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1.
The cell volume regulation of the lower segment cells of the Malpighian tubule of Rhodnius neglectus in anisosmotic media was evaluated by using videooptic techniques. When the medium osmolality was increased with addition of 100 mm mannitol the cells shrank to a minimum of 16.84±2.62% and subsequently swelled towards their initial volume undergoing a typical regulatory volume increase (RVI). Replacement of either K+ or Cl? or HCO 3 ? by Na+, gluconate and phosphate, respectively, abolished the RVI response. Furthermore, the substitution of Na+ by tetramethylammonium (TMA+) in isosmotic conditions led to cellular swelling and death. Addition of either amiloride 10?4 m, anthracene-9-COOH 5×10?4 m, furosemide 5×10?4 m or ethacrynic acid 5×10?5 m, also abolished RVI. On the other hand, addition of either Ba2+ 10?3 m, SITS 5× 10?4 m, ouabain 10?3 m or vanadate 10?3 m, did not change the RVI response. When the tubules were incubated in hyperosmotic media with EGTA 2 mm or verapamil 10?6 m, the RVI response was abolished. In contrast, a decrease of NaCl concentration from 129 to 79 mm induced a cell swelling to a maximum of 33.11+1.73%, but the cells maintained swollen, only partially regulating their volume. These results show that the proximal cells of Malpighian tubule of R. neglectus are able to regulate their volume in hyperosmotic but only partially regulating in hyposmotic solutions. The mechanisms in RVI involve Na+, K+, Cl?, Ca2+ and HCO 3 ? transport pathways and a ouabain-insensitive ATPase stimulated by Na+. This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP; Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq e Financiadora de Projetos e Pesquisas-FINEP.  相似文献   

2.
The uptake ofl-andd-aspartate was studied in astrocytes cultured from prefrontal cortex and in granule cells cultured from cerebellum. A high affinity uptake system forl- andd-aspartate was found in both cell types, and the two stereoisomers exhibited essentially the sameK m - andV max -values in bouth astrocytes (l-aspartate:K m 77 μM;V max 11.8 nmol×min?1×mg?1;d-aspartate:K m 83 μM;V max 14.0 nmol×min?1×mg?1) and granule cells (l-aspartate:K m 32 μM;V max 2.8 nmol ×min?1×mg?1;d-aspartate:K m 26 μM;V max 3.0 nmol×min?1×mg?1). To investigate whetherl-glutamate,l-aspartate andd-aspartate use the same uptake system a detailed kenetic analysis was performed. The uptake kinetics of each one of the three amino acids was studied in the presence of the two other amino acids, and no essential differences between the uptake characteristics of the amino acids were found. In addition to the uptake studies the release ofD-aspartate from cerebellar granule cells was investigated and compared withl-glutamate release. A Ca2+-dependent, K+-induced release was found for both amino acids.  相似文献   

3.
Uptake of amino acids by actidione-treated yeast cells   总被引:1,自引:0,他引:1  
The active uptake ofl-aspartic acid, glycine andl-lysine by actidione-treated cells ofSaccharomyces cerevisiae was found to be inhibited by anaerobic conditions in the absence of a source of energy, only facilitated diffusion persisting. Similarly, metabolic inhibitors (iodoacetamide, sodium fluoride and potassium sorbate) inhibited the uptake very substantially. 2,4-Dinitrophenol and sodium azide appeared to inhibit the movement of the transport carrier itself, while uranyl ions showed a complex interaction pattern, ranging from inhibition at concentrations of 10?6–10?4 m, to stimulation at concentrations of 3×10?4–10?3 m, to pronounced inhibition at higher concentrations. The uptake was pH-dependent with optima forl-aspartic acid near pH 4, for glycine near pH 5, forl-lysine near pH 6.5.  相似文献   

