Carlos Calvo-Méndez

The role of polyamine metabolism in dimorphism of Yarrowia lipolytica.

We have devised a convenient procedure to induce the yeast-to-mycelium transition of Yarrowia lipolytica in conditions which avoid the occurrence of the reverse process during the period of study. Yeast cells in late exponential phase were resuspended in water and cooled down to 4 degrees C for at least 15 min, then heat-shocked by inoculation into a pre-warmed (30 degrees C) medium containing N-acetyl-D-glucosamine. Under these conditions, yeast cells developed into large branching filaments which continued elongating for more than 24 h. Further, ornithine decarboxylase (ODC) activity and polyamine cell pools increased compared to those of cells maintained in glucose medium, which continued yeast-like growth. Addition of ODC inhibitors blocked mycelial development, but only if added during a critical initial period after which they had no effect. At effective concentrations, ODC inhibitors had no significant effect on cell growth. Comparative studies of intact and permeabilized cells suggest that this selective effect is probably due to the location of ODC in more than one cell compartment, one of them being inaccessible to the drugs. Blocking of the morphological transition by ODC inhibitors was specifically reversed by putrescine, and by growing the cells in the presence of 5-azacytidine. It is suggested that the effect of the latter compound is related to its capacity to inhibit DNA methylation, indicating a relationship between polyamines and DNA methylation at the onset of the differentiation process.

Cr(VI) reduction in a chromate-resistant strain of Candida maltosa isolated from the leather industry

A Cr(VI)-resistant yeast was isolated from tanning liquors from a leather factory in Leon, Guanajuato, Mexico. Based on morphological and physiological analyses and the D1/D2 domain sequence of the 26S rDNA, the yeast was identified as Candida maltosa. Resistance of the strain to high Cr(VI) concentrations and its ability to chemically reduce chromium was studied. When compared to the three laboratory yeasts Candida albicans, Saccharomyces cerevisiae and Yarrowia lipolytica, the C. maltosa strain was found to tolerate chromate concentrations as high as 100 μg/ml. In addition to this phenotypic trait, the C. maltosa strain showed ability to reduce Cr(VI). Chromate reduction occurred both in intact cells (grown in culture medium or in soil containing chromate) as well as in cell-free extracts. NADH-dependent chromate reductase activity was found associated with soluble protein and, to a lesser extent, with the membrane fraction.

Chitinase activity in encysting Entamoeba invadens and its inhibition by allosamidin.

Chitinase activity was measured in extracts of Entamoeba invadens cells as a function of time of encystation in axenic conditions using 4-MU(Ch)3 as substrate. Encystment was paralleled by chitinase activity which showed a peak after about 72 h of cultivation where cysts accounted for 63% of cell population. Thereafter, activity fell off rapidly, whereas encystment continued, reaching 80% at the end of the experiment (96 h). Comparison of activity between cysts and the total cell population in 48- and 72-h-old encysting cultures suggested that chitinase may start to accumulate in the pre-cyst forms. About 70% of the enzyme was recovered in the supernatant following low-speed centrifugation of whole extracts. Most of this activity represented soluble chitinase since it was not sedimented by further centrifugation at 105,000 x g. A minor proportion of enzyme activity remained associated to the buffer-washed, high-speed sediment. In addition to 4-MU(Ch)3, chitinase activity was also measured following the hydrolysis of other substrates such as nascent, preformed or colloidal chitin. Like other chitinases, the cyst enzyme preferred nascent over preformed chitin as substrate. Digestion of the former yielded GlcNAc and minor amounts of (GlcNAc)2 as products. Allosamidin strongly inhibited hydrolysis of the fluorogenic substrate by the amebic chitinase in vitro with a Ki of 0.065 microM. IC50 values were 0.085 microM and 0.16 microM at 5 microM and 10 microM 4-MU(Ch)3, respectively. When added to the axenic medium, the drug markedly retarded encystment though it was partially recovered after longer periods of incubation.

Inhibition of the yeast-mycelial transition and the phorogenesis of Mucorales by diamino butanone.

Diamino butanone (DAB), a competitive inhibitor of ornithine decarboxylase (ODC) a key enzyme in polyamine biosynthesis, inhibited the yeast to hyphae transition in Mucor rouxii, induced by transfer from anaerobiosis to aerobiosis, but not the opposite phenomenon. Addition of DAB to anaerobic yeast cells brought about a decrease in ODC and polyamine levels. In these conditions, the aerobic shift produced only a weak increase in ODC activity and no change in polyamine levels. DAB also blocked phorogenesis in M. rouxii and in Phycomyces blakesleeanus. At the effective concentrations DAB did not affect cell growth of either fungus. It is suggested that low, constant levels of ODC and polyamines are necessary for cell growth, and that high transient levels are required during the differentiative steps. DAB, at the concentrations used, affects this last process, but does not interfere with the maintenance level of polyamines.

Regulation of ornithine decarboxylase activity in Mucor bacilliformis and Mucor rouxii

Abstract Ornithine decarboxylase (ODC) from Mucor bacilliformis and Mucor rouxii was studied. Enzymatic activity was maximal at pH 7.2–7.4 and at 30°C. The K m was 0.17 m M for the M. bacilliformis enzyme. Putrescine was a competitive inhibitor of ODC with a K i of 2–3 m M . Enzymatic activity was undetectable in sporangiospores but increased rapidly during the first stages of spore swelling, reaching the highest levels during germ tube or bud emergence, and then decreased. Incubation at 30°C inhibited spore germination in M. bacilliformis and prevented development of ODC activity. More ODC activity was present in mycelial than in yeast cells. Morphological transition of yeast cells into hyphae by an anaerobic-aerobic shift induced a rapid and transient increase in ODC activity. Similar results were obtained when the morphogenetic transformation of M. rouxii was induced by CO 2 elimination in an anaerobic environment. Transfer of mycelial cells to anaerobiosis resulted in a rapid decrease in enzyme activity. Changes in ODC activity were accompanied by a change in the pool of polyamines. The possible role of ODC in growth and cell differentiation in Mucor is discussed.

