium-doped nZVI (Fe(0)- and Fe/Pd-alginate) were immobilized in the alginate bead where ferric and barium ions are used as the cross-linking cations of the bead. According to TEM (transmission electron microscopy), the size of the immobilized ZVI was as small as a few nanometers. From the surface analysis of the Fe/Pd-alginate, it is found that the immobilized nZVI has the core-shell structure. The core is composed of single crystal Fe(0), while most of irons on the surface are oxidized to Fe(3+). When 50 g/L of Fe/Pd-alginate (3.7 g Fe/L) was introduced to the aqueous solution, >99.8% of TCE was removed and the release of metal from the support was <3% of the loaded iron. The removal of TCE by Fe/Pd-alginate followed pseudo-first-order kinetics. The observed pseudo-first-order reaction constant (k(obs)) of Fe/Pd-alginate was 6.11 h(-1) and the mass normalized rate constant (k(m)) was 1.6 L h(-1) g(-1). The k(m) is the same order of magnitude with that of iron nanoparticles. In conclusion, it is considered that Fe/Pd-alginate can be used efficiently in the treatment of chlorinated solvent. 2009 Elsevier B.V. All rights reserved.

The study of the structural properties of very low viscosity sodium alginate by small-angle neutron scattering

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

Badita, C. R., E-mail: ramona@tandem.nipne.ro; University of Bucharest, Faculty of Physics, Atomiştilor 405, CP MG - 11, RO – 077125, Bucharest-Magurele; Aranghel, D., E-mail: daranghe@nipne.ro

2016-03-25

Sodium alginate is a linear polymer extract from brown algae and it is used in the biomedical, food, cosmetics and pharmaceutical industries as solution property modifiers and gelling agents. But despite the extensive studies of the alginate gelation process, still some fundamental questions remain unresolved. The fractal behavior of very low viscosity sodium alginate solutions and their influence on the critical gelation of alginate induced by Ca{sup 2+} ions were investigated using Small-Angle Neutron Scattering (SANS) measurements. SANS data are interpreted using both standard linear plots and the Beaucage model. The scattering intensity is dependent by alginate concentration and Ca{supmore » 2+} concentration. From a critical concentration of 1.0 % w/w our polymer swelled forming spherical structures with rough surfaces. Also the addition of the salt induces the collapse and the appearance of the aggregation and clusters formation.« less

Effects of process variables on the encapsulation of oil in ca-alginate capsules using an inverse gelation technique.

PubMed

Abang, Sariah; Chan, Eng-Seng; Poncelet, Denis

2012-01-01

The objective of this study was to investigate the effects of process variables on the encapsulation of oil in a calcium alginate membrane using an inverse gelation technique. A dispersion of calcium chloride solution in sunflower oil (water-in-oil emulsion) was added dropwise to the alginate solution. The migration of calcium ions to the alginate solution initiates the formation of a ca-alginate membrane around the emulsion droplets. The membrane thickness of wet capsules and the elastic modulus of dry capsules increased following first-order kinetics with an increasing curing time. An increase in the calcium chloride concentration increased the membrane thickness of wet capsules and the elastic modulus of dry capsules. An increase in the alginate concentration decreased the mean diameter of wet capsules but increased the elastic modulus of dry capsules.

PECTATE LYASE-LIKE10 is associated with pollen wall development in Brassica campestris.

PubMed

Jiang, Jingjing; Yao, Lina; Yu, Youjian; Lv, Meiling; Miao, Ying; Cao, Jiashu

2014-11-01

PECTATE LYASE-LIKE10 (PLL10) was previously identified as one of the differentially expressed genes both in microspores during the late pollen developmental stages and in pistils during the fertilization process in Chinese cabbage (Brassica campestris ssp. chinensis). Here, antisense-RNA was used to study the functions of BcPLL10 in Chinese cabbage. Abnormal pollen was identified in the transgenic lines (bcpll10-4, -5, and -6). In fertilization experiments, fewer seeds were harvested when the antisense-RNA lines were used as pollen donor. In vivo and in vitro pollen germination assays less germinated pollen tubes were observed in bcpll10 lines. Scanning electron microscopy observation verified that the tryphine materials were over accumulated around the pollen surface and sticked them together in bcpll10. Moreover, transmission electron microscopy observation revealed that the internal endintine was overdeveloped and predominantly occupied the intine, and disturbed the normal proportional distribution of the two layers in the non-germinal furrow region; and no obvious demarcation existed between them in the germinal furrow region in the bcpll10 pollen. Collectively, this study presented a novel PLL gene that played an important role during the pollen wall development in B. campestris, which may also possess potential importance for male sterility usage in agriculture. © 2014 Institute of Botany, Chinese Academy of Sciences.

Lifting bloody footwear impressions using alginate casts followed by chemical enhancement.

PubMed

Wiesner, Sarena; Izraeli, Elad; Shor, Yaron; Domb, Avi

2013-05-01

A method for lifting bloody footwear impressions using alginate casts and enhancing the lifted impressions with amido black is presented. On rough or dark substrates, background interferences may conceal significant details of footwear impressions. Illumination with alternative light sources and chemically enhancing the bloody footwear impressions may reveal additional details, but sometimes, lifting footwear impressions prior to enhancing is the only way to expose hidden details (by using blood reagents not adequate on the original). Several cast formulations were tested for lifting the footwear impressions. The best results were achieved using Aroma fine®. Enhancement of the footwear impressions was attempted with several reagents prior to lifting, during the casting process, and on the lifted footwear impressions. Applying amido black to footwear impressions lifted with alginate produced the sharpest and most detailed footwear impressions. Alginate castings followed by chemical enhancement with amido black may produce high-quality footwear impressions for comparison. © 2013 American Academy of Forensic Sciences.