4.
This investigation was prompted by the findings that (1) dibucaine-resistant homozygotes and heterozygotes for plasmacholinesterase also exhibit resistance to fluoride inhibition, (2) the differentiation of dibucaine-resistant from the fluoride-resistant genotypes is ambiguous with the method of Harris and Whittaker, (3) the plasmacholinesterase inhibition by Na fluoride (FN) is markedly influenced by the temperature. Therefore, we modified their method by increasing (1) the temperature of the reaction from 25C to 37C and (2) the concentration of Na fluoride from 5.0×10?5 m to 2.5×10?4 m. With this method, genetically normal individuals have a mean FN±sd=77.0±3.22 while atypical dibucaine-resistant homozygotes have a mean FN±sd=43.0±10.0 and atypical dibucaine-resistant heterozygotes 67.0±5.37. Since a linear correlation was observed between DN and FN by our new method, a fluoride number 2 sd lower than the predicted FN from the DN can distinctly identify the fluoride-resistant plasmacholinesterase genotype E 1 f .  相似文献   

5.
d-galactose is an attractive substrate for bioconversion. Herein, Escherichia coli was metabolically engineered to convert d-galactose into d-galactonate, a valuable compound in the polymer and cosmetic industries. d-galactonate productions by engineered E. coli strains were observed in shake flask cultivations containing 2 g L?1 d-galactose. Engineered E. coli expressing gld coding for galactose dehydrogenase from Pseudomonas syringae was able to produce 0.17 g L?1 d-galactonate. Inherent metabolic pathways for assimilating both d-galactose and d-galactonate were blocked to enhance the production of d-galactonate. This approach finally led to a 7.3-fold increase with d-galactonate concentration of 1.24 g L?1 and yield of 62.0 %. Batch fermentation in 20 g L?1 d-galactose of E. coli ?galK?dgoK mutant expressing the gld resulted in 17.6 g L?1 of d-galactonate accumulation and highest yield of 88.1 %. Metabolic engineering strategy developed in this study could be useful for industrial production of d-galactonate.  相似文献   

6.
Activation of the human red cell calcium ATPase by calcium pretreatment   总被引:1,自引:0,他引:1  
Some kinetic parameters of the human red cell Ca2+-ATPase were studied on calmodulin-free membrane fragments following preincubation at 37°C. After 30 min treatment with EGTA(1 mm) plus dithioerythritol (1 mm), a V max of about 0.4 μmol Pi/mg × hr and a K s of 0.3 μm Ca2+ were found. When Mg2+ (10 mm) or Ca2+(10 μm) were also added during preincubation, V maxbut not Kwas altered. Ca2+ was more effective than Mg2+, thus increasing V max to about 1.3 μmol Pi/mg × hr. The presence of both Ca2+ and Mg2+ during pretreatment decreasedKto 0.15 μm, while having no apparent effect on V max. Conversely, addition of ATP (2 mm) with either Ca2+ or Ca2+ plus Mg2+increased Vmax without affecting K. Preincubation with Ca2+ for periods longer than 30 min further increased Vmaxand reduced Kto levels as low as found with calmodulin treatment. The Ca2+ activation was not prevented by adding proteinase inhibitors (iodoacetamide, 10 mm; leupeptin, 200 μm; pepstatinA, 100 μm; phenylmethanesulfonyl fluoride, 100 μm). The electrophoretic pattern of membranes preincubated with or without Mg2+, Ca2+ or Ca2+ plus Mg2+ did not differ significantly from each other. Moreover, immunodetection of Ca2+-ATPase by means of polyclonal antibodiesrevealed no mobility change after the various treatments. The above stimulation was not altered by neomycin (200 μm), washing with EGTA (5 mm) or by both incubating and washing with delipidized serum albumin (1 mg/ml), or omitting dithioerythritol from the preincubation medium. On the other hand, the activation elicited by Ca2+ plus ATP in the presence of Mg2+ was reduced 25–30% by acridine orange (100 μm), compound 48/80 (100 μm) or leupeptin (200 μm) but not by dithio-bis-nitrobenzoic acid (1 mm). The fluorescence depolarization of 1,6-diphenyl-and l-(4-trimethylammonium phenyl)-6-phenyl 1,3,5-hexatriene incorporated into membrane fragments was not affected after preincubating under the different conditions. The results show that proteolysis, fatty acid production, an increased phospholipid metabolism or alteration of membrane fluidity are not involved in the Ca2+ effect. Ca2+ preincubation may stimulate the Ca2+-ATPase activity by stabilizing or promoting the E1 conformation.  相似文献   

