Evidence for voltage-dependent Cl− permeability in mouse pancreatic β-cells

Microdissected -cell-rich pancreatic islets fromob/ob-mice were used in studies of transmembrane³⁶Cl^– efflux. The mean rate coefficient for³⁶Cl^– efflux was stable at 0.158 min^–1 during the initial 10 min. Depolarization of the -cell plasma membrane by acute increases in extracellular K⁺ (5–130mM) stimulated the³⁶Cl^– efflux in a concentration-dependent manner. Glucose-induced (20mM) and K⁺-induced increases in³⁶Cl^– efflux were largely overlapping, but even at 135.9 mM K⁺, glucose slightly further enhanced the³⁶Cl^– efflux rate. The data suggest (1) that pancreatic -cells are equipped with a voltage-dependent Cl^– permeability, (2) that glucose-induced increase in Cl^– permeability may, at least partly, be mediated by primary membrane depolarization, and (3) that glucose in addition may activate other mechanisms for -cell Cl^– transport.     

Optimization of cellulase-free xylanase production by a novel yeast strain

Summary A novel yeast strain, NCIM 3574, isolated from a decaying wood produced up to 570 IU ml^–1 of xylanolytic enzymes when grown on medium containing 4% xylan. The yeast strain also produced xylanase activity (40–50 IU ml^–1) in the presence of soluble carbon sources like xylose or arabinose. No xylanase activity was detected when the organism was grown on glucose. The crude xylanase preparation showed no activity towards cellulolytic substrates but low levels of -xylosidase (0.1 IU ml^–1) and -l-arabinofuranosidase (0.05 IU ml^–1) were detected. The temperature and pH optima for the crude xylanase preparation were 55°C and 4.5 respectively. The crude xylanase produced mainly xylose from xylan within 5 min. Prolonged hydrolysis of xylan produced xylobiose and arabinose, in addition to xylose, as the end products. The presence of arabinose as one of the end products in xylan hydrolysate could be due to the low levels of arabinofuranosidase enzyme present in the crude fermentation broth.     

Liquefaction of dulse (Palmaria palmata (L.) Kuntze) by a commercial enzyme preparation and a purified endo ,β-1,4-D-xylanase

Liquefaction of dry and freshPalmaria palmata by food grade enzyme preparations and a purified endo--1,4-D-xylanase was studied.The endo--1,4-D-xylanase (EC 3.2.1.8) was purified to homogeneity from a commercial food grade enzyme prepared fromAspergillus niger. It has a molecular weight of 22 500, a pI of 3.5, is inactive toward corn arabinoxylan,p-nitrophenyl--D-xylose, carboxymethyl cellulose but shows a weak activity toward microcrystalline cellulose. It hydrolyzes oat and dulse xylan equally well in seawater and deionized water essentially into xylose and xylobiose. It is stable between pH 5.5 to 9.0 and 0 to 30 °C and its activity is optimal at pH 4.5–5.5 and 40–60 °C. It has a K_m of 2.2 and 2.8 mg ml^-1 and V_max of 3600 and 3900 nkat mg^-1 of protein on oat and dulse xylan, respectively.Acetate buffer, deionized water and seawater alone extracted 62.6 to 64.5 % of the dry weight of dry dulse, but the use of commercial food grade enzyme preparations or the purified xylanase improved liquefaction to 81.2–87.1 %. Xylose and galactose were the only sugars present in the soluble extracts. Deionized and seawater extracted 58.8–52.7 and 39.1–42.2% of the dry weight of the fresh algae collected in fall and summer, respectively. Only galactose was found in the seawater extract, while some xylose with galactose were measured in the deionized water extract of the fresh autumn algal sample. Purified and crude xylanase improved liquefaction of fresh algae to 79.8–81.4 and 71.9–77.9% of the fresh dry weight (fall and summer, respectively) in deionized and seawater, respectively, and increased the xylose content of the soluble fractions. Polysaccharides in the soluble residues were composed of 1,3/1,4-linked xylose, 1-linked galactose (floridoside) and 1,4-linked glucose (cellulose) and contained essentially 1,4-linked xylose and 1,4-linked glucose in insoluble fractions obtained after enzymatic treatment.The use of xylanase-containing food grade enzyme preparations improves liquefaction ofPalmaria palmata, particularly from fresh alga. This study indicates that processing such as drying may modify markedly the solubility ofP. palmata cell wall polysaccharides, which would imply the existence of some organization and/or other components in the fresh cell wall that lower xylan solubility in seawater.     

