Effect of sodium nitroprusside on H+-ATPase activity and ATP concentration in Candida albicans

ATP hydrolysis by plasma membrane H+-ATPase from Candida albicans has been investigated in presence of nitric oxide and various nutrients (sugars and amino acids). Sodium nitroprusside (SNP) was used as nitric oxide donor. It was found that ATP concentration decreased in SNP treated cells which was more in presence of sugars like glucose, xylose and 2-deoxy-D-glucose and amino acids as compared to their respective controls. The activity of H+-ATPase from plasma membrane decreased by 70 % in SNP treated cells. Both in vivo and in vitro treatments of SNP showed almost similar effects of decrease in ATPase activity. Effect of SNP was more pronounced in presence of nutrients. Interestingly, it was observed that vanadate did not show any independent effect in presence of nitric oxide. Several workers have reported similar type of results with other P-type ATPases. For the first time, it was observed in the present study that in presence of nitric oxide, H+-ATPase activity decreased like other P-type ATPases. Our study indicated that NO had a significant effect on ATP synthesis and activity of H+- ATPase. In the presence of NO, the ATP concentration was decreased indicating it affected mitochondrial electron transport chain. It may be concluded that NO, not only affects (inhibit) mitochondrial electron transport chain but also interferes with H+- ATPase of plasma membrane by changing its conformation resulting in decreased activity.     

Involvement of the relA gene product and feedback inhibition in the regulation of DUP-N-acetylmuramyl-peptide synthesis in Escherichia coli.

The regulation of uridine diphosphate-N-acetylmuramyl-peptide (UDP-MurNAc-peptide) synthesis was studied by labeling Escherichia coli strains auxotrophic for lysine and diaminopimelate with [3H]diaminopimelate for 15 min under various conditions. The amounts of [3H]diaminopimelate incorporated into UDP-MurNAc-tripeptide and -pentapeptide by a stringent (rel+) strain were the same in the presence or absence of lysine. Chloramphenicol-treated rel+ cells showed a 2.8-fold increase in labeled UDP-MurNAc-pentapeptide. An isogenic relaxed (relA) strain deprived of lysine showed a 2.7-fold increase in UDP-MurNAc-pentapeptide. Thus, UDP-MurNAc-pentapeptide synthesis is regulated by the relA gene. D-Cycloserine treatment of rel+ and relA strains caused a depletion of intracellular UDP-MurNAc-pentapeptide. Labeled UDP-MurNAc-tripeptide accumulated in D-cycloserine-treated cells of the rel+ and relA strains, suggesting that UDP-MurNAc-pentapeptide is a feedback inhibitor of UDP-MurNAc-peptide synthesis. In lysine-deprived cells, D-cycloserine treatment caused 41- and 71-fold accumulations of UDP-MurNAc-tripeptide in rel+ and relA strains, respectively. A 124-fold increase in UDP-MurNAc-tripeptide occurred in lysine-deprived rel+ cells treated with both chloramphenicol and D-cycloserine. These results indicate that both the relA gene product and feedback inhibition are involved in regulating UDP-MurNAc-peptide synthesis during amino acid deprivation.     

Energetics of alanine, lysine, and proline transport in cytoplasmic membranes of the polyphosphate-accumulating Acinetobacter johnsonii strain 210A.

Amino acid transport in right-side-out membrane vesicles of Acinetobacter johnsonii 210A was studied. L-Alanine, L-lysine, and L-proline were actively transported when a proton motive force of -76 mV was generated by the oxidation of glucose via the membrane-bound glucose dehydrogenase. Kinetic analysis of amino acid uptake at concentrations of up to 80 microM revealed the presence of a single transport system for each of these amino acids with a Kt of less than 4 microM. The mode of energy coupling to solute uptake was analyzed by imposition of artificial ion diffusion gradients. The uptake of alanine and lysine was driven by a membrane potential and a transmembrane pH gradient. In contrast, the uptake of proline was driven by a membrane potential and a transmembrane chemical gradient of sodium ions. The mechanistic stoichiometry for the solute and the coupling ion was close to unity for all three amino acids. The Na+ dependence of the proline carrier was studied in greater detail. Membrane potential-driven uptake of proline was stimulated by Na+, with a half-maximal Na+ concentration of 26 microM. At Na+ concentrations above 250 microM, proline uptake was strongly inhibited. Generation of a sodium motive force and maintenance of a low internal Na+ concentration are most likely mediated by a sodium/proton antiporter, the presence of which was suggested by the Na(+)-dependent alkalinization of the intravesicular pH in inside-out membrane vesicles. The results show that both H+ and Na+ can function as coupling ions in amino acid transport in Acinetobacter spp.     