7.
Small-conductance chloride channels in human peripheral T lymphocytes   总被引:4,自引:0,他引:4  
During whole-cell patch-clamp recording from normal (nontransformed) human T lymphocytes a chloride current spontaneously activated in >98% of cells (n > 200) in the absence of applied osmotic or pressure gradients. However, some volume sensitivity was observed, as negative pressure pulses reduced the current. With iso-osmotic bath and pipette solutions the peak amplitude built up (time constant ≈23 sec at room temperature), a variable-duration plateau phase followed, then the current ran down spontaneously (time constant ≈280 sec). The anion permeability sequence, calculated from reversal potentials was I?, Br? > NO 3 ? , Cl? > CH3SO 3 ? , HCO 3 ? > CH3COO? > F? > aspartate, gluconate, SO 4 2? and there was no measurable monovalent cation permeability. The Cl? current was independent of time during long voltage steps and there was no evidence of voltage-dependent gating; however, the current showed intrinsic outward rectification in symmetrical Cl? solutions. The conductance of the channels underlying the whole-cell current was calculated from fluctuation analysis, using power-spectral density and variance-vs.-mean analysis. Both methods yielded a single channel conductance of about 0.6 pS at ?70 mV (close to the normal resting potential of T lymphocytes). The power spectral density function was best fit by the sum of two Lorentzian functions, with corner frequencies of 30 and 295 Hz, corresponding to mean open times of 0.54 and 5.13 msec. The pharmacological profile included rapid block by external application of flufenamic acid (50 μm), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 100 μm, [6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5-y1) oxy] acetic acid (IAA-94, 250 μm) or 100 μm 1,9-dideoxyforskolin. The stilbene derivatives DIDS (4,4′-diisothiocyano-2,2′ di-sulphonic acid stilbene, 500 μm) and SITS (4-acetamido-4′-isothiocyano-2, 2′-disulphonic acid stilbene, 500 μm) prevented buildup of Cl? current after a 30-min preincubation at 500 μm. When tested in a mitogenic assay, DIDS, flufenamic acid, NPPB and IAA-94 all inhibited T-cell proliferation, suggesting a physiological function in addition to the observed volume sensitivity.  相似文献   

8.
A recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus was purified as a single 68 kDa band with an activity of 76 U mg?1. The molecular mass of the native enzyme was 204 kDa as a trimer. The maximum activity for l-fucose isomerization was at pH 7 and 75°C in the presence of 1 mM Mn2+. Its half-life at 70°C was 6.1 h. For aldose substrates, the enzyme displayed activity in decreasing order for l-fucose, with a k cat of 11,910 min?1 and a K m of 140 mM, d-arabinose, d-altrose, and l-galactose. These aldoses were converted to the ketoses l-fuculose, d-ribulose, d-psicose, and l-tagatose, respectively, with 24, 24, 85, 55% conversion yields after 3 h.  相似文献   

9.
Bifidobacterium longum NRRL B-41409 l-arabinose isomerase (l-AI) was overexpressed in Lactococcus lactis using a phosphate depletion inducible expression system. The resting L. lactis cells harboring the B. longum l-AI were used for production of d-tagatose from d-galactose in the presence of borate buffer. Multivariable analysis suggested that high pH, temperature and borate concentration favoured the conversion of d-galactose to d-tagatose. Almost quantitative conversion (92 %) was achieved at 20 g L?1 substrate and at 37.5 °C after 5 days. The d-tagatose production rate of 185 g L?1 day?1 was obtained at 300 g L?1 galactose, at 1.15 M borate, and at 41 °C during 10 days when the production medium was changed every 24 h. There was no significant loss in productivity during ten sequential 24 h batches. The initial d-tagatose production rate was 290 g L?1 day?1 under these conditions.  相似文献   

