The effect of mycophenolic acid on the cell cycle ofCandida albicans

Mycophenolic acid inhibited the growth ofCandida albicans. Cultures exposed to a concentration of 8.4 g ml^–1 mycophenolic acid were found to exhibit cell cycle arrest with two or more buds. Nuclear staining revealed that these were nucleate implying a possible defect in cytokinesis.The results are discussed in relation to the possible mode of action of mycophenolic acid.     

Genistein effects on growth and cell cycle ofCandida albicans

Microbial virulence is generally considered to be multifactorial with infection resulting from the sum of several globally regulated virulence factors. Estrogen may serve as a signal for global virulence induction in Candida albicans. Nonsteroidal estrogens and estrogen receptor antagonists may therefore have interesting effects on yeast and their virulence factors. Growth of C. albicans was monitored by viable plate counts at timed intervals after inoculation into yeast nitrogen broth plus glucose. To determine if increased growth of yeast in the presence of estradiol was due to tyrosine kinase-mediated signaling, we measured growth in the presence of genistein, estradiol or genistein plus estradiol and compared these conditions to controls, which were not supplemented with either compound. Unexpectedly, genistein stimulated growth of C. albicans. In addition, genistein was found to increase the rate of germination (possibly reflecting release from G(0) into G(1) cell cycle phase) and also increased Hsp90 expression, demonstrated by a dot blot technique which employed a commercial primary antibody detected with chemiluminescence with horseradish peroxidase-labeled secondary antibody. These biological effects may be attributable to genistein's activity as a phytoestrogen. In contrast, nafoxidine suppressed growth of Candida and mildly diminished Hsp90 expression. This study raises the possibility of receptor cross-talk between estrogen and isoflavinoid compounds, and antiestrogens which may affect the same signaling system, though separate targets for each compound were not ruled out.     

Dimorphism-associated changes in amino acid transport of Candida albicans

Abstract Amino acid uptake was followed during pH-regulated dimorphism of Candida albicans . It was observed that transport activities of various amino acids differed with the morphological phenotype. The uptake rates of l-alanine , l -phenylalanine and of l -lysine were lower and those of l -methionine were higher in elongated hypha (germ tube), while the rates of glycine, l -glutamic acid and l -proline were similar in bud and hyphal phenotypes. Minimum threshold of amino acids transport activity is required at the time of phenotypic commitment in a diverging population of Candida albicans .     

Characterization of amino acid transport during erythroid cell differentiation

We have studied the changes in amino acid transport in fetal erythroid cells isolated from rat fetal liver at different gestation days. Our results show that System A transport as measured by the Na+-dependent uptake of 2-(methylamino)isobutyric acid (MeAIB) was conspicuous at day 13 but virtually disappeared between days 16 and 18. In contrast, the activity of System ASC measured by the Na+-dependent uptake of MeAIB-insensitive threonine uptake increased after day 14 and was optimal between days 16 and 18. This transport system regressed in activity with further maturation, but remained conspicuously saturable in the matured red blood cell. Interestingly, the newly discovered Na+-independent System asc (Vadgama, J. V., and Christensen, H.N. (1985) J. Biol. Chem. 260, 2912-2921), selective for the uptake of test substrates threonine, serine, and alanine, was present in these erythroid cells. Its activity increased during gestation days 16-18. System L transport was present simultaneously with the Na+-independent System asc. As we had previously demonstrated for the pigeon red blood cell, these two transport systems are kinetically independent as confirmed with inhibition studies and the special selectivity of System L to trans stimulation. Tryptophan uptake could be attributed predominantly to System L, as also observed for the nucleated pigeon red blood cells and certain other cells. Arginine showed its familiar Na+-independent mode of uptake as a cation throughout the interval of study. An exceptional Na+-dependent component of arginine uptake emerged after day 14, peaked at day 18, and then disappeared on further maturation of the erythroid cell.     