Identification of a GDP-Fuc:Galβ1–3GalNAc-R (Fuc to Gal)α1–2 fucosyltransferase and a GDP-Fuc:Galβ1–4GlcNAc (Fuc to GlcNAc)α1–3 fucosyltransferase in connective tissue of the snailLymnaea stagnalis

Connective tissue of the freshwater pulmonateLymnaea stagnalis was shown to contain fucosyltransferase activity capable of transferring fucose from GDP-Fuc in 1–2 linkage to terminal Gal of type 3 (Gal1–3GalNAc) acceptors, and in 1–3 linkage to GlcNAc of type 2 (Gal1–4GlcNAc) acceptors. The 1–2 fucosyltransferase was active with Gal1–3GalNAc1-OCH₂CH=CH₂ (K _m=12 mM,V _max=1.3 mU ml^–1) and Gal1–3GalNAc (K _m=20 mM,V _max=2.1 mU ml^–1), whereas the 1–3 fucosyltransferase was active with Gal1–4GlcNAc (K _m=23 mM,V _max=1.1 mU ml^–1). The products formed from Gal1–3GalNAc1-OCH₂CH=CH₂ and Gal1–4GlcNAc were purified by high performance liquid chromatography, and identified by 500 MHz¹H-NMR spectroscopy and methylation analysis to be Fuc1–2Gal1–3GalNAc1-OCH₂CH=CH₂ and Gal1–4(Fuc1–3)GlcNAc, respectively. Competition experiments suggest that the two fucosyltransferase activities are due to two distinct enzymes.Abbreviations 2Fuc-T 1–2 fucosyltransferase - 3Fuc-T 1–3 fucosyltransferase - MeO-3Man 3-O-methyl-D-mannose - MeO-3Gal 3-O-methyl-D-galactose     

A cellulosic exopolysaccharide produced from sugarcane molasses by a Zoogloea sp.

An extracellular polysaccharide producing bacterium Zoogloea sp. was isolated from an agro-industrial environment in the north-eastern region of Brazil. The extracellular polysaccharide produced from sugarcane molasses was hydrolysed with trifluoroacetic acid (mild and strong conditions) giving 88% of soluble material. The main monosaccharides present in the soluble fraction were glucose (87.6%), xylose (8.6%), mannose (0.8%), ribose (1.7%), galactose (0.1%), arabinose (0.4%) and glucuronic acid (0.8%). Methylation analysis of the polysaccharide showed mainly 2,3,6-tri-O-methylhexitol (74.7%) and 2,3,-di-O-methylhexitol (17.7%). Enzyme hydrolysis of the polysaccharide with a cellulase confirmed the presence of (1→4)-β- -glucopyranosyl residues.     