10.
d-Tagatose 3-epimerase family enzymes can efficiently catalyze the epimerization of free keto-sugars, which could be used for d-psicose production from d-fructose. In previous studies, all optimum pH values of these enzymes were found to be alkaline. In this study, a d-psicose 3-epimerase (DPEase) with neutral pH optimum from Clostridium bolteae (ATCC BAA-613) was identified and characterized. The gene encoding the recombinant DPEase was cloned and expressed in Escherichia coli. In order to characterize the catalytic properties, the recombinant DPEase was purified to electrophoretic homogeneity using nickel-affinity chromatography. Ethylenediaminetetraacetic acid was shown to inhibit the enzyme activity completely; therefore, the enzyme was identified as a metalloprotein that exhibited the highest activity in the presence of Co2+. Although the DPEase demonstrated the most activity at a pH ranging from 6.5 to 7.5, it exhibited optimal activity at pH 7.0. The optimal temperature for the recombinant DPEase was 55 °C, and the half-life was 156 min at 55 °C. Using d-psicose as the substrate, the apparent K m, k cat, and catalytic efficiency (k cat/K m) were 27.4 mM, 49 s?1, and 1.78 s?1 mM?1, respectively. Under the optimal conditions, the equilibrium ratio of d-fructose to d-psicose was 69:31. For high production of d-psicose, 216 g/L d-psicose could be produced with 28.8 % turnover yield at pH 6.5 and 55 °C. The recombinant DPEase exhibited weak-acid stability and thermostability and had a high affinity and turnover for the substrate d-fructose, indicating that the enzyme was a potential d-psicose producer for industrial production.  相似文献   

11.
Four potential dehydrogenases identified through literature and bioinformatic searches were tested for l-arabonate production from l-arabinose in the yeast Saccharomyces cerevisiae. The most efficient enzyme, annotated as a d-galactose 1-dehydrogenase from the pea root nodule bacterium Rhizobium leguminosarum bv. trifolii, was purified from S. cerevisiae as a homodimeric protein and characterised. We named the enzyme as a l-arabinose/d-galactose 1-dehydrogenase (EC 1.1.1.-), Rl AraDH. It belongs to the Gfo/Idh/MocA protein family, prefers NADP+ but uses also NAD+ as a cofactor, and showed highest catalytic efficiency (k cat/K m) towards l-arabinose, d-galactose and d-fucose. Based on nuclear magnetic resonance (NMR) and modelling studies, the enzyme prefers the α-pyranose form of l-arabinose, and the stable oxidation product detected is l-arabino-1,4-lactone which can, however, open slowly at neutral pH to a linear l-arabonate form. The pH optimum for the enzyme was pH 9, but use of a yeast-in-vivo-like buffer at pH 6.8 indicated that good catalytic efficiency could still be expected in vivo. Expression of the Rl AraDH dehydrogenase in S. cerevisiae, together with the galactose permease Gal2 for l-arabinose uptake, resulted in production of 18 g of l-arabonate per litre, at a rate of 248 mg of l-arabonate per litre per hour, with 86 % of the provided l-arabinose converted to l-arabonate. Expression of a lactonase-encoding gene from Caulobacter crescentus was not necessary for l-arabonate production in yeast.  相似文献   

12.
The gene coding for d-psicose 3-epimerase (DPEase) from Clostridium sp. BNL1100 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by Ni-affinity chromatography. It was a metal-dependent enzyme and required Co2+ as optimum cofactor. It displayed catalytic activity maximally at pH 8.0 and 65 °C (as measured over 5 min). The optimum substrate was d-psicose, and the K m, turnover number (k cat), and catalytic efficiency (k cat/K m) for d-psicose were 227 mM, 32,185 min?1, and 141 min?1 mM?1, respectively. At pH 8.0 and 55 °C, 120 g d-psicose l?1 was produced from 500 g d-fructose l?1 after 5 h.  相似文献   

