Eugenol and carvone as relaxants of arsenic and mercury hypercontracted rat trachea.

Exposure to arsenic and mercury is known to cause respiratory problems in both humans and animals. In this study, we elicit and compare maximum contraction caused by As(III) and Hg(II) when the pollutants are fully equilibrated with contractile machinery in resting mode. Hypercontraction of 27% and 69% was obtained following exposure of tracheal rings to 25 µM As(III) and 6 nM Hg(II) for 40 min, respectively. Co-incubation of tracheal rings with pollutants and verapamil, sodium nitroprusside or apocynin indicates that major contributors to As(III) and Hg(II) caused hypercontraction are reactive oxygen species (ROS) elevation and nitric oxide (NO) depletion. Changes in calcium influx have minor contribution in As(III) and Hg(II) caused increased contraction of tracheal tissues. Eugenol and carvone caused relaxation of 38% and 45% in pollutant unexposed rings, 56% and 49% in As(III)-exposed tracheal rings, and 54% and 47% in Hg(II)-exposed tracheal rings. Pathway delineation studies indicate that the major effect of eugenol originates from quenching of ROS whereas that of carvone originates from the blockage of extracellular calcium influx. Both molecules also show a minor stimulatory effect on NO generation. In line with their suggested mode of relaxation, eugenol is found to better ameliorate both As(III)- and Hg(II)-caused hypercontraction. Carvone, though a better relaxant than eugenol, comes out as poor ameliorator of both As(III)- and Hg(II)-caused hypercontraction, as the pathway on which it acts is not elevated following exposure to these pollutants.

Cinnamic aldehydes affect hydrolytic enzyme secretion and morphogenesis in oral Candida isolates.

Effect of cinnamaldehyde (CD), 4-hydroxy-3-methoxy cinnamaldehyde (HMCD) and 3,5-dimethoxy-4-hydroxy cinnamaldehyde (HDMCD) on growth and virulence factors of standard (Candida albicans 90028) and 26 oral isolates of C. albicans has been investigated. Growth was significantly inhibited by all three compounds in both solid and liquid medium, no systematic difference was observed between various isolates. MIC90 ranged from 125 to 450 µg/ml for CD, 100-250 µg/ml for HMCD and 62.5-125 µg/ml for HDMCD. All oral isolates were found to be proteinase and phospholipase secretors, both proteinase and phospholipase secretion was significantly inhibited by all the three tested molecules. No systematic difference in secretion or its inhibition was observed between standard and oral isolates as also between various isolates. Average drop in proteinase and phospholipase secretion caused by ½ MIC of CD was 33% and 28%, HMCD; 46% and 44%, HDMCD; 59% and 54%. The standard strain and all the 26 oral isolates displayed morphogenesis under triggering experimental conditions; no difference was seen between standard and various isolates. In the absence of test compounds hyphae development at 300 min was 83% for standard strain whereas average hyphae development for oral isolates was 85%. Average hyphal transition was suppressed by all tested compounds. At ½ MIC concentration at 300 min average hyphal transition of standard and oral isolates was CD; 49% and 57%, HMCD; 45% and 38%, HDMCD; 5% and 5%. Average haemolytic activity of the three tested compounds varied from 10 to 15% at their highest MIC compared to 20% shown by fluconazole at typical MIC of 30 µg/ml.

Proton pumping ATPase mediated fungicidal activity of two essential oil components.

This work evaluates the antifungal activity of two essential oil components against 28 clinical isolates (17 sensitive, 11 resistant) and 3 standard laboratory strains of Candida. Growth of the organisms was significantly effected in both solid and liquid media at different test compound concentrations. The minimum inhibitory concentrations (MICs) of Isoeugenol (compound 1) against 31 strains of Candida ranged 100-250 µg/ml and those of o -methoxy cinnamaldehyde (compound 2) ranged 200-500 µg/ml, respectively. Insight studies to mechanism suggested that these compounds exert antifungal activity by targeting H(+)-ATPase located in the membranes of pathogenic Candida species. At their respective MIC(90) average inhibition of H(+)-efflux for standard, clinical and resistant isolates caused by compound 1 and compound 2 was 70%, 74%, 82% and 42%, 42% and 43%. Respective inhibition of H(+)-efflux by fluconazole (5 µg/ml) was 94%, 92% and 10%. Inhibition of H(+)-ATPase leads to intracellular acidification and cell death. SEM analysis of Candida cells showed cell membrane breakage and alterations in morphology. Haemolytic activity on human erythrocytes was studied to exclude the possibility of further associated cytotoxicity.