Acute modulation of myocardial function by angiotensin 1–7

Angiotensin 1–7 is a bioactive heptapeptide of the renin–angiotensin system. Its cardiovascular actions have recently acquired growing relevance, mainly due to its counter-regulatory actions in the angiotensin cascade. The aim of the present study was to evaluate the actions of angiotensin 1–7 on myocardial function. Increasing concentrations of angiotensin 1–7 (10⁻⁹ to 10⁻⁵ M) were added to rabbit right papillary muscles: (1) in baseline conditions with intact endocardial endothelium (EE); (2) after selective removal of the EE with Triton X-100 (1 s, 0.01%); (3) with intact EE in the presence of the Mas receptor antagonist A-779, the AT₁ receptor antagonist ZD-7155, the AT₂ receptor antagonist PD-123,319 or the nitric oxide synthesis inhibitor NG-nitro-l-arginine (l-NA). Concerning the effects on contractility, we observed a significant decrease on active tension, dT/dt_max, peak shortening and dL/dt_max of −10.5 ± 3.6%, −8.0 ± 3.0%, −5.3 ± 2.6% and −5.7 ± 2.3%, respectively. There was no change on relaxation parameters, namely dT/dt_min or dL/dt_min. Time to half relaxation was significantly decreased. The presence of ZD-7155 or PD-123,319 did not change these effects. However, angiotensin 1–7 effects on myocardial properties were abolished after selective EE removal and in the presence of A-779 or l-NA. In conclusion, in this animal species, angiotensin 1–7 through its binding to Mas receptor induces a negative inotropic effect modulated by the EE and nitric oxide and independent of AT₁ or AT₂ receptors activation. As the effects described in the present work were influenced by the endocardial endothelium, they may be disrupted in situations associated to endothelial dysfunction, as in heart failure or myocardial ischemia.     

Thiolation and nitrosation of cysteines in biological fluids and cells

Summary. Thiols (RSH) are potent nucleophilic agents, the rates of which depend on the pKa of the sulfhydryl. Unlike compounds having other nucleophile moieties (–OH or –NH₂), RSH are involved in reactions, such as conjugations, redox and exchange reactions. Although protein SH groups (PSH) react like non-protein thiols (NPSH), the biochemistry of proteins is much more complex for reasons such as steric hindrance, charge distribution and accessibility of PSH to the solvent (protein conformation). The reaction rates and types of end-products of PSH vary a lot from protein to protein. The biological problem is even more complex because in all compartments and tissues, there may be specific competition between thiols (namely between GSH and PSH), regulated by the properties of antioxidant enzymes. Moreover, PSH are divided biologically into essential and non-essential and their respective influence in the various biological systems is unknown. It follows that during phenomena eliciting a prompt thiol response (oxidative stress), the antioxidant PSH response and reaction mechanisms vary considerably from case to case. For example, in spite of a relatively low pKa that should guarantee good antioxidant capacity, PSH of albumin has much less propensity to form adducts with conjugating agents than NPSH; moreover, the structural characteristics of the protein prevent albumin from forming protein disulfides when exposed to oxidants (whereas protein-thiol mixed disulfides are formed in relative abundance). On the other hand, proteins with a relatively high reactivity, such rat hemoglobin, have much greater antioxidant capacity than GSH, but although human hemoglobin has a pKa similar to GSH, for structural reasons it has less antioxidant capacity than GSH.When essential PSH are involved in S-thiolation and S-nitrosation reactions, a similar change in biological activity is observed. S-thiolated proteins are a recurrent phenomenon in oxidative stress elicited by reactive oxygen species (ROS). This event may be mediated by disulfides, that exchange with PSH, or by the protein intermediate sulfenic acid that reacts with thiols to form protein-mixed disulfides. During nitrosative stress elicited by reactive nitrogen species (RNS), depending on the oxygen concentration of the system, nitrosation reactions of thiols may also be accompanied by protein S-thiolation. In this review we discuss a number of cell processes and biochemical modifications of enzymes that indicate that S-thiolation and S-nitrosation may occur simultaneously in the same protein in the presence of appropriate interactions between ROS and RNS.     

Glucose repression of xylitol production in Candida tropicalis mixed-sugar fermentations

Glucose repressed xylose utilization inCandida tropicalis pre-grown on xylose until glucose reached approximately 0–5 g l^–1. In fermentations consisting of xylose (93 g l^–1) and glucose (47 g l^–1), xylitol was produced with a yield of 0.65 g g^–1 and a specific rate of 0.09 g g^–1 h^–1, and high concentrations of ethanol were also produced (25 g l^–1). If the initial glucose was decreased to 8 g l^–1, the xylitol yield (0.79 g g^–1) and specific rate (0.24 g g^–1 h^–1) increased with little ethanol formation (<5 g l^–1). To minimize glucose repression, batch fermentations were performed using an aerobic, glucose growth phase followed by xylitol production. Xylitol was produced under O₂ limited and anaerobic conditions, but the specific production rate was higher under O₂ limited conditions (0.1–0.4 vs. 0.03 g g^–1 h^–1). On-line analysis of the respiratory quotient defined the time of xylose reductase induction.     

