Evaluation of industrial yeasts for pathogenicity.

Eleven yeasts representative of species of industrial interest were compared with Candida albicans for their potential pathogenicity for untreated and cortisone-treated mice. Only C. tropicalis produced a progressive infection similar to that produced by C. albicans. Candida lipolytica, Torulopsis spp., and Hansenula polymorpha were not recovered from mice 6 days after inoculation. Kluyveromyces fragilis, C. pseudotropicalis, C. utilis, C. guilliermondii and C. maltosa were recovered from mice but did not produce evidence of infection.     

Candida adherence phenomena, from commensalism to pathogenicity.

Molecules present in the most external layers of Candida cells are essential for the adherence to host surfaces, playing a pivotal role in the pathophysiology of candidiasis. Receptors for fibrinogen, fibronectin and other components of the extracellular matrix have been described in Candida surfaces. Their expression may be influenced by particular host conditions, and these changes may be important in the transition from commensalism to pathogenicity. Surface proteins are also essential in the interactions of the fungal cell with the various constitutive, inducible defense host mechanisms that act during candidiasis.     

2-Oxoamide inhibitors of phospholipase A2 activity and cellular arachidonate release based on dipeptides and pseudodipeptides

A series of 2-oxoamides based on dipeptides and pseudodipeptides were synthesized and their activities towards two human intracellular phospholipases A₂ (GIVA cPLA₂ and GVIA iPLA₂) and one human secretory phospholipase A₂ (GV sPLA₂) were evaluated. Derivatives containing a free carboxyl group are selective GIVA cPLA₂ inhibitors. A derivative based on the ethyl ester of an ether pseudodipeptide is the first 2-oxoamide, which preferentially inhibits GVIA iPLA₂. The effect of 2-oxoamides on the generation of arachidonic acid from RAW 264.7 macrophages was also studied and it was found that selective GIVA cPLA₂ inhibitors preferentially inhibited cellular arachidonic acid release; one pseudodipeptide gave an IC₅₀ value of 2 μM.     

Annexins in cardiac tissue: cellular localization and effect on phospholipase activity

Stimulation of cardiac phospholipid metabolism has diverse biological effects, ranging from subtle changes in cellular function to severe cellular damage. Accordingly, knowledge of the factors governing the activity of cardiac phospholi pases is of great biological importance. A possible role of annexins, intracellular proteins that bind to membranes in a calcium dependent manner, as modulators of phospholipase activity has been proposed. In this study we investigated the cell type specific distribution of Annexin V and VIII in the heart. Recombinant Annexin V was used to examine the effect of this type of Annexin on cardiac phospholipase activity. Western blot analysis shows that annexin V is abundantly present in the heart. Using isolated myocytes and cultured cardiac endothelial and fibroblast-like cells, it is demonstrated that the localization of Annexin V is confined to non-myocytes. No positive bands matching the Mw of recombinant Annexin VIII are found in any of the cell types examined.In vitro studies demonstrate that recombinant Annexin V potently inhibits the activity of cardiac membrane-bound phospholipases, acting on their natural sur rounding substrate, in a calcium dependent manner. Interestingly, annexin V also inhibits triacylglycerol hydrolysis. In conclusion, the expression of annexins is cell-type specific and suggests a cell-type specific function of these proteins in the heart. The absence of Annexin V in cardiac myocytes dismisses involvement of this annexin in cardiomyocyte phospholipid metabolism. The presence of Annexin V in cardiac endothelial and fibroblasts suggests a regulating role in the phospholipid homeostasis of non-myocyte cell types in the heart. (Mol Cell Biochem116: 95–101, 1992)     

Antibacterial activity of snake, scorpion and bee venoms: a comparison with purified venom phospholipase A2 enzymes