Elucidation of Mechanisms in Cu (II) Caused Hypercontraction of Rat Tracheal Rings.

Airway smooth muscle contraction is one of the primary factors involved in the initiation and progression of asthma which in turn is regulated by increased cytosolic Ca2+ concentration from intracellular stores and through transmembrane ion channels. Calcium-independent factors such as reactive oxygen species (ROS) generation, nitric oxide (NO) depletion and cyclooxygenase (COX) pathways also contribute to tracheal smooth muscle contraction. Studies on copper toxicity suggest significance of this essential micronutrient overdose in acute respiratory disorders, allergic asthma and ciliary motion in tracheal explants. However, the mechanism of copper caused hypercontraction upon direct exposure to tracheal smooth muscle is largely unknown. In this study we investigate the effect of copper exposure on isolated tracheal rings and relative contributions of various factors in acetylcholine-induced contractions. Results obtained suggest that rise in intracellular calcium concentration via voltage-operated Ca2+ channel (VOCC), store-operated Ca2+ channel (SOCC), stretch-activated channels (SAC) and TRP channel (transient receptor potential channel) activation is the major factor in copper-mediated hypercontraction. ROS generation or COX-dependent pathways do not appear to significantly contribute to Cu2+ caused hypercontraction.

Interesting anticandidal effects of anisic aldehydes on growth and proton-pumping-ATPase-targeted activity.

Attention has been drawn to evaluate the antifungal activity of p-anisaldehyde (1), o-anisaldehyde (2) and m-anisaldehyde (3). To put forward this approach, antifungal activity has been assessed in thirty six fluconazole-sensitive and eleven fluconazole-resistant Candida isolates. Growth and sensitivity of the organisms were significantly effected by test compounds at different concentrations. The rapid irreversible action of compound-1, compound-2 and compound-3 on fungal cells suggested a membrane-located target for their action. We investigated their effect on H(+) ATPase mediated H(+)-pumping by various Candida species. All the compounds inhibit H(+)- ATPase activity at their respective MIC(90) values. Inhibition of H(+) ATPase leads to intracellular acidification and cell death. Scanning electron microscopy analysis revealed deep wrinkles, deformity and flowed content. Furthermore, it was also observed that position of methoxy group attached to the benzene ring decides antifungal activity of the compound. The present study indicates that compound-1, compound-2 and compound-3 have significant antifungal activity against Candida, including azole-resistant strains, advocating further investigation for clinical applications in the treatment of fungal infections.

Effect of garlic-derived allyl sulphides on morphogenesis and hydrolytic enzyme secretion in Candida albicans.

The effect of diallyl sulphide (DAS) and diallyl disulphide (DADS) on secretion of hydrolytic enzymes and dimorphism has been investigated in two strains of Candida albicans. Minimum inhibitory concentration (MIC) of DADS and DAS was determined to be 500 µg/ml and 40 µg/ml, respectively for a clinical isolate (accession #3043) and 450 µg/ml and 50 µg/ml, respectively, for a reference strain (ATCC 90028). At one-half of the minimum inhibitory concentration (MIC), DAS and DADS inhibited proteinase secretion by 24% and 35%, respectively, in the clinical strain, and 28% and 44%, respectively, in the reference strain. Inhibition of phospholipase secretion at one-half MIC of DAS and DADS was 27% and 60%, respectively, in the clinical strain and 31% and 64%, respectively, for the reference strain. Hyphal induction at 300 min in the reference strain was 15% (at one-half MIC of DAS) and 5% (at one-half the MIC of DADS) as compared to control (90% hyphal formation). Hyphal induction in the clinical strain was 16% (one-half the MIC of DAS) and 8% (one-half the MIC of DADS) compared to 95% in the control. To conclude, both DAS and DADS significantly inhibit proteinase, phospholipase secretion and dimorphism in C. albicans. These compounds can therefore be explored for their therapeutic potential against C. albicans.

Hepatocellular carcinoma with persistent hepatitis B virus infection shows unusual downregulation of Ras expression and differential response to Ras mediated signaling.