Effect of ornithine decarboxylase inhibitors on the germination of sporangiospores of mucorales

Of several known inhibitors of ornithine decarboxylase activity, only 1,4-diamino-2-butanone (DAB) inhibited spore germination of Mucor rouxii. Drug-exposed cells went through the phase of spherical growth attaining huge cell volumes, but were unable to germinate. The same phenomenon occurred irrespective of the growth medium in several species of Mucorales. DAB barely inhibited the biosynthesis of proteins and DNA during the phase of spherical growth. Higher levels of inhibition were observed at later growth periods. The addition of DAB to the growth medium decreased the cellular levels of polyamines. Removal of DAB or addition of putrescine reversed the inhibitory effect. Cycloheximide and actinomycin D completely blocked the reversal of the inhibition of germination after DAB removal or addition of putrescine, whereas hydroxyurea gave variable intermediate values of inhibition. It is suggested that the DAB effect can be interpreted as a requirement of high levels of polyamines for the change of growth patterns from spherical to apical in the fungi studied.

Biosynthesis of glycoproteins in Candida albicans : activity of dolichol phosphate mannose synthase and protein mannosylation in a mixed membrane fraction

A mixed membrane fraction (MMF) was isolated from yeast cells of Candida albicans with the ability to synthesize dolichol phosphate mannose (Dol-P-Man) from GDP-Man and dolichol phosphate (Dol-P) and transfer the sugar to proteins. Temperature of incubation (20-37 degrees C) did not affect the synthesis of Dol-P-Man but protein mannosylation occurred better at physiological temperatures (28 degrees C and 37 degrees C). Most of the sugar (87-93%) in the mannoproteins was O-linked as judged by its release by beta-elimination. Mannose was identified as the sole product after this treatment. Following incubation of MMF with the sugar donor, parallel levels of Dol-P-Man and mannosylated proteins were detected up to 30 min. Thereafter, Dol-P-Man levels reached a steady value whereas mannoproteins rapidly accumulated. Lipid-linked oligosaccharides were also detected in incubation mixtures, though in much lower amounts than those of Dol-P-Man or mannoproteins. Dol-P-Man synthase activity increased proportionally in response to increasing concentrations of either of the two enzyme substrates. A Km value of 0.36 microM for GDP-Man was calculated. MMF failed to use exogenous Dol-P-Man for protein glycosylation. Specific inhibition of Dol-P-Man synthesis with amphomycin was concomitant with a parallel decrease in protein mannosylation, indicating that most of the sugar is transferred to protein via the carrier lipid. Results are discussed in terms of the role of Dol-P-Man in protein glycosylation in C. albicans.

Biosynthesis of chitosan in membrane fractions fromMucor rouxii by the concerted action of chitin synthetase and a particulate deacetylase

Mixed membrane fractions fromMucor rouxii synthesize two different products when incubated with uridine diphosphateN-acetylglucosamine: one containing an average of 2200–3000N-acetylglucosamine units (chitin), and a second one of about equal size containing, in addition toN-acetylglucosamine, a large proportion of glucosamine units (chitosan). Biosynthesis of both polymers requires chitin synthetase, but biosynthesis of the second, partially deacetylated product also requires the action of a membrane-bound deacetylase. The deacetylase was absent from purified chitosomes and chitosomal subunits. Evidence was obtained that during chitosan biosynthesis, chain formation and deacetylation occur simultaneously. It is hypothesized that the role of the membrane-bound deacetylase activity is to initiate the deacetylation of a proportion of nascent chitin chains which may be thus recognized by an extracellular soluble deacetylase responsible for their further deacetylation.

Ornithine decarboxylase activity in Entamoeba invadens.

Growth of E. invadens was paralleled by a concomitant increase in ornithine decarboxylase activity which peaked after 5 days of cultivation in TYI-S-33 medium. Over this period, enzyme activity increased about nine-fold with respect to that present at the start of incubation. Thereafter and coinciding with the onset of the stationary growth phase, enzyme activity started to decline reaching trace levels after 8 days of cultivation. Most of the enzyme remained soluble following centrifugation of amoeba homogenates at 105,000 g. alpha-Difluoromethylornithine failed to affect ornithine decarboxylase activity in vitro and amoeba growth. The enzyme was markedly inhibited by polyamines (putrescine, spermidine and spermine) and 1,4-diamino-2-butanone, a putrescine-analog. The latter arrested proliferation of cells, an effect that could not be reversed by polyamines which by themselves also inhibited growth to a low but significant extent. Our results indicate that polyamine biosynthesis from ornithine is required for growth of E. invadens and that this function is rapidly abolished following entry into the stationary growth phase.

Bursera fagaroides, effect of an ethanolic extract on ornithine decarboxylase (ODC) activity in vitro and on the growth of Entamoeba histolytica.

The effect of an ethanolic extract from the stem bark of Bursera fagaroides on ornithine decarboxylase (ODC) activity in vitro and on the growth of Entamoeba histolytica was evaluated. For this purpose, increasing concentrations of the extract, up to 8.0mg/mL, were added to amoeba cultures or ODC reaction mixtures, which were incubated at 37 degrees C. Metronidazole and G418 were added as controls. After 1.5 and 72 h, the ODC activity in vitro and growth, respectively, were determined. Results revealed a strong inhibition of growth with IC(50) values on the order of 0.05 mg/mL. ODC activity, on the other hand, was inhibited by 12% and 50% at concentrations of 4.0 and 8.0mg/mL, respectively.