13.
Poly-lactic acid (PLA) derived from renewable resources is considered to be a good substitute for petroleum-based plastics. The number of poly l-lactic acid applications is increased by the introduction of a stereocomplex PLA, which consists of both poly-l and d-lactic acid and has a higher melting temperature. To date, several studies have explored the production of l-lactic acid, but information on biosynthesis of d-lactic acid is limited. Pulp and corn stover are abundant, renewable lignocellulosic materials that can be hydrolyzed to sugars and used in biosynthesis of d-lactic acid. In our study, saccharification of pulp and corn stover was done by cellulase CTec2 and sugars generated from hydrolysis were converted to d-lactic acid by a homofermentative strain, L. delbrueckii, through a sequential hydrolysis and fermentation process (SHF) and a simultaneous saccharification and fermentation process (SSF). 36.3 g L?1 of d-lactic acid with 99.8 % optical purity was obtained in the batch fermentation of pulp and attained highest yield and productivity of 0.83 g g?1 and 1.01 g L?1 h?1, respectively. Luedeking–Piret model described the mixed growth-associated production of d-lactic acid with a maximum specific growth rate 0.2 h?1 and product formation rate 0.026 h?1, obtained for this strain. The efficient synthesis of d-lactic acid having high optical purity and melting point will lead to unique stereocomplex PLA with innovative applications in polymer industry.  相似文献   

14.
On the basis of allozyme and cultivation data, and of additional herbarium material, a taxonomic and nomenclatural revision ofTaraxacum sect.Piesis A.J. Richards exKirschner et?těpánek is provided. The section is made up of halophilous, sexually reproducing taxa. InT. stenocephalum Boiss. etKotschy,T. pindicum Kirschner et?těpánek, sp. nov., andT. perenne Kirschner et?těpánek, sp. nov., a tetraploid chromosome number has been recorded, representing the only known case of sexuality at the tetraploid level in the genus. The complex ofT. stenocephalum, includes some geographically and morphologically extreme populations treated as subspecies: subsp.gumusanicum (Soest)Kirschner et?těpánek, comb. nov., subsp.magnum Kirschner et?těpánek, subsp. nov., and subsp.daralagesicum (Schischk.)Kirschner et?těpánek, comb. nov. In addition toT. bessarabicum (Hornem.)Hand.-Mazz., a widely distributed Eurasian species,T. stenocephalum, a complex centred in Transcaucasia and Anatolia, andT. pachypodum H. Lindb., a North African endemic, four new species are described:T. salsum Kirschner et?těpánek, sp. nov., a diploid endemic confined to E Crimea,T. perenne Kirschner et?těpánek, sp. nov., a tetraploid sexual species known only from SW Crimea,T. pindicum Kirschner et?těpánek, sp. nov., a remarkable tetraploid endemic to the Pindos Mts., Greece, andT. salsitatis Kirschner, ?těpänek etYirdirimli, sp. nov., an Anatolian diploid species. Furthermore, a hybrid betweenT. salsum andT. bessarabicum from Crimea (documented on the basis of allozyme data elsewhere) is given a binomial,T. xmesohalobium Kirschner et?těpánek, nothosp. nov.  相似文献   

15.
l-DOPA (3,4-dihydroxyphenyl-l-alanine) is the most widely used drug for treatment of Parkinson’s disease. In this study Yarrowia lipolytica-NCIM 3472 biomass was used for transformation of l-tyrosine to l-DOPA. The process parameters were optimized using response surface methodology (RSM). The optimum values of the tested variables for the production of l-DOPA were: pH 7.31, temperature 42.9 °C, 2.31 g l?1 cell mass and 1.488 g l?1 l-tyrosine. The highest yield obtained with these optimum parameters along with recycling of the cells was 4.091 g l?1. This optimization of process parameters using RSM resulted in 4.609-fold increase in the l-DOPA production. The statistical analysis showed that the model was significant. Also coefficient of determination (R2) was 0.9758, indicating a good agreement between the experimental and predicted values of l-DOPA production. The highest tyrosinase activity observed was 7,028 U mg?1 tyrosine. l-DOPA production was confirmed by HPTLC and HPLC analysis. Thus, RSM approach effectively enhanced the potential of Y. lipolytica-NCIM 3472 as an alternative source to produce l-DOPA.  相似文献   

