Influence of phenothiazines, phenazines and phenoxazine on cation transport in Candida albicans.

AIMS: (i) To analyse the increase in calcium ion uptake caused by several cationic dyes on Candida albicans, (ii) to postulate a mechanism, (iii) to define the effects of Zn ions on the phenomenon, and (iv) to propose the use of the dyes or their derivatives against C. albicans. METHODS AND RESULTS: Cells were grown in yeast peptone dextrose medium and starved. We measured the hydrophobic solvent/water partition coefficients and the dyes uptake by the cells and found no correlation with their hydrophobicity. Most of the dyes caused an increase in K+ efflux (in correlation with a decrease in 86 Rb+ uptake), and a raise in Ca2+ uptake except for those used as Zn salts, but not of their HCl salts. Respiration and acidification of the medium were modified only with few dyes and interestingly, when exposing cultures to nile blue, neutral red and toluidine blue ZnCl2 a decrease in C. albicans growth was observed. CONCLUSIONS: We propose a general mechanism for the stimulation of Ca2+ uptake by the dyes used. Some of the dyes tested might be used as agents against C. albicans, probably combined with other agents. Moreover, the effects of Zn ions on Ca2+ uptake and on cell growth open possibilities of further studies, not only of their effects, but also of the mechanism of Ca2+ transport in C. albicans and other yeasts. SIGNIFICANCE AND IMPACT OF THE STUDY: This study, in conjunction with previously published results, contribute to the basic research regarding ion transport in C. albicans and the role of zinc in this process. Besides, suggests the additional use of dyes, along with other antifungals agents, as combined therapy against candidiasis. Derived dyes from those used also might be possible therapeutic agents against this disease.

In the ovine pituitary, CXCR4 is localized in gonadotropes and somatotropes and increases with elevated serum progesterone.

The pituitary is the central endocrine regulator of reproduction and in addition to various hormones regulating its actions, other molecules, such as chemokines, influence pituitary physiology as well. Despite reports over 2 decades ago that chemokines regulate the pituitary, much of the basic biology discerning chemokine action in the pituitary is unclear. A small number of chemokines and their receptors have been localized to the pituitary, yet chemokine ligand 12 (CXCL12) and its receptor, CXCR4, have received the most attention as both are increased in human pituitary adenomas. This chemokine duo was also reported in normal human and rat pituitary, suggestive of a functional role and that this chemokine axis might function in pituitaries from other mammalian species. To date, reports of CXCL12 and CXCR4 in pituitary from livestock are lacking, and research on pituitary during pregnancy in any mammalian species is limited. Moreover, progesterone regulates CXCR4 expression in a tissue-dependent manner, but whether differing concentrations of progesterone reaching the pituitary modulate CXCL12 or CXCR4 is not known. To address these gaps, our first objective was to determine if CXCL12 and CXCR4 expression and protein abundance differ in sheep pituitary during early gestation (days 20, 25, and 30 of gestation) compared to nonpregnant ewes. The second objective was to determine if CXCL12 or CXCR4 production was altered in the ovine pituitary when circulating progesterone concentrations are elevated. The expression of CXCL12 messenger RNA decreased on day 20 of gestation compared to nonpregnant ewes; CXCL12 protein was similar across all days tested. In nonpregnant and pregnant ewes, CXCR4 was localized to somatotropes and gonadotropes on all days tested. Abundance of CXCR4 increased in the pituitary tissue of pregnant ewes with elevated circulating progesterone compared with pregnant ewes with normal circulating progesterone concentrations (control). The present study details CXCL12 and CXCR4 in normal ovine pituitary and reveals that gonadotropes and somatotropes may be regulated by CXCL12/CXCR4, underscoring this signaling axis as a potential new class of modulator in endocrine functions.

Glycolytic sequence and respiration of Debaryomyces hansenii as compared to Saccharomyces cerevisiae.

The fermentation and respiration activities of Debaryomyces hansenii were compared with those of Saccharomyces cerevisiae grown to stationary phase with high respiratory activity. It was found that: (a) glucose consumption, fermentation and respiration were lower than for S. cerevisiae; (b) fasting produced a much smaller decrease of respiration; (c) glucose consumed and not transformed to ethanol was higher; (d) in S. cerevisiae, full oxygenation prevented ethanol production but this effect was reversed by CCCP, whereas D. hansenii still showed some ethanol production under aerobiosis, which was moderately increased by CCCP. ATP levels were similar in the two yeasts. Levels of glycolytic intermediaries after glucose addition, and enzyme activities, indicated that the main difference and limiting step to explain the lower fermentation of D. hansenii is phosphofructokinase activity. Respiration and fermentation, which are lower in D. hansenii, compete for the re-oxidation of reduced nicotinamide adenine nucleotides; this competition, in turn, seems to play a role in defining the fermentation rates of the two yeasts. The effect of CCCP on glucose consumption and ethanol production also indicates a role of ADP in both the Pasteur and Crabtree effects in S. cerevisiae but not in D. hansenii. D. hansenii shows an alternative oxidase, which in our experiments did not appear to be coupled to the production of ATP.