Molecular cloning and expression in yeast of 2,3–oxidosqualene– triterpenoid cyclases from Arabidopsis thaliana

A vast array of triterpenes are found in living organisms in addition to lanosterol and cycloartenol, which are involved in sterol biosynthesis in non–photosynthetic and photosynthetic eukaryotes respectively. The chemical structure of these triterpenes is determined by a single step catalysed by 2,3–oxidosqualene–triterpene cyclases. The present study describes cloning and functional expression in yeast of several OS–triterpene cyclases. Three Arabidopsis thaliana cDNAs encoding proteins (ATLUP1, ATLUP2, ATPEN1) 57%, 58% and 49% identical to cycloartenol synthase from the same plant were isolated. Expression of these cDNAs in yeast showed that the recombinant proteins catalyse the synthesis of various pentacyclic triterpenes. Whereas ATLUP1 is essentially involved in the synthesis of lupeol, ATLUP2 catalyses the production of lupeol, – and –amyrin (in a 15:55:30 ratio). ATLUP2 is therefore a typical multifunctional enzyme. Under the same conditions, ATPEN1 did not lead to any product. Systematic sequencing of the Arabidopsis genome has led to genomic sequences encoding proteins identical to the above triterpene synthases. ATLUP1 and ATLUP2 are representative of a small subfamily (A) of at least five genes, whereas ATPEN1 is representative of a subfamily (B) of at least seven genes. The number of introns is characteristic of each subfamily. Whereas genes of family A possess 17 exons and 16 introns, genes of the subfamily B contain 14 exons and 13 introns. The size of each exon is remarkably conserved within each subfamily whereas that of each intron appears to be highly variable. Organization of the genes, sequences and functions of the deduced proteins are discussed in evolutionary terms.     

Apoplastic expression of the xylanase and β(1–3, 1–4) glucanase domains of the xyn D gene from Ruminococcus flavefaciens leads to functional polypeptides in transgenic tobacco plants

We are investigating the possibilities of transgenic plants as bioreactors for the production of industrial enzymes using cell wall-hydrolysing enzymes as first examples. Within the frame work of this work two distinct domains of the xynD gene from Ruminococcus flavefaciens encoding a xylanase (XYLD-A) and a (1–3, 1–4)glucanase (XYLD-C) were separately cloned into a plant expression vector which would target the proteins into the apoplast. Transgenic tobacco plants were obtained expressing xylan-hydrolysing as well as lichenan-hydrolysing activities. Despite similar steady-state levels of the respective mRNAs xylan hydrolysis rates were between 40 and 170 mol min–1 m^–2 leaf area depending on the transgenic plant while (1–3, 1–4)glucan degradation was much more effective ranging between 200 and 2000 mol min^–1 m^–2. The high activity levels of the XYLD-C expressing plants were reflected on the protein level. XYLD-C accumulated in the intercellular space and was one of the most prominent bands in protein gels. Despite their apoplastic location as confirmed by activity measurements using intercellular fluids the transgenic plants had not undergone any phenotypic alteration.     

Male-sterile mutation alters Zea m 1 (β-expansin 1) accumulation in a maize mutant