Refeeding syndrome in a young woman with argininosuccinate lyase deficiency.

PubMed

Stuy, M; Chen, G-F; Masonek, J M; Scharschmidt, B F

2015-09-01

A severely chronically protein and calorie restricted young woman with argininosuccinate lyase deficiency developed transient refeeding syndrome (RFS) and hyperammonemia after modest diet liberalization following initiation of glycerol phenylbutyrate (GPB). The patient required IV supportive care and supplementation with potassium, magnesium and calcium. She is now doing well on GPB and an appropriate maintenance diet. Susceptibility to RFS should be considered in chronically nutritionally restricted patients with metabolic disorders after liberalization of diet.

Refeeding syndrome in a young woman with argininosuccinate lyase deficiency☆

PubMed Central

Stuy, M.; Chen, G.-F.; Masonek, J.M.; Scharschmidt, B.F.

2015-01-01

A severely chronically protein and calorie restricted young woman with argininosuccinate lyase deficiency developed transient refeeding syndrome (RFS) and hyperammonemia after modest diet liberalization following initiation of glycerol phenylbutyrate (GPB). The patient required IV supportive care and supplementation with potassium, magnesium and calcium. She is now doing well on GPB and an appropriate maintenance diet. Susceptibility to RFS should be considered in chronically nutritionally restricted patients with metabolic disorders after liberalization of diet. PMID:26937403

Potentiometric titration for determining the composition and stability of metal(II) alginates and pectinates in aqueous solutions

NASA Astrophysics Data System (ADS)

Kaisheva, N. Sh.; Kaishev, A. Sh.

2015-07-01

The compositions and stabilities of Cu2+, Mn2+, Pb2+, Ca2+, Zn2+, Cd2+, Co2+, and Ni2+ alginates and pectinates are determined in aqueous solutions via titrimetry and potentiometry with calculations performed using Bjerrum's method, the curve intersection technique, and the equilibrium shift method. It is found that the interaction between Cu2+ and polyuronides is a stepwise process and, depending on the ligand concentration and the method of determination, Cu2+ alginate can be characterized by its ML, ML2, and ML3 compositions (where M is the metal ion and L is the structural unit of polyuronide) and stability constants logβ = 2.65, 5.00-5.70, and 7.18-7.80, respectively. The compositions of Cu2+ pectinates are ML and ML2 with logβ = 3.00 and 7.64-7.94, respectively. It is concluded that Pb2+, Ca2+, Mn2+, Zn2+, Cd2+, Co2+, and Ni2+ ions form only alginates and pectinates of ML2 composition with logβ values of 3.45 (Pb2+ alginate), 2.20 (Ca2+ alginate), 1.06 (Mn2+ alginate), 3.51 (Pb2+ pectinate), 2.35 (Ca2+ pectinate), and 1.24 (Mn2+ pectinate). The pectinates are shown to be more stable than the alginates, the most stable compounds being those formed by polyuronides and Cu2+. The least stable are those with Mn2+.

Targeting sphingosine-1-phosphate lyase as an anabolic therapy for bone loss.

PubMed

Weske, Sarah; Vaidya, Mithila; Reese, Alina; von Wnuck Lipinski, Karin; Keul, Petra; Bayer, Julia K; Fischer, Jens W; Flögel, Ulrich; Nelsen, Jens; Epple, Matthias; Scatena, Marta; Schwedhelm, Edzard; Dörr, Marcus; Völzke, Henry; Moritz, Eileen; Hannemann, Anke; Rauch, Bernhard H; Gräler, Markus H; Heusch, Gerd; Levkau, Bodo

2018-05-01

Sphingosine-1-phosphate (S1P) signaling influences bone metabolism, but its therapeutic potential in bone disorders has remained unexplored. We show that raising S1P levels in adult mice through conditionally deleting or pharmacologically inhibiting S1P lyase, the sole enzyme responsible for irreversibly degrading S1P, markedly increased bone formation, mass and strength and substantially decreased white adipose tissue. S1P signaling through S1P 2 potently stimulated osteoblastogenesis at the expense of adipogenesis by inversely regulating osterix and PPAR-γ, and it simultaneously inhibited osteoclastogenesis by inducing osteoprotegerin through newly discovered p38-GSK3β-β-catenin and WNT5A-LRP5 pathways. Accordingly, S1P 2 -deficient mice were osteopenic and obese. In ovariectomy-induced osteopenia, S1P lyase inhibition was as effective as intermittent parathyroid hormone (iPTH) treatment in increasing bone mass and was superior to iPTH in enhancing bone strength. Furthermore, lyase inhibition in mice successfully corrected severe genetic osteoporosis caused by osteoprotegerin deficiency. Human data from 4,091 participants of the SHIP-Trend population-based study revealed a positive association between serum levels of S1P and bone formation markers, but not resorption markers. Furthermore, serum S1P levels were positively associated with serum calcium , negatively with PTH , and curvilinearly with body mass index. Bone stiffness, as determined through quantitative ultrasound, was inversely related to levels of both S1P and the bone formation marker PINP, suggesting that S1P stimulates osteoanabolic activity to counteract decreasing bone quality. S1P-based drugs should be considered as a promising therapeutic avenue for the treatment of osteoporotic diseases.

Review: efficacy of alginate supplementation in relation to appetite regulation and metabolic risk factors: evidence from animal and human studies.