16.
Ethylene glycol (EG) is an important platform chemical with steadily expanding global demand. Its commercial production is currently limited to fossil resources; no biosynthesis route has been delineated. Herein, a biosynthesis route for EG production from d-xylose is reported. This route consists of four steps: d-xylose?→?d-xylonate?→?2-dehydro-3-deoxy-d-pentonate?→?glycoaldehyde?→?EG. Respective enzymes, d-xylose dehydrogenase, d-xylonate dehydratase, 2-dehydro-3-deoxy-d-pentonate aldolase, and glycoaldehyde reductase, were assembled. The route was implemented in a metabolically engineered Escherichia coli, in which the d-xylose?→?d-xylulose reaction was prevented by disrupting the d-xylose isomerase gene. The most efficient construct produced 11.7 g?L?1 of EG from 40.0 g?L?1 of d-xylose. Glycolate is a carbon-competing by-product during EG production in E. coli; blockage of glycoaldehyde?→?glycolate reaction was also performed by disrupting the gene encoding aldehyde dehydrogenase, but from this approach, EG productivity was not improved but rather led to d-xylonate accumulation. To channel more carbon flux towards EG than the glycolate pathway, further systematic metabolic engineering and fermentation optimization studies are still required to improve EG productivity.  相似文献   

17.
The effects of indole-3-butyric acid (IBA) alone and in combination with l-arginine on the morphogenic and biochemical responses in shoot tip explants of the cherry rootstock M × M 14 (Prunus avium × Prunus mahaleb) were examined. The maximum root number per rooted explant (16), root fresh (FW) and dry (DW) weights, as well as the rooting percentage (100 %) were recorded when 2 mg l?1 IBA (alone) were applied. Including the lowest IBA concentration (0.5 mg l?1) with the lowest and highest l-arginine concentrations (0.5 and 2 mg l?1, respectively) resulted in the greatest root length. The maximum leaf chlorophyll concentration and shoot length of the initial explant were recorded when 0.5 mg l?1 IBA plus 2 mg l?1 l-arginine were applied. In addition, l-arginine in combination with IBA (1 and 2 mg l?1) was found to suppress shoot FW and DW. On the other hand, l-arginine enhanced the promoting effect of IBA on both root length and leaf chlorophyll concentration. The carbohydrate and proline concentrations in leaves were not significantly altered with the application of IBA alone or in combination with l-arginine. On the other hand, the carbohydrate and proline concentrations in roots were decreased with the application of 1 and 2 mg l?1 IBA with l-arginine, resulting in the suppression of the promoting effects of IBA. It is clear from the findings that l-arginine has a direct effect on the in vitro rooting of M × M 14 explants, is involved in the function of the photosythetic apparatus, influences leaf chlorophyll content, participates in carbohydrate biosynthesis and metabolism, and is involved in proline accumulation both in leaves and roots.  相似文献   

18.
19.
We show that the voltage-gated K+ and Ca2+ currents of rat osteoblastic cells are strongly modulated by arachidonic acid (AA), and that these modulations are very sensitive to the AA concentration. At 2 or 3 μm, AA reduces the amplitude and accelerates the inactivation of the K+ current activated by depolarization; at higher concentrations (≥5 μm), AA still blocks this K+ current, but also induces a very large noninactivating K+ current. At 2 or 3 μm, AA enhances the T-type Ca2+ current, close to its threshold of activation, whereas at 10 μm, it blocks that current. AA (1–10 μm) also blocks the dihydropyridine-sensitive L-type Ca2+ current. Thus, the effect of AA on Ca2+ entry through voltage-gated Ca2+ channels can change qualitatively with the AA concentration: at 2 or 3 μm, AA will favor Ca2+ entry through T channels, both by lowering the voltage-gated K+ conductance and by increasing the T current, whereas at 10 μm, AA will prevent Ca2+ entry through voltage-gated Ca2+ channels, both by inducing a K+ conductance and by blocking Ca2+ channels.  相似文献   

20.
l-dopa-l-Tyr was synthesized by Fmoc solid-phase peptide synthesis, purified by reversed-phase HPLC and characterized by using 1H, 13C NMR and ESI–MS analyses. The interaction of l-dopa-l-Tyr and l-dopa with ctDNA has been investigated respectively by UV–vis absorption and fluorescence spectroscopy. The results showed that both l-dopa and l-dopa-l-Tyr interacted with ctDNA through intercalative mode and l-dopa-l-Tyr showed a higher affinity for DNA. Meanwhile, compared with the free l-dopa, gel electrophoresis assay also demonstrated that l-dopa-l-Tyr interacted with DNA by intercalation.  相似文献   

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