Action of levorin on amino acid transport in Candida albicans]

It was shown that suppression by levorin of the leucine transport into the cells of C. albicans was due to replacement of intracellular K+ by Na+ induced by the antibiotic. The alanine transport was suppressed by levorin irrespective of the ratio of the monovalent cations concentration in the medium and inside the cell. The levorin effect on the protone escape from the cells was negligible and probably played no significant role in the mechanism of the amino acid transport suppression by the antibiotic.     

Control of the cell cycle ofCandida utilis by external conditions

A mathematical model of the cell cycle ofCandida utilis in a continuous culture was formulated with respect to dilution rate. It makes it possible to express the duration of morphological stages in minutes, separately for mother cells and daughter cells. These values were compared with equivalent parameters in batch cultures. Duration of the morphological stage with buds was much longer in batch cultures as compared with the same value determined for a continuous culture according to the mathematical model. When using cultivation apparatus with a higher aeration capacity the (S + G₂) phase, i.e. the stage bearing the bud, was reduced also in the batch cultures and approached the values determined for the continuous culture by means of the mathematical model.     

Differences between resting and insulin-stimulated amino acid transport in frog skeletal muscle

Summary We have compared some features of the resting and the insulin-stimulated uptake of -aminoisobutyrate (AIB) in frog skeletal muscle. We found a substantial difference between the two processes, namely, that resting AIB uptake is Na-independent while the insulin-stimulated fraction of the AIB uptake is Na-dependent.Since the amino acid transport systems in frog skeletal muscle are poorly characterized, we have also surveyed some of their properties. One of the most interesting findings of this survey is that both the uptake and efflux of AIB are inhibited by low concentrations of PCMBS (parachloro-mercury-benzene sulfonic acid 5×10^–5 m). In contrast, the carrier mediated transport of basic amino acids is neither inhibited by this mercurial agent nor accelerated by insulin.The action of PCMBS strongly suggests the presence of a critical sulfhydryl group in the amino acid carrier system utilized by AIB. This group is exposed to the outside solution since PCMBS penetrates cell membranes poorly, and in addition its inhibitory actions were reverted by agents that do not penetrate the cell membrane like albumin or glutathione.     

Involvement of transglutaminase in the formation of covalent cross-links in the cell wall ofCandida albicans

Activity of the enzyme glutaminyl-peptide-γ-glutamylyl-transferase (EC 2.3.2.13; transglutaminase), which forms the interpeptidic cross-link N^∈-(γ-glutamic)-lysine, was demonstrated in cell-free extracts obtained from both the yeast like and mycelial forms ofCandida albicans. Higher levels of enzymatic activity were observed in the cell wall fraction, whereas the cytosol contained only trace amounts of activity. Cystamine, a highly specific inhibitor of the enzyme, was used to analyze a possible role of transglutaminase in the organization of the cell wall structure of the fungus. Cystamine delayed protoplast regeneration and inhibited the yeast-to-mycelium transition and the incorporation of proteins into the cell wall. The incorporation of covalently bound high-molecular-weight proteins into the wall was sensitive to cystamine. Proteic epitopes recognized by two monoclonal antibodies, one of which is specific for the mycelial walls of the fungus, were also sensitive to cystamine. These data suggest that transglutaminase may be involved in the formation of covalent bonds between different cell wall proteins during the final assembly of the mature cell wall.     

A comparative study on cell wall antigens and cell surface hydrophobicity in clinical isolates ofCandida albicans

Characterization of common cell surface-bound antigens inCandida albicans strains, particularly those expressed in the walls of mycelial cells might be useful in the diagnosis of systemic candidiasis. Hence, antigenic similarities among wall proteins and mannoproteins fromC. albicans clinical serotype A and B isolates, were studied using polyclonal (mPAbs) and monoclonal (MAb 4C12) antibodies raised against wall antigens from the mycelial form of a commonC. albicans serotype A laboratory strain (ATCC 26555). Zymolyase digestion of walls isolated from cells of the different strains studied grown at 37°C (germination conditions), released, in all cases, numerous protein and mannoprotein components larger than 100 kDa, along with a 33–34 kDa species. The pattern of major antigens exhibiting reactivity towards the mPAbs preparation was basically similar for all the serotype A and B isolates, though minor strain-specific bands were also observed. The immunodeterminant recognized by MAb 4C12 was found to be absent or present in very low amounts inC. albicans isolates other than the ATCC 26555 strain, yet high molecular weight species similar in size (e.g., 260 kDa) to the wall antigen against which MAb 4C12 was raised, were observed, particularly in wall digests from serotype A strains. Cell surface hydrophobicity, an apparently important virulence factor inC. albicans, of the cell population of each serotype B strain was lower than that of the corresponding serotype A counterparts, which is possibly due to the fact that the former strains exhibited a reduced ability to form mycelial filaments under the experimental conditions used.Abbreviations CSH cell surface hydrophobicity - IIF indirect immunofluorescence     