PubMed

Georg Jensen, M; Pedersen, C; Kristensen, M; Frost, G; Astrup, A

2013-02-01

This review provides a critical update on human and animal studies investigating the effect of alginate supplementation on appetite regulation, glycaemic and insulinemic responses, and lipid metabolism with discussion of the evidence on potential mechanisms, efficacy and tolerability. Dependent on vehicle applied for alginate supplementation, the majority of animal and human studies suggest that alginate consumption does suppress satiety and to some extent energy intake. Only one long-term intervention trial found effects on weight loss. In addition, alginates seem to exhibit beneficial influence on postprandial glucose absorption and insulin response in animals and humans. However, alginate supplementation was only found to have cholesterol-lowering properties in animals. Several mechanisms have been suggested for the positive effect observed, which involve delayed gastric emptying, increased viscosity of digesta and slowed nutrient absorption in the small intestine upon alginate gel formation. Despite reasonable efficacy and tolerability from the acute or short-term studies, we still realize there is a critical need for development of optimal alginate types and vehicles as well as studies on further long-term investigation on alginate supplementation in humans before inferring that it could be useful in the management of obesity and the metabolic syndrome. © 2012 The Authors. obesity reviews © 2012 International Association for the Study of Obesity.

Bromo-oxidation reaction in enzyme-entrapped alginate hollow microfibers

PubMed Central

Asthana, Amit; Lee, Kwang Ho; Shin, Su-Jung; Perumal, Jayakumar; Butler, Lauren; Lee, Sang-Hoon; Kim, Dong-Pyo

2011-01-01

In this article, the authors present the fabrication of an enzyme-entrapped alginate hollow fiber using a microfluidic device. Further use of enzyme-entrapped alginate hollow fibers as a biocatalytic microchemical reactor for chemical synthesis is also deliberated in this article. To ensure that there is no enzyme leaching from the fiber, fiber surfaces were coated with chitosan. To confine the mobility of reactants and products within the porous hollow fibers the entire fibers were embedded into a transparent polydimethylsiloxane (PDMS) matrix which also works as a support matrix. A vanadium-containing bromoperoxidase enzyme isolated from Corallina confusa was used as a model enzyme to demonstrate the use of these alginate hollow-fiber reactors in bromo-oxidation of phenol red to bromophenol blue at different dye flow rates. Stability of the entrapped enzyme at different temperatures and the effect of the chitosan coating on the reaction conversion were also studied. It was observed that molecules as big as 27 kDa can be retained in the matrix after coating with chitosan while molecules with molecular-weight of around 378 Da can still diffuse in and out of the matrix. The kinetic conversion rate in this microfluidic bioreactor was more than 41-fold faster when compared with the standard test-tube procedure. PMID:21799723

Improvement of aromatic thiol release through the selection of yeasts with increased β-lyase activity.

PubMed

Belda, Ignacio; Ruiz, Javier; Navascués, Eva; Marquina, Domingo; Santos, Antonio

2016-05-16

The development of a selective medium for the rapid differentiation of yeast species with increased aromatic thiol release activity has been achieved. The selective medium was based on the addition of S-methyl-l-cysteine (SMC) as β-lyase substrate. In this study, a panel of 245 strains of Saccharomyces cerevisiae strains was tested for their ability to grow on YCB-SMC medium. Yeast strains with an increased β-lyase activity grew rapidly because of their ability to release ammonium from SMC in comparison to others, and allowed for the easy isolation and differentiation of yeasts with promising properties in oenology, or another field, for aromatic thiol release. The selective medium was also helpful for the discrimination between those S. cerevisiae strains, which present a common 38-bp deletion in the IRC7 sequence (present in around 88% of the wild strains tested and are likely to be less functional for 4-mercapto-4-methylpentan-2-one (4MMP) production), and those S. cerevisiae strains homozygous for the full-length IRC7 allele. The medium was also helpful for the selection of non-Saccharomyces yeasts with increased β-lyase activity. Based on the same medium, a highly sensitive, reproducible and non-expensive GC-MS method for the evaluation of the potential volatile thiol release by different yeast isolates was developed. Copyright © 2016 Elsevier B.V. All rights reserved.

Еvaluation of biocompatibility and antioxidant efficiency of chitosan-alginate nanoparticles loaded with quercetin.

PubMed

Aluani, Denitsa; Tzankova, Virginia; Kondeva-Burdina, Magdalena; Yordanov, Yordan; Nikolova, Elena; Odzhakov, Feodor; Apostolov, Alexandar; Markova, Tzvetanka; Yoncheva, Krassimira

2017-10-01

The present study deals with development and evaluation of the safety profile of chitosan/alginate nanoparticles as a platform for delivery of a natural antioxidant quercetin. The nanoparticles were prepared by varying the ratios between both biopolymers giving different size and charge of the formulations. The biocompatibility was explored in vitro in cells from different origin: cultivated HepG2 cells, isolated primary rat hepatocytes, isolated murine spleen lymphocytes and macrophages. In vivo toxicological evaluation was performed after repeated 14-day oral administration to rats. The study revealed that chitosan/alginate nanoparticles did not change body weight, the relative weight of rat livers, liver histology, hematology and biochemical parameters. The protective effects of quercetin-loaded nanoparticles were investigated in the models of iron/ascorbic acid (Fe 2+ /AA) induced lipid peroxidation in microsomes and tert-butyl hydroperoxide oxidative stress in isolated rat hepatocytes. Interesting finding was that the empty chitosan/alginate nanoparticles possessed protective activity themselves. The antioxidant effects of quercetin loaded into the nanoparticles formulated with higher concentration of chitosan were superior compared to quercetin encapsulated in nanoparticles with higher amount of sodium alginate. In conclusion, chitosan/alginate nanoparticles can be considered appropriate carrier for quercetin, combining safety profile and improved protective activity of the encapsulated antioxidant. Copyright © 2017 Elsevier B.V. All rights reserved.

Microencapsulation of islets within alginate/poly(ethylene glycol) gels cross-linked via Staudinger ligation

PubMed Central

Hall, Kristina K.; Gattás-Asfura, Kerim M.; Stabler, Cherie L.