Effect of phosphocreatine on H+ extrusion,pHi and dimorphism in Candida albicans

Candida albicans is an opportunistic pathogen. Its proliferation in human hosts is believed to be controlled by immunologic mechanisms. The plasma membrane of the fungus possesses an H(+)-ATPase (PM-ATPase) which actively extrudes protons to generate an electrochemical gradient which is used in co-transport of nutrients. This ATPase is associated with the growth, dimorphism and pathogenicity of the fungus. The physiological concentration of phosphocreatine (PCr) is 20-35 mM in skeletal muscles. H(+)-extrusion in Candida cells was strongly inhibited by PCr; 44% at 20 mM and 69% at 40 mM. H(+)-extrusion was stimulated 6.2-fold in the presence of 10 mM glucose. This glucose stimulated extrusion was inhibited significantly by PCr; 36% at 20 mM and 53% at 40 mM. The intracellular pH pattern of cells destined to differentiate was greatly altered in the presence of PCr. Evagination time for control cells was between 90-120 min. PCr, delayed dimorphism, reduced the population of cells differentiating to hyphae and also reduced the length of hyphae after each time interval. Only 60% differentiation was observed with 10 mM PCr and 40% for higher PCr concentration even after 210 min. Direct interaction of PM-ATPase and PCr has been demonstrated by difference spectrum measurement employing stopped flow spectrophotometer. It can be concluded that PCr may be playing a significant role in checking growth and pathogenesis of C. albicans.     

The effect of altered ergosterol content on the transport of various amino acids in Candida albicans.

Candida albicans cells have low levels of ergosterol when grown in ascorbic acid-supplemented media. When cells are grown in hydroquinone-supplemented media, the ergosterol levels became higher as compared to normal cells. The uptake of lysine, glycine, glutamic acid, proline, methionine and serine is reduced in hydroquinone-supplemented cells. In contrast to hydroquinone-supplemented cells, the rate and level of accumulation of these amino acids are higher in ascorbic acid-supplemented cells. Nystatin-resistant isolates of C. albicans with low ergosterol contents also exhibit an increased rate and level of accumulation of these amino acids. The uptake of phenylalanine and leucine remained unaffected by such a change in ergosterol levels brought about by different supplementation of the media. The results demonstrate a correlation between ergosterol levels and amino acids uptake. Contrary to various reports, the rate of K+ efflux does not seem to correlate with the amino acid uptake in C. albicans cells.     

Amino acid synthesis from glucose-U-14C in Glossina morsitans

Amino acid synthesis from glucose-U-¹⁴C was investigated in 2 day post-emergent and pregnant females of Glossina morsitans. This insect can synthesize alanine, aspartic acid, cystine, glutamic acid, glycine, proline, and serine from glucose. Arginine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, taurine, threonine, and valine showed no radioactivity and hence may be classified as nutritionally indispensable amino acids. Although tyrosine and hydroxyproline were not synthesized from glucose, they are at least partially dispensable nutrients for this insect because their synthesis from phenylalanine has been demonstrated. After the labelled glucose injection the highest radioactivity was recovered in the proline fraction. This is probably related to its rôle as an important energy reserve for flight. The radioactive amino acids recovered from females and from their offspring following glucose-U-¹⁴C injection were similar to those recovered from younger females. Radioactivity was also detected in the expired CO₂ and the excreta. The amino acids alanine, arginine, cystine, glycine, histidine, leucine/isoleucine, lysine, methionine, proline, and valine were identified in the excreta, of which arginine and histidine were in the largest amounts. Only excreted alanine, glycine, and proline showed radioactivity.     