Requirements of Cyc2p and the porin, Por1p, for ionic stability and mitochondrial integrity in Saccharomyces cerevisiae.

It was previously demonstrated that Cyc2p from Saccharomyces cerevisiae is a mitochondrial protein; that the cyc2-Delta2 deletion lacking the entire gene causes a diminution to only approximately 20% of the normal levels of cytochrome c due to a partial deficiency in mitochondrial import of apo-cytochrome c; that the deletion causes a defective mitochondrial function, as revealed by diminished growth on media containing nonfermentable carbon sources; and that this defect is exacerbated in hyper-ionic KCl media and at higher incubation temperatures, but is suppressed on media containing sorbitol, a nonionic compound. We report that por1-Delta strains lacking the mitochondrial porin, Por1p, but not por2-Delta strains lacking the related porin, share some phenotypes similar to the cyc2-Delta2 strain, including hypersensitivity to KCl in glycerol medium. Moreover, spontaneous swelling in the presence of ATP was detected in mitochondria from the cyc2-Delta2 strain, while swelling could be detected in mitochondria from the other strains only after the addition of KCl. Thus, highly unspecific membrane permeation may be triggered by ATP in the cyc2-Delta2 strain. We suggest that Por1p and Cyc2p, in addition to their own unique functions, serve to maintain the osmotic stability of mitochondria, but by different mechanisms.

Andrographis paniculata (Ness) induces relaxation of uterus by blocking voltage operated calcium channels and inhibits Ca(+2) influx.

The possible relaxation of uterine smooth muscle by Andrographis paniculata dried extract via a blockade of voltage operated calcium channels was investigated in rats. Uterine horns pretreated with oestradiol were incubated in Ca(+2) -free Jalon's solution and stimulated with KCl (20-60 mM) in order to produce depolarization of the membrane. The isometric contractile response to 1 mM or cumulative concentrations of CaCl2 were blockaded by 0.2, 0.4 and 0.8 mg/mL of A. paniculata. The maximum contractile response induced by acetylcholine was moderately antagonized by A. paniculata. The possible blockade of Ca(+2) entry by A. paniculata was evaluated with (45)Ca(+2) uptake in uterine rings incubated with free-Ca(+2)-Ringer's solution high in K+ (KCl 40 mM). The influx was completely blockaded with 0.4 mg/mL of A. paniculata. These results strongly suggest that A. paniculata blockades voltage operated calcium channels inhibiting the entry of Ca(+2) from the external medium.

Andrographis paniculata (Nees) selectively blocks voltage-operated calcium channels in rat vas deferens.

The possible blockade of voltage-operated calcium channels (VOCs) by Andrographis paniculata dried extract in vas deferens smooth muscle was investigated in rats. The tissues were incubated in Ca(2+)-free Kreb's solution and stimulated with KCl (40 mM) to produce depolarisation of the membrane. The isometric contractile response to cumulative concentrations of CaCl(2) was effectively blockaded by 0.2 and 0.4 mg/ml A. paniculata. In other experiments, the maximum contractile response induced by norepinephrine was not antagonised by 0.2, 0.4 or 0.8 mg/ml A. paniculata. The possible blockade of Ca(2+) entry by A. paniculata was evaluated with 45Ca(2+) uptake in vas deferens treated with reserpine (5 and 2.5 mg/kg) 48 and 24 h before the experiments. Epididymal segments were incubated with Ca(2+)-free Kreb's solution with KCl, 25 and 50 mM. The influx was completely blockaded with 0.4 mg/ml A. paniculata. These results suggest that A. paniculata selectively blockades VOCs, hence inhibiting the 45Ca(2+) influx.

Testicular toxicity assessment of Andrographis paniculata dried extract in rats.

The possible testicular toxicity of Andrographis paniculata, Nees (Acanthaceae) standardized dried extract was evaluated in male Sprague Dawley rats for 60 days. No testicular toxicity was found with the treatment of 20, 200 and 1000 mg/kg during 60 days as evaluated by reproductive organ weight, testicular histology, ultrastructural analysis of Leydig cells and testosterone levels after 60 days of treatment. It is concluded that Andrographis paniculata dried extract did not produce subchronic testicular toxicity effect in male rats.