2010-01-01

Functionalized alginate and PEG polymers were used to generate covalently linked alginate-PEG (XAlgPEG) microbeads of high stability. The cell-compatible Staudinger ligation scheme was used to chemoselectively cross-link phosphine-terminated poly(ethylene glycol) (PEG) to azide-functionalized alginate, resulting in XAlgPEG hydrogels. XAlgPEG microbeads were formed by co-incubation of the two polymers, followed by ionic cross-linking of the alginate using barium ions. The enhanced stability and gel properties of the resulting XAlgPEG microbeads, as well as the compatibility of these polymers for the encapsulation of islets and beta cells lines, were investigated. Our data show that XAlgPEG microbeads exhibit superior resistance to osmotic swelling compared to traditional barium cross-linked alginate (Ba-Alg) beads, with a 5-fold reduction in observed swelling, as well as resistance to dissolution via chelation solution. Diffusion and porosity studies found XAlgPEG beads to exhibit properties comparable to standard Ba-Alg. Our data found XAlgPEG microbeads to be highly cell compatible with insulinoma cell lines, as well as rat and human pancreatic islets, where the viability and functional assessment of cells within XAlgPEG were comparable to Ba-Alg controls. The remarkable improved stability, as well as demonstrated cellular compatibility, of XAlgPEG hydrogels makes them an appealing option for a wide variety of tissue engineering applications. PMID:20654745

Preparation and Characterization of Azadirachtin Alginate-Biosorbent Based Formulations: Water Release Kinetics and Photodegradation Study.

PubMed

Flores-Céspedes, Francisco; Martínez-Domínguez, Gerardo P; Villafranca-Sánchez, Matilde; Fernández-Pérez, Manuel

2015-09-30

The botanical insecticide azadirachtin was incorporated in alginate-based granules to obtain controlled release formulations (CRFs). The basic formulation [sodium alginate (1.47%) - azadirachtin (0.28%) - water] was modified by the addition of biosorbents, obtaining homogeneous hybrid hydrogels with high azadirachtin entrapment efficiency. The effect on azadirachtin release rate caused by the incorporation of biosorbents such as lignin, humic acid, and olive pomace in alginate formulation was studied by immersion of the granules in water under static conditions. The addition of the biosorbents to the basic alginate formulation reduces the rate of release because the lignin-based formulation produces a slower release. Photodegradation experiments showed the potential of the prepared formulations in protecting azadirachtin against simulated sunlight, thus improving its stability. The results showed that formulation prepared with lignin provided extended protection. Therefore, this study provides a new procedure to encapsulate the botanical insecticide azadirachtin, improving its delivery and photostability.

Plant defense genes are regulated by ethylene

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

Ecker, J.R.; Davis, R.W.

One of the earliest detectable events during plant-pathogen interaction is a rapid increase in ethylene biosynthesis. This gaseous plant stress hormone may be a signal for plants to activate defense mechanisms against invading pathogens such as bacteria, fungi, and viruses. The effect of ethylene on four plant genes involved in three separate plant defense response pathways was examined; these included (i and ii) genes that encode L-phenylalanine ammonia-lyase (EC 4.3.1.5) and 4-coumarate:CoA ligase (4-coumarate:CoA ligase (AMP-forming), EC 6.2.1.12), enzymes of the phenylpropanoid pathway, (iii) the gene encoding chalcone synthase, an enzyme of the flavonoid glycoside pathway, and (iv) the genesmore » encoding hydroxyproline-rich glycoprotein, a major protein component(s) of plant cell walls. Blot hybridization analysis of mRNA from ethylene-treated carrot roots reveals marked increases in the levels of phenylalanine ammonia-lyase mRNA, 4-coumarate CoA ligase mRNA, chalcone synthase mRNA, and certain hydroxyproline-rich glycoprotein transcripts. The effect of ethylene on hydroxyproline-rich glycoprotein mRNA accumulation was different from that of wounding. Ethylene induces two hydroxyproline-rich glycoprotein mRNAs (1.8 and 4.0 kilobases), whereas wounding of carrot root leads to accumulation of an additional hydroxyproline-rich mRNA (1.5 kilobases). These results indicate that at least two distinct signals, ethylene and a wound signal, can affect the expression of plant defense-response genes.« less

Encapsulated eucalyptus oil in ionically cross-linked alginate microcapsules and its controlled release.

PubMed

Noppakundilograt, Supaporn; Piboon, Phianghathai; Graisuwan, Wilaiporn; Nuisin, Roongkan; Kiatkamjornwong, Suda

2015-10-20

Sodium alginate microcapsules containing eucalyptus oil were prepared by oil-in-water emulsification via Shirasu porous glass (SPG) membrane and cross-linked by calcium chloride (CaCl2). SPG membrane pore size of 5.2μm was used to control the size of eucalyptus oil microdroplets. Effects of sodium alginate, having a mannuronic acid/guluronic acid (M/G) ratio of 1.13, eucalyptus oil and CaCl2 amounts on microdroplet sizes and size distribution were elucidated. Increasing sodium alginate amounts from 0.1 to 0.5% (wv(-1)) sodium alginate, the average droplets size increased from 42.2±2.0 to 48.5±0.6μm, with CVs of 16.5±2.2 and 30.2±4.5%, respectively. CaCl2 successfully gave narrower size distribution of cross-linked eucalyptus oil microcapsules. The optimum conditions for preparing the microcapsules, oil loading efficiency, and controlled release of the encapsulated eucalyptus oil from the microcapsules as a function of time at 40°C were investigated. Release model for the oil from microcapsules fitted Ritger-Peppas model with non-Fickian transport mechanism. Copyright © 2015 Elsevier Ltd. All rights reserved.

Understanding Which Residues of the Active Site and Loop Structure of a Tyrosine Aminomutase Define Its Mutase and Lyase Activities.