Population study of cell cycle in a continuous culture ofCandida utilis

The mean lengths of G1, S, G2 and M phases of the cell cycle were determined on the basis of the population distribution ofCandida utilis grown in a continuous culture under steady-state conditions by using an original mathematical method. The length of the G2 phase was proportional to that of G1; the length of M was effectively independent of the growth rate. The length of S was proportional to the mean number of mitochondria in the cell.     

Differences in amino acid transport in isonuclear lines of cytoplasmic male-sterile and male-fertile petunia

Summary Two pairs of isonuclear lines of cytoplasmic male-sterile (CMS) and fertile (F) petunia cells grown in suspension culture in the presence or absence of amino acid sources were examined for uptake of 11 amino acids and adenosine. Cells from CMS lines exhibited a significant lower rate of uptake than F cells. These differences, for various amino acids, are a result of lower affinity (high Km) values and of lower maximal velocities. Although the uptake of most of the amino acids examined was affected by the availability of energy in the cell, the differences in uptake seem to be less dependent on the energy status of the cell.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 3351-E, 1991 series     

Influence of methylfenpropidine on growth, sterol content and fatty acid composition ofCandida albicans

The effect of methylfenpropidine on growth, lipid contents, sterol and fatty acid composition was investigated in 5 strains ofCandida albicans. The sensitivity of the strains decreased in the order: wild strains >erg ⁺ ade nys ^R >ade nys ^R erg (defective Δ⁸⁻⁷-isomerase) >ade nys ^R erg (defective Δ⁵-desaturase). The presence of the inhibitor influenced fecosterol isomerization, episterol dehydrogenation, zymosterol transmethylation, ignosterol reduction and squalene epoxidation. Methylfenpropidine also induced changes in fatty acid composition, causing a reduction of the palmitic and oleic acid content with a concomitant elevation of stearic, linoleic and linolenic acid levels. The lipid unsaturation index slightly increased. Morphological changes of wild strains were observed after the fungicide treatment.     

Differential membrane phospholipid synthesis during the cell cycle of Caulobacter crescentus.

The pattern of phospholipid synthesis during the cell cycle of Caulobacter crescentus has been determined. Although the phospholipid composition of swarmer and stalked cells was indistinguishable in continuously labeled cultures if the two cell types were pulse-labeled for a short time period, marked differences in the pattern of phospholipid synthesis were detected. Pulse-labeled swarmer cells exhibited a higher proportion of phosphatidic acid and a lower proportion of phosphatidylglycerol. In addition, minor phospholipids were detected in the swarmer cells that were not detected in stalked cells. Stalked cells that developed directly from swarmer cells showed that same phospholipid profile as the swarmer cells. The switch to the second phospholipid profile was observed to occur at the predivisional cell stage. Because cell division then yielded a swarmer cell with a different phospholipid profile than its sibling stalked cell, the cell division process may trigger a mechanism which alters the pattern of phospholipid synthesis.     