The effect of sugar,amino acid,metal ion,and NaCl on model Maillard reaction under pH control

Summary. The color intensities was determined of Maillard reaction products (MRPs) prepared by heating each of five sugars (maltose, fructose, glucose, arabinose, and xylose) with each of 12 amino acids (aspartic acid, glutamic acid, alanine, leucine, isoleucine, valine, proline, serine, cysteine, phenylalanine, arginine, and lysine). The remaining percentages of glucose and rate of change of color intensity due to the addition of a metal ion and NaCl were monitored for nine MRPs that had been formed between glucose and each of nine amino acids (aspartic acid, glutamic acid, alanine, valine, serine, cysteine, phenylalanine, arginine, and lysine). Model MRPs were prepared in a block heater at 100°C for 1–12h with the pH value controlled at 6.5. The resulting color intensity of each MRPs formed from the basic amino acids was greater due to the higher reactivity than those from the acidic amino acids. The remaining percentage of glucose in each MRPs from the basic amino acids was lower than those from the acidic amino acids. The MRPs from the nonpolar amino acids showed an intermediate color intensity and remaining percentages of glucose between those formed from the basic and acidic amino acids. Browning tended to be accelerated in the presence of metal ions, especially Fe²⁺ and Cu²⁺, although it was affected by the property of the amino acid and heating time as well as by the type of metal ion. On the other hand, browning was greatly inhibited by a high concentration of NaCl.     

Uptake and excretion of amino acids and utilization of glucose by Schistosoma japonicum eggs

The utilization of amino acids and glucose in the external nutrients and the excretion of nitrogenous compounds by Schistosoma japonicum eggs were investigated with the eggs cultured in a chemically defined medium (MEMSE-J). Of the 15 amino acids in MEMSE-J, arginine and glutamine markedly decreased in concentration during cultivation of S. japonicum eggs. The nitrogenous excretory products of developing eggs were demonstrated to be at least four amino acids (alanine, proline, glutamic acid and ornithine), urea and ammonia. Glucose was consumed at an estimated rate of 32 ng/living egg/day during the period of egg growth and differentiation. When 14C-labelled glucose was included in the culture medium, the radioactivity was incorporated into three amino acids (alanine, proline and glutamic acid), which were excreted by S. Japonicum eggs. The results were discussed with reference to the possible role in stimulating fibrosis in the granuloma of schistosomiasis.     

The role of glucose in the pH regulation of germ-tube formation in Candida albicans

It has been reported that Candida albicans can form germ-tubes only in the narrow pH range of 6-8, and that by changing only the pH one can regulate germ-tube formation. We found that the pH minimum for germ-tube formation could be dramatically lowered by eliminating the glucose present in many induction solutions. Lee's medium lacking glucose, ethanol, N-acetyl-D-glucosamine, and proline induced germ-tubes at pH values as low as 3 under most conditions. The presence of as little as 1 mM-glucose in these induction solutions was sufficient to cause the cells to grow either as yeasts with multiple buds or as pseudohyphae when the pH was 3.7. However, when C. albicans was grown in any of the above induction solutions (with the exception of ethanol), containing 200 mM-glucose buffered at pH 5.8, not only were germ-tubes formed, but their rate of formation and length were also increased. Preincubation of the cells in a solution buffered at pH 3.7 and containing 200 mM-glucose, before exposure to induction solutions lacking glucose at pH 3.7 or at pH 5.8, did not inhibit germ-tube formation. Likewise, addition of glucose after 45 min exposure to an induction solution was without effect. Theophylline and dibutyryl cAMP did not counteract the action of glucose. Other sugars which suppressed germ-tube formation at low pH were fructose, galactose, mannose, xylose, gluconic acid and the nonmetabolizable sugar 3-O-methylglucose. These results indicate that pH does not directly regulate dimorphism in C. albicans, and that glucose or its metabolites may play an important role.     

Studies on the isolation and chemical properties of porcine follicle-stimulating hormone.

Porcine follicle-stimulating hormone (pFSH) has been prepared from acetone dried pituitary glands. The acetone powder was initially extracted in pH 5 ethanol-acetate buffer (40% ethanol and acetate at 0.5 ionic strength) containing 0.02% PMSF. The gonadotropin rich fraction, obtained in the 80% ethanol precipitate, was dissolved in 0.12 M NH4HCO3, heated at 60 degrees for 3 min, cooled, and centrifuged. The supernatant was lyophilized for chromatography on QAE-Sephadex, then Sephadex G-100. The pFSH obtained has a biological activity of 15 X NIH-FSH-P-1 and 2.1% sialic acid. The amino acid analysis is characterized by high lysine, aspartic acid, and glutamic acid with notable amounts of threonine, proline, and half-cystine.     