PubMed

Attanayake, Gayanthi; Walter, Tyler; Walker, Kevin D

2018-05-30

Site-directed mutations and substrate analogues were used to gain insights into the branch-point reaction of the 3,5-dihydro-5-methylidene-4 H-imidazol-4-one (MIO)-tyrosine aminomutase from Oryza sativa ( OsTAM). Exchanging the active residues of OsTAM (Y125C/N446K) for those in a phenylalanine aminomutase TcPAM altered its substrate specificity from tyrosine to phenylalanine. The aminomutase mechanism of OsTAM surprisingly changed almost exclusively to that of an ammonia lyase making cinnamic acid (>95%) over β-phenylalanine [Walter, T., et al. (2016) Biochemistry 55, 3497-3503]. We hypothesized that the missing electronics or sterics on the aryl ring of the phenylalanine substrate, compared with the sizable electron-donating hydroxyl of the natural tyrosine substrate, influenced the unexpected lyase reactivity of the OsTAM mutant. The double mutant was incubated with 16 α-phenylalanine substituent analogues of varying electronic strengths and sterics. The mutant converted each analogue principally to its acrylate with ∼50% conversion of the p-Br substrate, making only a small amount of the β-amino acid. The inner loop structure over the entrance to the active site was also mutated to assess how the lyase and mutase activities are affected. An OsTAM loop mutant, matching the loop residues of TcPAM, still chiefly made >95% of the acrylate from each substrate. A combined active site:loop mutant was most reactive but remained a lyase, making 10-fold more acrylates than other mutants did. While mutations within the active site changed the substrate specificity of OsTAM, continued exploration is needed to fully understand the interplay among the inner loop, the substrate, and the active site in defining the mutase and lyase activities.

Development of a novel colorimetric sensor based on alginate beads for monitoring rainbow trout spoilage.

PubMed

Majdinasab, Marjan; Hosseini, Seyed Mohammad Hashem; Sepidname, Marziyeh; Negahdarifar, Manizheh; Li, Peiwu

2018-05-01

Alginate is a non-toxic, renewable, and linear copolymer obtained from the brown algae Laminaria digitata that can be easily shaped into beads. Its good gel forming properties have made it useful for entrapping food and pharmaceutical ingredients. In this study, alginate beads were used in a novel application as a colorimetric sensor in food intelligent packaging. Colorimetric sensor was developed through entrapping red cabbage extract as a pH indicator in alginate beads. The pH indicator beads were used in rainbow trout packaging for monitoring fillets spoilage. Color change of beads during fish storage was measured using the CIELab method. The alginate bead colorimetric sensor is validated by measuring total volatile basic nitrogen (TVB-N) levels and microbial populations in fish samples. Moreover, peroxide value (PV) and thiobarbituric acid reactive substances (TBARS) were evaluated during storage. Results indicated that increasing the bacterial population during storage and production of proteolytic enzymes resulted in protein degradation, accumulation of volatile amine compounds, increase in the pH and finally color change of alginate beads. The values of TVB-N, pH, PV and TBARS increased with time of storage. The results of TVB-N and microbial growth were in accordance with color change of beads and CIELab data. Therefore, the proposed system enjoys a high sensitivity to pH variations and is capable of monitoring the spoilage of fish or other protein-rich products through its wide range of color changes. The alginate beads containing the red cabbage extract can, thus, be used as a low-cost colorimetric sensor for intelligent packaging applications.

Removal of acetaminophen in water by laccase immobilized in barium alginate.

PubMed

Ratanapongleka, Karnika; Punbut, Supot

2018-02-01

This research has focused on the optimization of immobilized laccase condition and utilization in degradation of acetaminophen contaminated in aqueous solution. Laccase from Lentinus polychrous was immobilized in barium alginate. The effects of laccase immobilization such as sodium alginate concentration, barium chloride concentration and gelation time were studied. The optimal conditions for immobilization were sodium alginate 5% (w/v), barium chloride 5% (w/v) and gelation time of 60 min. Immobilized laccase was then used for acetaminophen removal. Acetaminophen was removed quickly in the first 50 min. The degradation rate and percentage of removal increased when the enzyme concentration increased. Immobilized laccase at 0.57 U/g-alginate showed the maximum removal at 94% in 240 min. The removal efficiency decreased with increasing initial acetaminophen concentration. The K m value for immobilized laccase (98.86 µM) was lower than that of free laccase (203.56 µM), indicating that substrate affinity was probably enhanced by immobilization. The immobilized enzyme exhibited high activity and good acetaminophen removal at pH 7 and temperature of 35°C. The activation energies of free and immobilized laccase for degradation of acetaminophen were 8.08 and 17.70 kJ/mol, respectively. It was also found that laccase stability to pH and temperature increased after immobilization. Furthermore, immobilized laccase could be reused for five cycles. The capability of removal and enzyme activity were retained above 70%.

Alginate Immobilization of Metabolic Enzymes (AIME) for High ...

EPA Pesticide Factsheets

Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays DE DeGroot, RS Thomas, and SO SimmonsNational Center for Computational Toxicology, US EPA, Research Triangle Park, NC USAThe EPA’s ToxCast program utilizes a wide variety of high-throughput screening (HTS) assays to assess chemical perturbations of molecular and cellular endpoints. A key criticism of using HTS assays for toxicity assessment is the lack of xenobiotic metabolism (XM) which precludes both metabolic detoxification as well as bioactivation of chemicals tested in vitro thereby mischaracterizing the potential risk posed by these chemicals. To address this deficiency, we have developed an extracellular platform to retrofit existing HTS assays with XM activity. This platform utilizes the S9 fraction of liver homogenate encapsulated in an alginate gel network which reduces the cytotoxicity caused by direct addition of S9 to cells in culture. Alginate microspheres containing encapsulated human liver S9 were cross-linked to solid supports extending from a 96-well plate lid and were assayed using a pro-luciferin substrate specific for CYP3A4 (IPA). We demonstrate that S9 was successfully encapsulated and remained enzymatically active post-encapsulation with 5-10X the CYP3A4 activity as compared to 1 µg solubilized human liver S9. Ketoconazole, a known inhibitor of human CYP3A4, inhibited CYP3A4 activity in a concentration-dependent manner (IC50: 0.27 µM) and inhibiti

In-situ photopolymerization of monodisperse and discoid oxidized methacrylated alginate microgels in a microfluidic channel

DOE PAGES

Wang, Shuo; Jeon, Oju; Shankles, Peter G.; ...