gaMS-2 is a gametophytic male-sterile mutant of maize, with sterile pollen grains developmentally blocked at the binucleate stage. To characterise differentially expressed proteins in gaMS-2 pollen, we compared protein profiles of anthers and mature pollen from heterozygous GaMS-2/gaMS-2 plants and wild type (wt) plants by two-dimensional electrophoresis (2-DE). A basic protein present at a greatly reduced level in GaMS-2/gaMS-2 anthers was subsequently identified by tandem mass spectrometry as Zea m 1 (a glycoprotein of 31 kDa), the major group-1 allergen of maize pollen and a member of the -expansin 1 family. Moreover, we show that Zea m 1 can be deglycosylated by peptide N-glycosidase F. After deglycosylation, four major isoforms—Zea m 1a (more acetic), Zea m 1b, Zea m1c and Zea m 1d (more basic)—can be discriminated in wt anther in 2-DE immunoblots probed with a monoclonal antibody against the group-1 pollen allergen, whereas all the isoforms, especially Zea m 1a, exist at reduced levels in GaMS-2/gaMS-2 anthers. Furthermore, the reduced Zea m 1 accumulation in the mutant appears to occur in immature pollen but not in anther sporophytic tissues. Finally, we separated sterile pollen grains (at the mononucleate stage) from fertile ones using 42% Percoll solution, and found that Zea m 1 is barely detectable in sterile pollen grains. Together, our results indicate that a reduced Zea m 1 level is associated with the sterile phenotype of gaMS-2.W. Wang and M. Scali contributed equally to this study     

Xylose chemostat isolates of Saccharomyces cerevisiae show altered metabolite and enzyme levels compared with xylose, glucose, and ethanol metabolism of the original strain

The efficient conversion of xylose-containing biomass hydrolysate by the ethanologenic yeast Saccharomyces cerevisiae to useful chemicals such as ethanol still remains elusive, despite significant efforts in both strain and process development. This study focused on the recovery and characterization of xylose chemostat isolates of a S. cerevisiae strain that overexpresses xylose reductase- and xylitol dehydrogenase-encoding genes from Pichia stipitis and the gene encoding the endogenous xylulokinase. The isolates were recovered from aerobic chemostat cultivations on xylose as the sole or main carbon source. Under aerobic conditions, on minimal medium with 30 g l^–1 xylose, the growth rate of the chemostat isolates was 3-fold higher than that of the original strain (0.15 h^–1 vs 0.05 h^–1). In a detailed characterization comparing the metabolism of the isolates with the metabolism of xylose, glucose, and ethanol in the original strain, the isolates showed improved properties in the assumed bottlenecks of xylose metabolism. The xylose uptake rate was increased almost 2-fold. Activities of the key enzymes in the pentose phosphate pathway (transketolase, transaldolase) increased 2-fold while the concentrations of their substrates (pentose 5-phosphates, sedoheptulose 7-phosphate) decreased correspondingly. Under anaerobic conditions, on minimal medium with 45 g l^–1 xylose, the ethanol productivity (in terms of cell dry weight; CDW) of one of the isolates increased from 0.012 g g^–1 CDW h^–1 to 0.017 g g^–1 CDW h^–1 and the yield from 0.09 g g^–1 xylose to 0.14 g g^–1 xylose, respectively.     

Synthesis of novel 3-pyridinecarbonitriles with amino acid function and their fluorescence properties

Summary. A variety of N-[(4,6-diaryl-3-pyridinecarbonitrile)-2-yl] amino acid esters 2–4 were synthesized through the reaction of 2-bromo-3-pyridinecarbonitriles 1 with the appropriate -amino acid ester hydrochloride in refluxing dioxane in the presence of triethylamine as dehydrohalogenating agent. Similarly, N-glycylglycine analogues 5 were obtained through the reaction of 1 with the dipeptide ester. On the other hand, attempts were made towards the construction of amino acid derivatives 7 through the reaction of 1 with aqueous solution -amino acids 6 in refluxing pyridine, but were unsuccessful, and instead the unexpected 2-amino-3-pyridinecarbonitriles 8 were isolated. The fluorescence properties of the newly synthesized pyridines 2–5 were evaluated. Some of the prepared compounds show considerable antibacterial activity.     