Molecular cloning of a pea H1 histone cDNA

A pea (Pisum sativum, var. Little Marvel) H1 histone cDNA has been isolated from a lambda gt11 expression vector library. This cDNA has been sequenced and shown to represent the entire protein-coding region of the mRNA. The deduced protein sequence is 265 amino acids long (28018 Da) and contains 70 lysines and 3 arginines. The structure of the encoded protein is comparable to animal lysine-rich histones. The central region, which has an amino acid composition similar to that found in the globular domains of animal lysine-rich histones, is flanked by an amino-terminal region rich in lysine, glutamic acid and proline and by a carboxyl-terminal region rich in lysine, alanine, valine and proline. Despite the structural similarities, the protein has little sequence homology with animal lysine-rich histones. This H1 protein is unusual because 12 of the first 40 amino acids are glutamic acid.     

Nutrient uptake by Candida albicans: the influence of cell surface mannoproteins.

Numerous ultrastructural and biochemical analyses have been performed to characterize the cell wall composition and structure of Candida albicans. However, little investigation has focused on how subtle differences in cell wall structure influence the intracellular transport of amino acids and monosaccharides. In this study C. albicans 4918 and ATCC 10231 were grown in culture conditions capable of modifying surface mannoproteins and induced surface hydrophobic or hydrophilic yeast cell wall states. Subcultures of these hydrophobic and hydrophilic yeasts were subsequently incubated with one of seven L-[3H] amino acids: glycine, leucine, proline, serine, aspartic acid, lysine, or arginine. The transport of [3H] mannose and [3H] N-acetyl-D-glucosamine were also investigated. This study revealed significant strain differences (P < or = 0.05) between hydrophilic and hydrophobic yeast transport of these nutrients throughout a 2 h incubation. Hydrophilic cultures of 4918 and ATCC 10231 transported nearly two times more (pmol mg-1 dry weight) proline, mannose, and N-acetyl-D-glucosamine than hydrophobic yeast. Hydrophobic cultures preferentially incorporated serine and aspartic acid in both these strains. Strain variation was indicated with the transport of leucine, lysine, and arginine, as follows: experiments showed that hydrophilic 4918 cultures selectively transported leucine, lysine, and arginine, whereas, the hydrophobic ATCC 10231 cultures incorporated these amino acids.     

Embryotoxicity of the nitric oxide donor sodium nitroprusside in preimplantation bovine embryos in vitro

Many early pregnancy complications are associated with an imbalance in pro- and anti-inflammatory cytokines, resulting in alterations in nitric oxide (NO) profile. Since very little is known about the modus operandi of this free radical in early embryos, this study characterised NO embryotoxicity in terms of bovine embryo development and metabolism. Embryos were generated by in vitro maturation and fertilisation of oocytes aspirated from abattoir-derived ovaries. Zygote to blastocyst rates were measured in SOFaaBSA in the presence and absence of the NO donor sodium nitroprusside (SNP) over the 0-50 microM range (n=10 per group). Since concentrations <10 microM SNP depressed blastocyst rate, blastocyst cell numbers (determined by bisbenzimide staining; n=22 and 20), glucose, pyruvate, lactate (measured ultramicrofluorometrically) and amino acid profiles (quantified by HPLC; n=28 and 23) were assessed at 0 and 10 microM SNP. SNP depressed cell numbers, reduced pyruvate and glucose uptake, perturbed quantitative tyrosine, threonine, phenylalanine, lysine, glycine, tryptophan, methionine and valine profiles, and decreased retention into the negative range (P<0.05). Qualitative asparagine and lysine profiles were affected by SNP, while proportional amino acid production and consumption were increased and decreased, respectively (P<0.05). These findings indicate that SNP (presumably through increases in NO profile): (i) fails to improve bovine embryo development in vitro, (ii) exerts toxic effects, likely through ATP starvation induced by cytochrome c oxidase (oxidative phosphorylation) and glyceraldehyde-3-phosphate dehydrogenase (glycolysis) inhibition, and (iii) may affect albumin endocytosis/hydrolysis or protein biosynthesis, rather than causing a loss of intracellular amino acids or simply depressing their metabolism.     