2016-02-03

Here, we present a simple microfluidic technique to in-situ photopolymerize (by 365 nm ultraviolet) monodisperse oxidized methacrylated alginate (OMA) microgels using a photoinitiator (VA-086). By this technique, we generated monodisperse spherical OMA beads and discoid non-spherical beads with better shape consistency than ionic crosslinking methods do. We found that a high monomer concentration (8 w/v %), a high photoinitiator concentration (1.5 w/v %) and absence of oxygen are critical factors to cure OMA microgels. This photopolymerizing method is an alternative to current methods to form alginate microgels and is a simpler approach to generate non-spherical alginate microgels.

Dye decolorization and detoxification potential of Ca-alginate beads immobilized manganese peroxidase.

PubMed

Bilal, Muhammad; Asgher, Muhammad

2015-12-10

In view of compliance with increasingly stringent environmental legislation, an eco-friendly treatment technology of industrial dyes and effluents is a major environmental challenge in the color industry. In present study, a promising and eco-friendly entrapment approach was adopted to immobilize purified manganese peroxidase (MnP) produced from an indigenous strain of Ganoderma lucidum IBL-05 on Ca-alginate beads. The immobilized MnP was subsequently used for enhanced decolorization and detoxification of textile reactive dyes). MnP isolated from solid-state culture of G. lucidum IBL-05, presented highest immobilization yield (83.9 %) using alginate beads prepared at optimized conditions of 4 % (w/v) sodium alginate, 2 % (w/v) Calcium chloride (CaCl2) and 0.5 mg/ml enzyme concentration. Immobilization of MnP enhanced optimum temperature but caused acidic shift in optimum pH of the enzyme. The immobilized MnP showed optimum activity at pH 4.0 and 60 °C as compared to pH 5.0 and 35 °C for free enzyme. The kinetic parameters K(m) and V(max) of MnP were significantly improved by immobilization. The enhanced catalytic potential of immobilized MnP led to 87.5 %, 82.1 %, 89.4 %, 95.7 % and 83 % decolorization of Sandal-fix Red C4BLN, Sandal-fix Turq Blue GWF, Sandal-fix Foron Blue E2BLN, Sandal-fix Black CKF and Sandal-fix Golden Yellow CRL dyes, respectively. The insolubilized MnP was reusable for 7 repeated cycles in dye color removal. Furthermore, immobilized MnP also caused a significant reduction in biochemical oxygen demand (BOD) (94.61-95.47 %), chemical oxygen demand (COD) (91.18-94.85 %), and total organic carbon (TOC) (89.58-95 %) of aqueous dye solutions. G. lucidum MnP was immobilized in Ca-alginate beads by entrapment method to improve its practical effectiveness. Ca-alginate bound MnP was catalytically more vigorous, thermo-stable, reusable and worked over wider ranges of pH and temperature as compared to its free counterpart. Results of cytotoxicity like

Design and performance of a sericin-alginate interpenetrating network hydrogel for cell and drug delivery

PubMed Central

Zhang, Yeshun; Liu, Jia; Huang, Lei; Wang, Zheng; Wang, Lin

2015-01-01

Although alginate hydrogels have been extensively studied for tissue engineering applications, their utilization is limited by poor mechanical strength, rapid drug release, and a lack of cell adhesive ability. Aiming to improve these properties, we employ the interpenetrating hydrogel design rationale. Using alginate and sericin (a natural protein with many unique properties and a major component of silkworm silk), we develop an interpenetrating polymer network (IPN) hydrogel comprising interwoven sericin and alginate double networks. By adjusting the sericin-to-alginate ratios, IPNs’ mechanical strength can be adjusted to meet stiffness requirements for various tissue repairs. The IPNs with high sericin content show increased stability during degradation, avoiding pure alginate’s early collapse. These IPNs have high swelling ratios, benefiting various applications such as drug delivery. The IPNs sustain controlled drug release with the adjustable rates. Furthermore, these IPNs are adhesive to cells, supporting cell proliferation, long-term survival and migration. Notably, the IPNs inherit sericin’s photoluminescent property, enabling bioimaging in vivo. Together, our study indicates that the sericin-alginate IPN hydrogels may serve as a versatile platform for delivering cells and drugs, and suggests that sericin may be a building block broadly applicable for generating IPN networks with other biomaterials for diverse tissue engineering applications. PMID:26205586

Design and performance of a sericin-alginate interpenetrating network hydrogel for cell and drug delivery

NASA Astrophysics Data System (ADS)

Zhang, Yeshun; Liu, Jia; Huang, Lei; Wang, Zheng; Wang, Lin

2015-07-01

Although alginate hydrogels have been extensively studied for tissue engineering applications, their utilization is limited by poor mechanical strength, rapid drug release, and a lack of cell adhesive ability. Aiming to improve these properties, we employ the interpenetrating hydrogel design rationale. Using alginate and sericin (a natural protein with many unique properties and a major component of silkworm silk), we develop an interpenetrating polymer network (IPN) hydrogel comprising interwoven sericin and alginate double networks. By adjusting the sericin-to-alginate ratios, IPNs’ mechanical strength can be adjusted to meet stiffness requirements for various tissue repairs. The IPNs with high sericin content show increased stability during degradation, avoiding pure alginate’s early collapse. These IPNs have high swelling ratios, benefiting various applications such as drug delivery. The IPNs sustain controlled drug release with the adjustable rates. Furthermore, these IPNs are adhesive to cells, supporting cell proliferation, long-term survival and migration. Notably, the IPNs inherit sericin’s photoluminescent property, enabling bioimaging in vivo. Together, our study indicates that the sericin-alginate IPN hydrogels may serve as a versatile platform for delivering cells and drugs, and suggests that sericin may be a building block broadly applicable for generating IPN networks with other biomaterials for diverse tissue engineering applications.