BACKGROUND AND AIM: Persistent infection with hepatitis B virus (HBV) is a major etiological risk factor for hepatocellular carcinoma (HCC). The host cellular components involved in the progression of the carcinoma are still unclear. In the present study we aimed to evaluate Ras mediated signaling in hepatocellular carcinoma with persistent HBV infection. METHODS: To gain insight into the role of Ras mediated signaling in HBV mediated carcinogenesis we evaluated Ras functionality by mutation analysis, reverse transcription-polymerase chain reaction, immunohistochemistry (IHC), Ras-guanosine triphosphate bound functionality assay and Ras-mediated downstream signaling in a cohort of primary HCC tissues positive for HBV-DNA. RESULTS: Mutation in codon 12 of K-ras appeared to be an uncommon event in the pathogenesis of HCC. We found unusually low levels of Ras expression in HCC compared with those with normal liver and chronic liver disease (cirrhosis and chronic hepatitis). Considerable heterogeneity was found with respect to Ras-mediated signaling events (pRaf, pMAPK and pAKT). The hepatoma cell line (Hep3B) with integrated HBV showed upregulation in expression and activation of Ras and its downstream signaling in comparison to HBV a negative cell line (HepG2). The contrasting result between the cell lines and primary tumors is worthy of note. CONCLUSIONS: The unusual finding on downregulation of Ras expression in primary HCC tumors in the present study together with tumor heterogeneity with respect to Ras-mediated signaling events prompts a new role of the wild type K-Ras as a possible growth suppressor and a stochastic model for progression of hepatic cancer.

Antifungal activity of α-methyl trans cinnamaldehyde, its ligand and metal complexes: promising growth and ergosterol inhibitors.

Antifungal effectivity and utility of cinnamaldehyde is limited because of its high MIC and skin sensitivity. In this study, α-methyl trans cinnamaldehyde, a less irritating derivative, have been self coupled and complexed with Co(II) and Ni(II) to generate N, N'-Bis (α-methyl trans cinnamadehyde) ethylenediimine [C(22)H(24)N(2)], [Co(C(44)H(48)N(4))Cl(2)] and [Ni(C(44)H(48)N(4))Cl(2)]. Ligand and complexes were characterized on the basis of FTIR, ESI-MS, IR and (1)HNMR techniques. Synthesized ligand [L] and complexes were investigated for their MICs, inhibition of ergosterol biosynthesis and H(+) extrusion against three strains of Candida: C. albicans 44829, C. tropicalis 750 and C. krusei 6258. Average of three species MIC of methyl cinnamaldehyde is 317 µg/ml (2168 µM). Compared to methyl cinnamaldehyde ligand [L], Co(II) and Ni(II) complex are found to be 4.48, 17.78 and 21.46 times more effective in liquid medium and 2.73, 8.93 and 10.38 times more effective in solid medium. At their respective MIC(90) average inhibition of ergosterol biosynthesis caused by methyl cinnamaldehyde, ligand [L], Co(II) and Ni(II) complex, respectively was 80, 78, 90 and 93%. H(+) extrusion was also significantly inhibited but did not co-relate well with MIC(90). Results indicate ergosterol biosynthesis as site of action of α-methyl cinnamaldehyde, synthesized ligand and complexes. α-methyl cinnamaldehyde and ligand did not show any toxicity against H9c2 rat cardiac myoblast cell, whereas Co(II) and Ni(II) complexes on an average produced 19% cellular toxicity.

Curcumin as a promising anticandidal of clinical interest.

Curcumin, an important Asian spice, is part of many Indian food preparations. This work evaluates the antifungal activity of curcumin against 14 strains of Candida (10 clinical and 4 standard). Curcumin displayed antifungal properties against all tested Candida strains, with minimum inhibitory concentrations (MICs) varying from 250 to 2000 µg·mL⁻¹. The in vitro effect of curcumin on growth, sterol content, proteinase secretion, and H+ extrusion by plasma membrane ATPase was investigated for 2 standard strains Candida albicans ATCC 10261 and Candida glabrata ATCC 90030 and compared with the effect of fluconazole. At MIC, curcumin inhibited H+ extrusion in 2 species of Candida by 42% and 32% in the absence of glucose and by 28% and 18% in the presence of glucose. Respective inhibition of H+ extrusion caused by the MIC of fluconazole was 85% and 89% in the absence of glucose and 61% and 66% in its presence. Ergosterol content decreased by 70% and 53% for the 2 strains following exposure to curcumin at MIC; comparative values for fluconazole at MIC were 93% and 98%. Curcumin and fluconazole decreased proteinase secretion by 49% and 53%, respectively, in C. albicans and by 39% and 46%, respectively, in C. glabrata. In conclusion, curcumin is found to be active against all tested clinical and standard strains but is less effective than fluconazole. Antifungal activity of curcumin might be originating from alteration of membrane-associated properties of ATPase activity, ergosterol biosynthesis, and proteinase secretion.