Effectors of amino acid transport processes in animal cell membranes

Various effectors, which act upon ion gradients, protein synthesis, membrane components or cellular functional groups, have been employed to provide insights into the nature of amino acid-membrane transport processes in animal cells. Such effectors, for example, include ions, hormones, metabolites and various organic reagents and their judicious use has allowed the following list of conclusions. Sodium ion has been found to stimulate amino acid transport in a wide variety of cell systems, although depending on the tissue and/or substrate, this ion may have no effect on such transport, or even inhibit it. Amino acid transport can be stimulated in some cell systems by other ions such as K+, Li+, H+ or Cl-. Both H+ and K+ have been found to be inhibitory in other systems. Amino acid transport is dependent in many cell systems upon an inwardly directed Na+ gradient and is stimulated by a membrane potential (negative cell interior). In some cell systems an inwardly directed Cl- and H+ gradient or an outwardly directed K+ gradient can energize transport. Structurally dissimilar effectors such as ouabain, Clostridium enterotoxin, aspirin and amiloride inhibit amino acid transport presumably through dissipation of the Na+ gradient. Inhibition by certain sugars or metabolic intermediates of the tricarboxylic acid cycle may compete with the substrate for the energy of the Na+ gradient or interact with the substrate at the carrier level either allosterically or at a common site. Stimulation of transport by other sugars or intermediates may result from their catabolism to furnish energy for transport. Insulin and glucagon stimulate transport of amino acids in a variety of cell systems by a mechanism which involves protein synthesis. Microtubules may be involved in the regulation of transport by insulin or glucagon. Some reports also suggest that insulin has a direct effect on membranes. In addition, a number of growth hormones and factors have stimulatory effects on amino acid transport which are also mediated by protein synthesis. Steroid hormones have been noted to enhance or diminish transport of amino acids depending on the nature of the hormone. These agents appear to function at the level of protein synthesis. While stimulation may involve increased carrier synthesis, inhibition probably involves synthesis of a labile protein which either decreases the rate of synthesis or increases the rate of degradation of a component of the transport system.(ABSTRACT TRUNCATED AT 400 WORDS)     

Early intermediates in the transport cycle of the neuronal excitatory amino acid carrier EAAC1

Electrogenic glutamate transport by the excitatory amino acid carrier 1 (EAAC1) is associated with multiple charge movements across the membrane that take place on time scales ranging from microseconds to milliseconds. The molecular nature of these charge movements is poorly understood at present and, therefore, was studied in this report in detail by using the technique of laser-pulse photolysis of caged glutamate providing a 100-micros time resolution. In the inward transport mode, the deactivation of the transient component of the glutamate-induced coupled transport current exhibits two exponential components. Similar results were obtained when restricting EAAC1 to Na(+) translocation steps by removing potassium, thus, demonstrating (1) that substrate translocation of EAAC1 is coupled to inward movement of positive charge and, therefore, electrogenic; and (2) the existence of at least two distinct intermediates in the Na(+)-binding and glutamate translocation limb of the EAAC1 transport cycle. Together with the determination of the sodium ion concentration and voltage dependence of the two-exponential charge movement and of the steady-state EAAC1 properties, we developed a kinetic model that is based on sequential binding of Na(+) and glutamate to their extracellular binding sites on EAAC1 explaining our results. In this model, at least one Na(+) ion and thereafter glutamate rapidly bind to the transporter initiating a slower, electroneutral structural change that makes EAAC1 competent for further, voltage-dependent binding of additional sodium ion(s). Once the fully loaded EAAC1 complex is formed, it can undergo a much slower, electrogenic translocation reaction to expose the substrate and ion binding sites to the cytoplasm.     

Morphogenesis and cell cycle progression in Candida albicans

Candida albicans, an opportunistic human pathogen, displays three modes of growth: yeast, pseudohyphae and true hyphae, all of which differ both in morphology and in aspects of cell cycle progression. In particular, in hyphal cells, polarized growth becomes uncoupled from other cell cycle events. Yeast or pseudohyphae that undergo a cell cycle delay also exhibit polarized growth, independent of cell cycle progression. The Spitzenk?rper, an organelle composed of vesicles associated with hyphal tips, directs continuous hyphal elongation in filamentous fungal species and also in C. albicans hyphae. A polarisome mediates cell cycle dependent growth in yeast and pseudohyphae. Regulation of morphogenesis and cell cycle progression is dependent upon specific cyclins, all of which affect morphogenesis and some of which function specifically in yeast or hyphal cells. Future work will probably focus on the cell cycle checkpoints involved in connecting morphogenesis to cell cycle progression.