The role of alginate in Pseudomonas aeruginosa EPS adherence, viscoelastic properties and cell attachment.

PubMed

Orgad, Oded; Oren, Yoram; Walker, Sharon L; Herzberg, Moshe

2011-08-01

Among various functions, extracellular polymeric substances (EPS) provide microbial biofilms with mechanical stability and affect initial cell attachment, the first stage in the biofilm formation process. The role of alginate, an abundant polysaccharide in Pseudomonas aeruginosa biofilms, in the viscoelastic properties and adhesion kinetics of EPS was analyzed using a quartz crystal microbalance with dissipation (QCM-D) monitoring technology. EPS was extracted from two P. aeruginosa biofilms, a wild type strain, PAO1, and a mucoid strain, PAOmucA22 that over-expresses alginate production. The higher alginate content in the EPS originating from the mucoid biofilms was clearly shown to increase both the rate and the extent of attachment of the EPS, as well as the layer's thickness. Also, the presence of calcium and elevated ionic strength increased the thickness of the EPS layer. Dynamic light scattering (DLS) showed that the presence of calcium and elevated ionic strength induced intermolecular attractive interactions in the mucoid EPS molecules. For the wild type EPS, in the presence of calcium, an elevated shift in the distribution of the diffusion coefficients was observed with DLS due to a more compacted conformation of the EPS molecules. Moreover, the alginate over-expression effect on EPS adherence was compared to the effect of alginate over-expression on P. aeruginosa cell attachment. In a parallel plate flow cell, under similar hydraulic and aquatic conditions as those applied for the EPS adsorption tests in the QCM-D flow cell, reduced adherence of the mucoid strain was clearly observed compared to the wild type isogenic bacteria. The results suggest that alginate contributes to steric hindrance and shielding of cell surface features and adhesins that are known to promote cell attachment. © 2011 Taylor & Francis

An alginate-antacid formulation localizes to the acid pocket to reduce acid reflux in patients with gastroesophageal reflux disease.

PubMed

Rohof, Wout O; Bennink, Roel J; Smout, Andre J P M; Thomas, Edward; Boeckxstaens, Guy E

2013-12-01

Alginate rafts (polysaccharide polymers that precipitate into a low-density viscous gel when they contact gastric acid) have been reported to form at the acid pocket, an unbuffered pool of acid that floats on top of ingested food and causes postprandial acid reflux. We studied the location of an alginate formulation in relation to the acid pocket and the corresponding effects on reflux parameters and acid pocket positioning in patients with gastroesophageal reflux disease (GERD). We randomly assigned patients with symptomatic GERD and large hiatal hernias to groups who were given either (111)In-labeled alginate-antacid (n = 8, Gaviscon Double Action Liquid) or antacid (n = 8, Antagel) after a standard meal. The relative positions of labeled alginate and acid pocket were analyzed for 2 hours by using scintigraphy; reflux episodes were detected by using high-resolution manometry and pH-impedance monitoring. The alginate-antacid label localized to the acid pocket. The number of acid reflux episodes was significantly reduced in patients receiving alginate-antacid (3.5; range, 0-6.5; P = .03) compared with those receiving antacid (15; range, 5-20), whereas time to acid reflux was significantly increased in patients receiving alginate-antacid (63 minutes; range, 23-92) vs those receiving antacid (14 minutes; range, 9-23; P = .01). The acid pocket was located below the diaphragm in 71% of patients given alginate-antacid vs 21% of those given antacid (P = .08). There was an inverse correlation between a subdiaphragm position of the acid pocket and acid reflux (r = -0.76, P < .001). In a study of 16 patients with GERD, we observed that the alginate-antacid raft localizes to the postprandial acid pocket and displaces it below the diaphragm to reduce postprandial acid reflux. These findings indicate the importance of the acid pocket in GERD pathogenesis and establish alginate-antacid as an appropriate therapy for postprandial acid reflux. Copyright © 2013 AGA Institute

Introduction of N-cadherin-binding motif to alginate hydrogels for controlled stem cell differentiation.

PubMed

Lee, Jae Won; An, Hyoseok; Lee, Kuen Yong

2017-07-01

Control of stem cell fate and phenotype using biomimetic synthetic extracellular matrices (ECMs) is an important tissue engineering approach. Many studies have focused on improving cell-matrix interactions. However, proper control of cell-cell interactions using synthetic ECMs could be critical for tissue engineering, especially with undifferentiated stem cells. In this study, alginate hydrogels were modified with a peptide derived from the low-density lipoprotein receptor-related protein 5 (LRP5), which is known to bind to N-cadherin, as a cell-cell interaction motif. In vitro changes in the morphology and differentiation of mouse bone marrow stromal cells (D1 stem cells) cultured in LRP5-alginate hydrogels were investigated. LRP5-alginate gels successfully induced stem cell aggregation and enhanced chondrogenic differentiation of D1 stem cells, compared to RGD-alginate gels, at low cell density. This approach to tailoring synthetic biomimetic ECMs using cell-cell interaction motifs may be critical in tissue engineering approaches using stem cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Design, fabrication and characterization of oxidized alginate-gelatin hydrogels for muscle tissue engineering applications.