Anticandidal activity of Cassia fistula and its effect on ergosterol biosynthesis.

CONTEXT: Cassia fistula Linn. (Caesalpiniaceae) has been used in folk medicine. Anthraquinone derivative rhein having antimicrobial properties is actively present in C. fistula fruit. Although, as yet there has been no study of its anticandidal potential. OBJECTIVE: The present study was conducted to determine the phytochemical composition of fruit pulp and seed extract and their effect on Candida albicans ATCC 10261, Candida glabrata ATCC 90030 and Candida tropicalis ATCC 750, respectively. MATERIALS AND METHODS: The fruit pulp and seed extracts were tested for phytochemicals by various standard methods and rhein was identified by thin-layer chromatography. The anticandidal activity was determined by minimum inhibitory concentration (MIC), growth curve studies, cytotoxicity and ergosterol estimation assay. RESULTS: The fruit pulp and seed extracts showed high content of phenolic compounds. Rhein was identified in both extracts, Rf 0.38. MICs of seed extract obtained with C. albicans, C. tropicalis and C. glabrata is 350, 300 and 300 µg/ml. However, for fruit pulp extract, these values significantly reduced to 150, 250 and 100 µg/ml, respectively. Comparative MIC values for fluconazole were 16, 16 and 04 µg/ml. At MICs, pulp reduced ergosterol content in cell membrane of C. albicans, C. tropicalis and C. glabrata by 54.42, 48.78 and 68.0%, seed extract by 38.11, 47.0 and 45.0%, whereas, fluconazole showed 93.56, 89.21 and 98.0%, respectively. DISCUSSION AND CONCLUSION: C. fistula fruit pulp and seed extract possessed anticandidal activity. The result was significantly correlated between the MICs, cytotoxicity and ergosterol inhibition. It was concluded that the crude extract is a promising source for anticandidal compounds.

Hepatitis B virus BCP, Precore/core, X gene mutations/genotypes and the risk of hepatocellular carcinoma in India.

The study aims to characterize mutations of the HBV genome involving BCP, Precore/core and X regions and also defines HBV genotypes in patients of hepatocellular carcinoma (HCC). The study involved 150 HBV-related HCC cases and 136 HBV-related chronic liver disease patients without HCC as controls. HBV DNA was subjected to mutational analysis using SSCP technique, genotyping by RFLP, and direct nucleotide sequencing. HBV DNA was found in 58.7% (88/150) of the HCC cases and 74.3% (101/136) of controls. HBV mutants were observed in 44.3% of HCC cases and 43.2% of controls. HBV/D was prevalent amongst the patients and controls, followed by HBV/A. The prevalence of the TT1504 mutation in the X gene, the V1753 and T1762/A1764 mutations in the BCP region, and G1914 mutation in the core gene were significantly higher in the HCC group than in the non-HCC group. Multivariate analyses showed that the TT1504, V1753, A1762T/G1764A, and the G1914 mutations and the patient's age, sex, and HBeAg status increased the risk of HCC development significantly. Also, patients with HCC had lower levels of serum albumin, viral load, and platelet counts but higher values of alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, bilirubin, and Alpha feto-protein than those of controls (P < 0.001 for all comparisons). HBV/D was the predominant genotype associated with HCC cases seen in India. The presence of different types of HBV mutations, age, sex, HBeAg status, and viral load was found to increase significantly the risk of HCC development in India.

Evolution of ergosterol biosynthesis inhibitors as fungicidal against Candida.