Purification and partial characterization of ad(−)-lactate dehydrogenase fromDesulfovibrio desulfuricans (ATCC 7757)

Summary A membrane-boundd(–)-lactate dehydrogenase (LDH), an important enzyme in carbon and energy metabolism in sulfate-reducing bacteria of the genusDesulfovibrio, was solubilized from the membrane fraction ofDesulfovibrio desulfuricans (ATCC 7757). The enzyme was purified 84-fold to a final specific activity of 525 nmol DCPIP-reduced/min/mg protein by ammonium sulfate precipitation, chloroform extraction, gel filtration with Sephadex G-150, and hydrophobic column chromatography withN-octylamine Sepharose 4B. The enzyme eluted off a Sephacryl S-300 column as a single peak with a molecular weight of 400 000±40 000 Da. Denaturing gel electrophoresis showed it to be composed of 5 protein bands. The oxidized and dithionite reduced spectra of LDH resembles the spectra ofc-type cytochromes found inDesulfovibrio species. The addition of lactate to LDH resulted in a partially reduced spectrum. The flavin/cytochromec/non-heme iron content per 400 000 Da LDH molecular weight was found to be 11.64.5. The LDH activity was specific ford(–)-lactate and had aK _m ford(–)-lactate of 4.3×10^–4 M. The pH optimum was between 6.5 and 8.5.     

Immobilization of glucosyltransferase fromAureobasidium

Summary Glucosyltransferase fromAureobasidium, which produces panose and isomaltose from maltose, was immobilized by alginate gel or DEAE-cellulose at high efficiency (71 and 41% respectively). Alkylamine porous silica was less efficient as a support. The enzymatic profiles of immobilized enzymes were almost identical to the native one except that their stabilities to extreme pH, metal ions and inhibitors were improved. Both immobilization procedures successfully produced high amounts of panose, 125 mg ml^–1 (alginate gel) or 141 mg ml^–1 (DEAE-cellulose), from 300 mg ml^–1 of maltose.     

Heterooligosaccharide synthesis catalyzed by α-glucosidase from Bacillus stearothermophilus

α-Glucosidase from Bacillus stearothermophilus was used as a catalyst for oligosaccharide synthesis by reversed hydrolysis. The yield of disaccharides and trisaccharides depended strongly on the units of enzyme activity added, and on the stability of the enzyme under reaction conditions. When glucose was the only saccharide present in the reaction mixture with α-glucosidase, isomaltose (51%), nigerose (25%), maltose (14%) and kojibiose (10%) were formed. In 50% glucose solution, disaccharide concentrations reached up to 400 mmol/l and trisaccharides were also produced. When other saccharides (mannose or xylose), in addition to glucose, were present in the reaction mixture, both homodisaccharides and heterodisaccharides were formed, their quantity being dependent on the glucose/saccharide acceptor ratios. The highest yields of oligosaccharides were observed with glucose alone, consistent with the observation that the enzyme stability was highest with glucose as the sole saccharide.     

Stimulatory effect of β-alanyl-L-histidinato zinc on cell proliferation is dependent on protein synthesis in osteoblastic MC3T3-E1 cells

The effect of -alanyl-L-histidinato zinc (AHZ) on bone metabolism was investigated in osteoblastic MC3T3-El cells. Cells were cultured for 3 days at 37°C in a CO₂ incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus various concentrations of AHZ or other reagents, and the cells were cultured further for appropriate periods of time. The presence of AHZ (10^–7–10^–5M) stimulated the proliferation of cells. AHZ (10^–6 and 10^–5M) increased deoxyribonucleic acid (DNA) content in the cells with 48hr-culture. This increase was completely blocked by the presence of cycloheximide (10^–6M) or hydroxyurea (10^–3M). Also, the presence of cycloheximide (10^–6M) completely inhibited the AHZ (10^–5M)-induced increase in the proliferation of cells. Meanwhile, parathyroid hormone (10^–7M), estrogen (10^–9M) and insulin (10^–M) significantly increased cellular DNA content. However, these hormonal effects clearly lowered in comparison with that of AHZ (10^–5M). Dibutyryl cyclic AMP (10^–4M) and zinc sulfate (10^–5M) did not cause a significant increase in cellular DNA content. The present results support the view that AHZ has a direct specific proliferative effect on osteoblastic cellsin vitro and that this effect is dependent on protein synthesis.     