PubMed

Baniasadi, Hossein; Mashayekhan, Shohreh; Fadaoddini, Samira; Haghirsharifzamini, Yasamin

2016-07-01

In this study, we reported the preparation of self cross-linked oxidized alginate-gelatin hydrogels for muscle tissue engineering. The effect of oxidation degree (OD) and oxidized alginate/gelatin (OA/GEL) weight ratio were examined and the results showed that in the constant OA/GEL weight ratio, both cross-linking density and Young's modulus enhanced by increasing OD due to increment of aldehyde groups. Furthermore, the degradation rate was increased with increasing OD probably due to decrement in alginate molecular weight during oxidation reaction facilitated degradation of alginate chains. MTT cytotoxicity assays performed on Wharton's Jelly-derived umbilical cord mesenchymal stem cells cultured on hydrogels with OD of 30% showed that the highest rate of cell proliferation belong to hydrogel with OA/GEL weight ratio of 30/70. Overall, it can be concluded from all obtained results that the prepared hydrogel with OA/GEL weight ratio and OD of 30/70 and 30%, respectively, could be proper candidate for use in muscle tissue engineering. © The Author(s) 2016.

Development of a cell culture surface conversion technique using alginate thin film for evaluating effect upon cellular differentiation

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

Nakashima, Y., E-mail: yuta-n@mech.kumamoto-u.ac.jp; Graduate School of Science and Engineering, Yamaguchi University, 2-16-1 Tokiwadai, Ube 755-8611; Tsusu, K.

2014-06-15

Here, we sought to develop a cell culture surface conversion technique that would not damage living cells. An alginate thin film, formed on a glass plate by spin coating of sodium alginate solution and dipping into calcium chloride solution, was used to inhibit adhesion of cells. The film could be removed by ethylenediaminetetraacetate (EDTA) at any time during cell culture, permitting observation of cellular responses to conversion of the culture surface in real time. Additionally, we demonstrated the validity of the alginate thin film coating method and the performance of the film. The thickness of the alginate thin film wasmore » controlled by varying the rotation speed during spin coating. Moreover, the alginate thin film completely inhibited the adhesion of cultured cells to the culture surface, irrespective of the thickness of the film. When the alginate thin film was removed from the culture surface by EDTA, the cultured cells adhered to the culture surface, and their morphology changed. Finally, we achieved effective differentiation of C2C12 myoblasts into myotube cells by cell culture on the convertible culture surface, demonstrating the utility of our novel technique.« less

Potential Use of Alginate-Based Carriers As Antifungal Delivery System

PubMed Central

Spadari, Cristina de Castro; Lopes, Luciana B.; Ishida, Kelly

2017-01-01

Fungal infections have become a major public health problem, growing in number and severity in recent decades due to an increase of immunocompromised patients. The use of therapeutic agents available to treat these fungal infections is limited by their toxicity, low bioavailability, antifungal resistance, and high cost of treatment. Thus, it becomes extremely important to search for new therapeutic options. The use of polymeric systems as drug carriers has emerged as a promising alternative to conventional formulations for antifungals. Alginate is a natural polymer that has been explored in the last decade for development of drug delivery systems due to its non-toxicity, biodegradability, biocompatibility, low cost, mucoadhesive, and non-immunogenic properties. Several antifungal agents have been incorporated in alginate-based delivery systems, including micro and nanoparticles, with great success, displaying promising in vitro and in vivo results for antifungal activities, reduction in the toxicity and the total drug dose used in the treatment, and improved bioavailability. This review aims at discussing the potential use and benefits of alginate-based nanocarriers and other delivery systems containing antifungal agents in the therapy of fungal infections. PMID:28194145

Alginate microencapsulation technology for the percutaneous delivery of adipose-derived stem cells.

PubMed

Moyer, Hunter R; Kinney, Ramsey C; Singh, Kimberly A; Williams, Joseph K; Schwartz, Zvi; Boyan, Barbara D

2010-11-01

Autologous fat is the ideal soft-tissue filler; however, its widespread application is limited because of variable clinical results and poor survival. Engineered fillers have the potential to maximize survival. Alginate is a hydrogel copolymer that can be engineered into spheres of <200 μm, thus facilitating mass transfer, allowing for subcutaneous injection, and protecting cells from shearing forces. Alginate powder was dissolved in saline, and adipose-derived stem cells (ADSCs) were encapsulated (1 million cells/mL) in alginate using an electrostatic bead generator. To assess effects of injection on cell viability, microspheres containing ADSCs were separated into 2 groups: the control group was decanted into culture wells and the injection group was mixed with basal media and injected through a 21-gauge needle into culture wells. Microbeads were cultured for 3 weeks, and cell number and viability were measured weekly using electron and confocal microscopy. To assess effects of percutaneous injection in vivo, twenty-four male nude mice were randomly separated into 2 groups and injected with either empty microcapsules or ADSC-laden microcapsules. Mice were harvested at 1 and 3 months, and the implants were examined microscopically to assess bead and cell viability. A flow rate of 5 mL/h and an electrostatic potential of 7 kV produced viable ADSC-laden microbeads of <200 μm. There were no differences in bead morphology and ADSC viability between microcapsules placed versus injected into tissue culture plates for up to 3 weeks. Microspheres implanted in a nude mouse model show durability up to 3 months with a host response around each individual sphere. ADSCs remained viable and showed signs of mitosis. ADSCs can be readily cultured, encapsulated, and injected in alginate microspheres. Stem cells suspended in alginate microspheres survive in vivo and are seen to replicate in vitro.