AIMS: Venoms of snakes, scorpions, bees and purified venom phospholipase A(2) (PLA(2)) enzymes were examined to evaluate the antibacterial activity of purified venom enzymes as compared with that of the crude venoms. METHODS AND RESULTS: Thirty-four crude venoms, nine purified PLA(2)s and two L-amino acid oxidases (LAAO) were studied for antibacterial activity by disc-diffusion assay (100 microg ml(-1)). Several snake venoms (Daboia russelli russelli, Crotalus adamanteus, Naja sumatrana, Pseudechis guttata, Agkistrodon halys, Acanthophis praelongus and Daboia russelli siamensis) showed activity against two to four different pathogenic bacteria. Daboia russelli russelli and Pseudechis australis venoms exhibited the most potent activity against Staphylococcus aureus, while the rest showed only a moderate activity against one or more bacteria. The order of susceptibility of the bacteria against viperidae venoms was -S. aureus > Proteus mirabilis > Proteus vulgaris > Enterobacter aerogenes > Pseudomonas aeruginosa and Escherichia coli. The minimum inhibitory concentrations (MIC) against S. aureus was studied by dilution method (160-1.25 microg ml(-1)). A stronger effect was noted with the viperidae venoms (20 microg ml(-11)) as compared with elapidae venoms (40 microg ml(-1)). The MIC were comparable with those of the standard drugs (chloramphenicol, streptomycin and penicillin). CONCLUSION: The present findings indicate that viperidae (D. russelli russelli) and elapidae (P. australis) venoms have significant antibacterial effects against gram (+) and gram (-) bacteria, which may be the result of the primary antibacterial components of laao, and in particular, the PLA(2) enzymes. The results would be useful for further purification and characterization of antibacterial agents from snake venoms. SIGNIFICANCE AND IMPACT OF THE STUDY: The activity of LAAO and PLA(2) enzymes may be associated with the antibacterial activity of snake venoms.     

Uteroglobin inhibits phospholipase A2 activity

Although progesterone is known to produce quiescence in the mammalian uterus, the mechanism of this effect is not clearly understood. Here, we report that uteroglobin, a progesterone-induced small molecular weight (16K) protein, inhibits phospholipase A2(PLA2) derived from porcine pancreas as well as from the RAW 264.7 macrophage cell line. We speculate that progesterone may exert its antimotility effects on the uterus via uteroglobin which, by inhibiting PLA2, decreases arachidonic acid release and subsequently reduces prostaglandin levels in this organ. This may explain why progesterone is so vital for the maintenance of pregnancy in almost all mammals.     

Flow cytometric determinations of cellular substances in algae,bacteria, moulds and yeasts

The practical use of flow cytometry is shown in several microbial assays. Recent technical improvements in the optics and electronics of flow cytometric systems as well as in staining techniques permit the measurements of minute cellular components such as the cellular DNA and the protein content of bacteria, algae, moulds and yeasts. Single cell ingredients can be measured by this assay according to their specific stainability. The cell DNA was stained by propidium iodide while the cell protein was fluorochromed by fluorescein-iso-thiocyanate. The DNA synthesis of Saccharomyces cerevisiae and Saccharomyces pastorianus runs discontinuously while the protein content increases continuously during the vegetative growth. The different stages of DNA synthesis of yeast cells can be divided into two gap phases, a synthesis and a mitosis period, corresponding to Howard and Pelc's model of DNA synthesis. Living and dead cells can be counted differentially after staining with Erythrosine B. The red fluorescence of the chlorophyll in algae can readily be used to determine the chlorophyll content of these cells.     

Amylolytic activity of yeasts

The total amylolytic activity of 14 selected fermentation type I members of the generaSaccharomyces, Zygosaccharomyces, Candida andTorulopsis was studied. Fractions with α-glucosidase activity, the specificty of which was tested for maltose and sucrose, were isolated on carboxymethyl-cellulose from the intracellular contents of two strains ofCandida albicans and one ofCandida stellatoidea. The fraction from the strainCandida albicans 29-3-109, which is more virulent for mice, displayed the greatest α-glucosidase activity, moderate activity was present in the strainCandida stellatoidea 29-64-1, while the lowest activity was found in the less virulent strain ofCandida albicans, 29-3-19.     

Ovine myometrial cytosol inhibits phospholipase A activity, in vitro

Myometrium obtained from pregnant ewes (30-80 days gestation) contains a factor which inhibits phospholipase A2 (PLA2) activity. The activity of this moiety was assessed using an in vitro porcine pancreatic PLA2 assay system. Inhibitory activity was associated with a 35-45000 dalton molecular weight fraction, heat-labile, sensitive to protease degradation and did not partition into organic solvents. These data are indicative that PLA2-inhibitory activity resides in a protein moiety. Dixon-plot analysis of myometrial-inhibitory activity was indicative that the inhibition of PLA2 activity was of a non-competitive nature (Ki = 4.1 +/- 0.7 micrograms/ml, ca 118 nmol/l). Myometrial phospholipase-inhibitory protein(s) may be involved in the suppression of eicosanoid biosynthesis by the uterine tissues throughout gestation thus inhibiting uterine contractile activity.     