Azoles target the ergosterol synthesizing enzyme lanosterol 14alpha-demethylase and are a widely applied class of antifungal agents. Unfortunately azoles are generally fungistatic, and resistance to fluconazole is emerging in several fungal pathogens. In contrast to the increasing number of agents for the treatment of invasive fungal infections, discoveries of new antifungal agents with therapeutic value in dermatomycoses are reported only rare. Attention has been drawn to the antimicrobial activity of plants and their active principles due to the challenge of growing incidences of drug-resistant pathogens. Eugenol and methyl eugenol were reported to possess antimycotic properties. To further explore the antifungal activity of these compounds, in vitro studies were conducted on various Candida isolates. Insight studies to mechanism suggested that both eugenol and methyl eugenol exerts their antifungal activity by targeting sterol biosynthesis. Furthermore, it was also observed that additional methyl group to eugenol increases its antifungal activity. The observed fungicidal characteristics of both eugenol and methyl eugenol indicate that both the compounds might be promising antifungal agents defining a new class of antimycotics.

Anticandidal activity of cinnamaldehyde, its ligand and Ni(II) complex: effect of increase in ring and side chain.

To increase efficacy of cinnamaldehyde as an antimycotic agent, N, N'- Bis (trans-cinnamadehyde) ethylenediimine [C(20)H(20)N(2)] and Ni(II) complex of the type [Ni(C(40)H(40)N(4))Cl(2)] have been synthesized. The ligand [P] and Ni(II) complex have been characterized on the basis of elemental analysis, FTIR, ESI- MS, IR, (1)H NMR, UV-Vis spectroscopic techniques, conductivity and magnetic measurements. MIC of cinnamaldehyde against clinical isolate of Candida albicans and Candida tropicalis was 400 microg/ml and 500 microg/ml, respectively. Synthesized ligand has markedly reduced MIC; 200 microg/ml and 300 microg/ml whereas Ni(II) complex of ligand displayed MIC of 90 microg/ml and 120 microg/ml. Growth and sensitivity of the organisms were effected by ligand & complex at significantly reduced concentration. Plasma membrane ATPase activity and ergosterol content have been investigated as site of action. Result obtained indicates ergosterol biosynthesis pathway as site of action of cinnamaldehyde, synthesized ligand and its Ni(II) complex.

Significance of anti-HBc screening of blood donors and its association with occult hepatitis B virus infection: Implications for blood transfusion.

BACKGROUND & OBJECTIVES: Expansions of blood donor screening and improved laboratory detection of viral markers have remarkably reduced the risk for infection with transfusion-transmitted viruses. This study was aimed to evaluate the presence of anti-HBc and to determine the presence or absence of HBV DNA in the serum samples from HBsAg negative, anti-HBc positive blood donors in a tertiary care hospital blood bank from Delhi. METHODS: A total of 2175 HBsAg negative, first time volunteer blood donors were included in the study from blood bank, Lok Nayak Hospital, New Delhi. The blood specimens from all these subjects were evaluated for anti-HBV-core antigen (anti-HBc) serology, anti-HBV-surface antigen (anti-HBs) titres and HBeAg. The presence of HBV DNA was evaluated by testing, through polymerase chain reaction (PCR) techniques. RESULTS: Of the 2175 HBsAg negative voluntary blood donors, 413 (19.8%) were tested to be positive for anti-HBc alone. Of these, 153 (group-I) were anti-HBs negative whereas group-II comprises a total of 260 anti-HBs positive cases i.e. 89 out of 413 had anti-HBs titres of 10-99 IU/l and the remaining 171 had anti-HBs titres of 100-500 IU/l. HBV DNA was detected in 7.5 per cent anti-HBc positive samples irrespective of anti-HBs status. INTERPRETATION & CONCLUSION: Our results showed that 18.9 per cent of our donor population was anti-HBc reactive, and hence inclusion of anti-HBc testing will lead to a high discard rate. The presence of HBV DNA in fairly high percentage of anti-HBc positive samples highlighted the need for a stringent and better screening system to prevent occult HBV infection.

Pre-steady state kinetics of ATP hydrolysis by Na,K-ATPase.

Fast reaction kinetics of ATP hydrolysis by Na,K-ATPase has been investigated by following absorption pattern of pH sensitive dye in stopped flow spectrophotometer. Distinct pre-steady state phase signal could be recorded with an initial decrease in acidity followed by increase in acidity. Average half time for H(+) absorption and peak alkalinity was, respectively, 30 ms and 60 ms. Under optimal Na(+) (120 mM) and K(+) (30 mM) concentrations, magnitude of both H(+) absorption and H(+) release are found to be about 1.0 H(+)/ATPase molecule. H(+) absorption and release decreased with decrease in Na(+) concentration, H(+) release was more affected. Both H(+) absorption and H(+) release are found to be independent of K(+) concentration in the pre-steady state phase. No H(+) absorption or release was observed following mixing of either ADP, Na(+) or K(+) alone with ATPase. Effect of delayed mixing of Na(+) or K(+) on two phases of pre-steady state cycle indicates that ATP hydrolytic cycle starts without K(+) ions if optimal Na(+) is present. ATP hydrolytic cycle does not start in the absence of Na(+) ions. Results obtained have been interpreted in terms of an extended kinetic scheme for Na,K-ATPase.