Mechanisms of nitric oxide release from nitrovasodilators in aqueous solution: reaction of the nitroprusside ion ([Fe(CN)5NO]2-) with L-ascorbic acid.

Nitric Oxide (NO) is a very diverse endogenous molecule and is responsible for a number of mammalian physiological activities. Sodium nitroprusside (SNP) is a long known hypotensive agent due to its ability to produce NO in situ. In this study, we have carried out a detailed investigation on the kinetics and mechanism of the decomposition of SNP by L-ascorbic acid to afford NO in aqueous medium. Spectrophotometric and electrochemical methods were employed in these studies. Experiments were performed under an argon atmosphere and reaction rates were strongly pH dependent and increases with pH. NO release increases up to about pH 7 after which it decreases at higher pHs. The two ionized forms of ascorbate present in aqueous solution reduce SNP in the order A2->HA->H2A (H2A=L-ascorbic acid) to release NO. The outer-sphere reduction of SNP by ascorbate involved three clear stages with NO being released in the last stage. Our kinetic results also show catalysis by group 1 alkali metal ions to increase down the group from Li+ to Cs+. A detail study presented here, strongly illustrate the possibility of the biological antioxidant, L-ascorbic acid, to play a vital role in the in situ metabolism of nitrosocompounds such as SNP to produce NO.     

一氧化氮参与调节盐胁迫下脱落酸诱导的小麦幼苗叶片脯氨酸的累积

不同浓度(0.01～5.00mmol/L)的外源一氧化氮(NO)供体硝普钠(SNP)以浓度依赖性的性式诱导150mmol/LNaCl胁迫下小麦(Triticum aestivum L.cv.Yangmai 158)幼苗叶片脯氨酸的累积.其中0.1 mmol/L的SNP效果最明显,而结合采用NO清除剂c-PTIO和血红蛋白的处理均分别逆转了该效应.研究结果还发现:0.1 mmol/L SNP诱导的脯氨酸累积还可能有利于盐胁迫下小麦幼苗的保水性;0.1 mmol/L的SNP显著激活了内源ABA的合成,而结合血红蛋白的处理则证实,在外源ABA诱导脯氨酸累积的过程中NO可能作用于ABA信号分子的下游,但NO和ABA信号分子在此诱导反应中不存在累积效应.进一步研究脯氨酸合成和降解的酶促反应途径,发现外源NO处理前4天内可能主要是通过提高△'-吡咯啉-5-羧酸合成酶(P5CS)的活性来促进脯氨酸的合成,以后直至第8天主要是通过抑制脯氨酸脱氢酶(ProDH)的活性来抑制脯氨酸的降解;ABA对于P5CS和ProDH活性的调节能力弱于NO.此外,Ca2 在NO诱导的盐胁迫下小麦叶片脯氨酸累积的信号分子途径中起重要的介导作用.     

Proline-induced germ-tube formation in Candida albicans: role of proline uptake and nitrogen metabolism

Proline-induced germ-tube formation and cell-cell aggregation in four strains of Candida albicans were completely inhibited when the pH of the medium was 5.0 or lower, whereas morphogenesis induced by N-acetylglucosamine (GlcNAc) was unaffected even at pH 4.5. The pH sensitivity of proline-induced germ-tube formation was not caused by a modulation of proline uptake, which was unchanged over the pH range 4.5-6.5. The proline uptake system was specific, constitutive and subject to ammonium repression, and only one permease was detected, with a Km of 179 microM. Cultures deprived of nitrogen in the presence of glucose were derepressed for proline uptake but the yeast-mycelial transition could not be mediated by either proline or GlcNAc. The inhibition of morphogenesis was reversed when the nitrogen starvation was relieved by the addition of ammonium ions, proline, or certain amino acids. These results indicate that the nitrogen status of the cells is critical for the morphogenesis of C. albicans.     