Ovine myometrial cytosol inhibits phospholipase A activity, in vitro

Myometrium obtained from pregnant ewes (30–80 days gestation) contains a factor which inhibits phospholipase A₂ (PLA₂) activity. The activity of this moiety was assessed using an in vitro porcine pancreatic PLA₂ assay system. Inhibitory activity was associated with a 35–45000 dalton molecular weight fraction, heat-labile, sensitive to protease degradation and did not partition into organic solvents. These data are indicative that PLA₂-inhibitory activity resides in a protein moiety. Dixon-plot analysis of myometrial-inhibitory activity was indicative that the inhibition of PLA₂ activity was of a non-competitive nature (K_i = 4.1 ± 0.7μg/ml, ca 118 nmol/1). Myometrial phospholipase-inhibitory protein(s) may be involved in the suppression of eicosanoid biosynthesis by the uterine tissues throughout gestation thus inhibiting uterine contractile activity.     

Mitochondrial phospholipase A2 activity and mitochondrial aging

The changes in mitochondrial phospholipid metabolism and energy-linked functions have been followed as coupled mitochondria are allowed to age in isotonic sucrose at 18 degrees C. Analysis of the aging process has provided an approach for studying the structure--function relationships within the mitochondrion without adding external agents to perturb the membrane structure. The initial event observed in this process of deterioration is a loss of respiratory control which is paralleled by diminishing levels of ATP. As ATP levels decline, so do the rates of reacylation of monoacyglycerophosphorylethanolamine and fatty acid oxidation. In most cases the previously inactive phospholipase A2 (EC 3.1.1.4, phosphatide-2-acyl-hydrolase) begins rapid hydrolysis of membrane phosphatidylethanolamine as ATP levels approach zero. The final energy-linked phenomenon observed to decline is the anilinonaphthalenesulfonic acid fluorescence response. Evidence is presented which suggests strongly that the activity of the mitochondrial phospholipase A2 on endogenous phospholipids is suppressed in tightly coupled mitochondria. This suppression is temporally linked to ATP levels in the mitochondria. Furthermore, this study demonstrates that mitochondria which are only slightly damaged have the potential to effect membrane repair through reacylation of monoacyl phospholipids.     

Intracellular phospholipase activity in rat heart: comparison between endogenous and exogenous substrates

Two methods were used to estimate the intracellular phospholipase activity in rat heart: one using exogenous radioactive substrate dispersed as unilamellar vesicles; the other using endogenous membrane hydrolysis and subsequent phospholipid and lysophospholipid separation by high-performance liquid chromatography and quantification by phosphorus determination. We found that the endogenous method provided a higher hydrolysis rate than the exogenous method and that phosphatidyl ethanolamine was a better substrate than phosphatidyl choline.     

Effect of cellular fatty acid composition on the phospholipase A2 activity of bone marrow-derived macrophages, and their ability to induce lucigenin-dependent chemiluminescence

Mouse bone marrow macrophages were obtained by cultivation in serum-free medium. Addition of specific fatty acids to the medium leads to macrophage populations which differ in their fatty acid composition. The fatty acid composition of the cellular membranes directly modulates functional abilities of the macrophages such as the generation of superoxide anion and phospholipase A2 activity in response to phorbol ester and zymosan. Both capacities were lowest in macrophages cultured serum-free without lipids. Incorporation of unsaturated fatty acids into macrophage phospholipids leads to an increase of O2- production as measured by lucigenin-dependent chemiluminescence and to an increased phospholipase A2 activity after challenge with phorbol ester or zymosan.     

Effect of sulfatide and gangliosides on phospholipase C and phospholipase A2 activity. A monolayer study

The effect of sulfatide and gangliosides GM1, GD1a and GT1b on the activity of phospholipase C from Clostridium perfringens on dilauroylphosphatidylcholine and of porcine pancreatic phospholipase A2 on dilauroylphosphatidic acid was studied in lipid monolayers containing different proportions of glycolipids under zero-order kinetics at various constant surface pressures. The presence of sulfatide in the monolayer increases the activity of phospholipase C at high surface pressures. Gangliosides shift the cut-off pressure to lower values and inhibit the action of phospholipase C. In mixed monolayers with dilauroylphosphatidic acid, sulfatide at a molar fraction of 0.5 increases the activity of phospholipase A2 at surface pressures below 18 mN/m and shows an inhibitory effect at higher pressures. Ganglioside GM1 at a molar fraction of 0.25 completely inhibits the enzyme above 20 mN/m and markedly reduces its activity at lower pressures. Gangliosides GD1a and GT1b abolish the enzyme activity at all pressures at molar fractions of 0.25 and 0.15, respectively. The modified velocity of the enzymatic reaction in the presence of glycosphingolipids is not due to an irreversible alteration of the catalytic activity.