Exploration of Fungal Lipase as Direct Target of Eugenol through Spectroscopic Techniques.

BACKGROUND: Fungal lipase dependent processes are important for their pathogenicity. Lipases can therefore be explored as direct target of promising herbal antifungals. OBJECTIVE: We explored Aspergillus niger lipase as a direct target of eugenol through spectroscopic techniques and compare results with Bovine Serum Albumin and lysozyme to comment on selectivity of eugenol towards lipase. METHODS: In vitro activity assays of lipase are used to determine concentration ranges. UV-Visible, Fluorescence and Circular dichroism spectroscopy were employed to determine binding constant, stoichiometric binding sites and structural changes in Lipase, BSA and lysozyme following incubation with varying concentrations of eugenol. RESULTS: In activity assays 50% inhibition of lipase was obtained at 0.913 mmoles/litre eugenol. UV-vis spectroscopy shows formation of lipase-eugenol, Bovine Serum Albumin-eugenol and lysozyme-eugenol complex well below this concentration of eugenol. Eugenol binding caused blue shift with Bovine Serum Albumin and lysozyme suggestive of compaction, and red shift with lipase. Negative ellipticity decreased with lipase but increased with Bovine Serum Albumineugenol and lysozyme-eugenol complexes suggesting loss of helical structure for lipase and compaction for Bovine Serum Albumin and lysozyme. Binding of eugenol to lipase was strong (Ka= 4.7 x 106 M-1) as compared to Bovine Serum Albumin and lysozyme. The number of stoichiometric eugenol binding sites on lipase was found to be 2 as compared to 1.37 (Bovine Serum Albumin) and 0.32 (lysozyme). Docking results also suggest strong binding of eugenol with lipase followed by Bovine Serum Albumin and lysozyme. CONCLUSION: Eugenol is found to be effective inhibitor and disruptor of secondary and tertiary structure of lipase, whereas its binding to Bovine Serum Albumin and lysozyme is found to be weak and less disruptive of structures suggesting selectivity of eugenol towards lipase.

Adenosine A3 Receptor: A promising therapeutic target in cardiovascular disease.

Cardiovascular complications are one of the major factors for early mortality in the present worldwide scenario and have become a major challenge in both developing and developed nations. It has thus become of immense importance to look for different therapeutic possibilities and treatments for the growing burden of cardiovascular diseases. Recent advancements in research have opened various means for better understanding of the complication and treatment of the disease. Adenosine receptors have become tool of choice in understanding the signaling mechanism which might lead to the cardiovascular complications. Adenosine A3 receptor is one of the important receptor which is extensively studied as a therapeutic target in cardiovascular disorder. Recent studies have shown that A3AR is involved in the amelioration of cardiovascular complications by altering the expression of A3R. This review focuses towards the therapeutic potential of A3AR involved in cardiovascular disease and it might help in better understanding of mechanism by which this receptor may prove useful in improving the complications arising due to various cardiovascular diseases. Understanding of A3AR signaling may also help to develop newer agonists and antagonists which might be prove helpful in the treatment of cardiovascular disorder.

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.

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.

Effect of nitric oxide on H+ -efflux in presence of various nutrients in Candida albicans.

In the present study tentative link has been established between H+ -efflux and effect of NO in presence of various nutrients (glucose, 2-deoxy-D-glucose, xylose, proline, glutamic acid and lysine) in C. albicans using sodium nitroprusside (SNP) as a potent source of NO. It was observed that there was a decreasing trend in pH with time, in control, while SNP treated cells showed an initial decline in pH for 10-15 min, followed by an increase in pH up to 30 min. In presence of glucose there was an enhancement in H+ -efflux by 9-fold whereas proline, glutamic acid and lysine showed enhancement by 3, 6 and 1.5-fold respectively. Similar trends in increase in pH after 15 min in SNP treated cells of Candida was observed in presence of all nutrients used. It was demonstrated for the first time that H+ -ATPase of C. albicans was affected by NO.