Link between free radicals and protein kinase C in glucose-induced alteration of vascular dilation

Development of vascular complications in diabetes has been linked to the quality of glucose regulation and characterized by endothelial dysfunction. The exact mechanism behind vascular complications in diabetes is poorly understood. However, alteration of nitric oxide (NO) biosynthesis or bioactivity is strongly implicated and the mechanism behind such alterations is still a subject for research investigations. In the present study, we tested the hypothesis that glucose-induced attenuation of vascular relaxation involves protein kinase C (PKC)-linked generation of free radicals. Vascular relaxation to acetylcholine (ACh; 10(-9)-10(-5) M), isoproterenol (10(-9)-10(-5) M), or NO donor, sodium nitropruside (SNP; 10(-9)-10(-6) M) was determined in phenylephrine (PE, 10(-7) M) pre-constricted aortic rings from Sprague-Dawley rats in the presence or absence of 30 mM glucose (30 min), L-nitro-arginine methyl ester (L-NAME; 10(-4) M for 15 min), a NO synthase inhibitor, or xanthine (10(-5) M), a free radical generator. ACh dose-dependently caused relaxation that was attenuated by L-NAME, glucose, or xanthine. Pre-incubation (15 min) of the rings with vitamin C (10(-4) M), an antioxidant or calphostin C (10(-6) M), a PKC inhibitor, restored the ACh responses. However, high glucose had no significant effects on SNP or isoproterenol-induced relaxation. ACh-induced NO production by aortic ring was significantly reduced by glucose or xanthine. The reduced NO production was restored by pretreatment with vitamin C or calphostin C in the presence of glucose, but not xanthine. These data demonstrate that oxidants or PKC contribute to glucose-induced attenuation of vasorelaxation which could be mediated via impaired endothelial NO production and bioavailability. Thus, pathogenesis of glucose-induced vasculopathy involves PKC-coupled generation of oxygen free radicals which inhibit NO production and selectively inhibit NO-dependent relaxation.     

Quantitative analysis of free amino acids and carbohydrates in spring barley anthers at different stages of maturity

The content of the carbohydrates glucose, fructose and sucrose was determined in spring barley anthers at different stages of maturity. During maturation the sucrose content of the anthers increased markedly. The following 17 free amino acids were detected in anthers of different stages of maturity: aspartic acid, glutamic acid, serine, alanine, arginine, leucine, isoleucine, lysine, α-aminobutyric acid, glutamine, proline, tyrosine, phenylalanine, valine, threonine, cystine and glycine. Quantitative analysis was only carried out in amino acids present in higher concentrations in the analysed samples. These were: aspartic acid, glutamic acid, α-aminobutyric acid, proline, serine, valine and glutamine, and a mixture of amino acids (leucine, isoleucine, valine and phenylalanine). The total content of free amino acids increased with increasing maturity of the anthers. However, not all amino acids followed contributed to this increase, but only proline, glutamic acid, aspartic acid and glutamine. A small difference was found in the variety Gopal in which the aspartic acid content did not increase significantly, but the content of the mixture of amino acids and serine did. With the exception of green anthers of the variety Firlbecks Union, proline was present in the highest concentration in all samples analysed.     

Inhibition by glucose of the synthesis of proline from glutamic acid

While in the absence of glucose, proline is not a required amino acid, in the presence of glucose the growth of Micrococcus pyogenes var. aureus in amino acid medium is proportional to the concentration of proline when all other amino acids and growth factors are present in amounts adequate for optimal growth. The data presented here and the ideas prevailing in the literature indicate that glutamic acid is a precursor of proline. Glucose inhibits the conversion of glutamic acid into proline, which in turn causes failure of growth. Thus, 1 μg. and 10 μg. glucose/ ml. cause 50% and 100% inhibition, respectively, of the growth dependent on the synthesis of proline. One μg. proline antagonizes completely the inhibition in the presence of 5,000 μg. glucose/ml.One μg. glycerol, 100 μg. pyruvate, 250 μg. lactate, or 100 μg. α-glycerophosphate/ml., individually, cause from 25 to 50% inhibition of the growth dependent on the synthesis of proline from glutamic acid. Five thousand μg./ml. either of malic, succinic, fumaric, α-keto-glutaric, cis-aconitic acid, or dihydroxyacetone, or 500 μg. citric acid/ml. fails to cause inhibition.Pyrrolidone carboxylic acid was found to substitute for glutamic acid but not for proline in tests with M. pyogenes var. aureus. Also, seven proline-less mutant strains of Escherichia coli were unable to utilize pyrrolidone carboxylic acid in place of proline. No evidence was obtained to indicate that pyrrolidone carboxylic acid could serve as a direct precursor of proline.