Xerotolerance in fission yeasts and the role of glycerol as compatible solute

The effect on growth of reducing the water activity (a _w) of a medium with various solutes has been investigated for 27 strains of fission yeasts (Schizosaccharomyces). The minimum-tolerated a _w (MTA) was dependent on both the nature of the solute and the species. When the strains of each species were grouped together, the lowest mean MTA values were found with glucose, fructose or glycerol as stressing solutes, being in the range 0.89–0.90 for S. pombe, S. malidevorans, S. octosporus and S. slooffiae, but in the range 0.92–0.94 for S. japonicus. With the non-metabolizable sugars sorbose and xylose and the salts NH₄Cl, KCl, and NaCl, the mean MTA values were in the range 0.96–0.985, except for (1) the single strain of S. slooffiae, which was more tolerant of NH₄Cl and KCl with values of 0.95 and 0.94, respectively, and (2) the strains of S. pombe, S. malidevorans and S. japonicus, which were less tolerant of NaCl with mean values of about 0.99. One strain of each species was examined for intracellular solutes when actively growing in the presence of near-limiting concentrations of stressing solute. With glucose, fructose or glycerol, all five strains contained substantial amounts of glycerol but no other polyol; with the other solutes no glycerol or other polyol was found, except for small amounts of glycerol in strains of S. octosporus and S. slooffiae stressed with NH₄Cl, KCl, or NaCl.Abbreviations MTA Minimum-tolerated water activity - a _w water activity - YEPG yeast extract, phosphate, glucose medium     

Effect of β-alanyl-L-histidinato zinc on protein components in osteoblastic MC3T3-El cells: Increase in osteocalcin,insulin-like growth factor-I and transforming growth factor-β

The effect of -alanyl-L-histidinato zinc (AHZ) on protein components in osteoblastic MC3T3-E1 cells was investigated. Cells were cultured for 3 days at 37°C in CO₂ incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus various concentrations of AHZ or other reagents, and the cells were cultured further 3 or 6 days. The homgenate of cells was analyzed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The presence of AHZ (10^–7 to 10^–5 M) caused an appreciable increase of many protein components in cells. Especially, the 67 killo-dalton (kDa) and 44 kDa proteins which are the major components from control cells were clearly increased by the presence of AHZ. Furthermore, the concentrations of osteocalcin, insulin-like growth factor-I and transforming growth factor- in the culture medium secreted from osteoblastic cells were markedly increased by the presence of AHZ (10^–6 and 10^–5 M). The effect of AHZ was a greater than that of zinc sulfate (10^–6 and 10^–5 M). The present findings suggest that AHZ can increase many proteins which are involved in the stimulation of bone formation and cell proliferation in osteoblastic cells.     

Biosynthesis of cyclic β-(1–3),β-(1–6) glucan in Bradyrhizobium spp.

Inner membranes of Bradyrhizobium japonicum strain USDA 110 produced in vitro soluble and insoluble -(1–3),-(1–6) glucans. The reaction proceeded through a 90 kDa inner membrane intermediate protein; used UDP-glucose as sugar donor and required Mg²⁺. Gel chromatography of soluble glucans resolved a cyclic -(1–3) glucan with a degree of polymerization of eleven from a family of -(1–3),-(1–6) glucans with variable degree of polymerization higher than eleven. Bradyrhizobium strains BR4406 and BR8404 isolated from tree legume nodules in Southeast Brazil produce -(1–3),-(1–6) glucans very similar to that of B. japonicum. A 100 kDa protein was identified in these strains as intermediates in the synthesis of these glucans. Inner membranes of B. japonicum USDA110, B. japonicum I17, and Bradyrhizobium strains BR4406 and BR8404 incubated with UDP-glucose were unable to synthesize -(1–2) glucan and lacked the 235 kDa intermediate protein known to be involved in the synthesis of -(1–2) glucan in Agrobacterium tumefaciens, Rhizobium meliloti and Rhizobium loti.Abbreviations EPS= exopolysaccharides - CPS= capsular polysaccharides - LPS= lipopolysaccharides - AMA= Yeast extract-mannitol medium - TY= tryptone-yeast extract - PMSF= phenyl methyl sulfonil fluoride

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