Purification and identification of bovine cheese whey fatty acids exhibiting in vitro antifungal activity.

Milk lipids contain several bioactive factors exhibiting antimicrobial activity against bacteria, viruses, and fungi. In the present study, we demonstrate that free fatty acids (FFA) derived from the saponification of bovine whey cream lipids are active in vitro at inhibiting the germination of Candida albicans, a morphological transition associated with pathogenicity. This activity was found to be significantly increased when bovine FFA were enriched in non-straight-chain FFA. At low cell density, this non-straight-chain FFA-enriched fraction was also found to inhibit in a dose-dependant manner the growth of both developmental forms of C. albicans as well as the growth of Aspergillus fumigatus. Using an assay-guided fractionation, the main components responsible for these activities were isolated. On the basis of mass spectroscopic and gas chromatographic analysis, antifungal compounds were identified as capric acid (C10:0), lauroleic acid (C12:1), 11-methyldodecanoic acid (iso-C13:0), myristoleic acid (C14:1n-5), and gamma-linolenic acid (C18:3n-6). The most potent compound was gamma-linolenic acid, with minimal inhibitory concentration values of 5.4 mg/L for C. albicans and 1.3 mg/L for A. fumigatus, in standardized conditions. The results of this study indicate that bovine whey contains bioactive fatty acids exhibiting antifungal activity in vitro against 2 important human fungal pathogens.

Modulation of juvenile brook trout (Salvelinus fontinalis) cellular immune system after Aeromonas salmonicida challenge.

In fish, the first line of defence against infectious microorganisms is based on non-specific cellular immune mechanisms (innate immunity). In this study, we measured the non-specific immune parameters (natural cytotoxic cells (NCC) activity, lymphoproliferation, percentage of phagocytosis and phagocytic activity) in brook trout (Salvelinus fontinalis) infected by a virulent strain of Aeromonas salmonicida. Eight days post-infection, the mortality of infected fish reached 70%. A transient immunostimulation of the NCC activity was noticed 24h post-infection, but there was no significant difference at 48 h. Then, infection of brook trout with A. salmonicida induced a biphasic immune response. At 24h post-infection, lymphoproliferation was drastically depressed but returned to control level at 96 h. A slight increase in the percentage of phagocytosis and the phagocytic activity was noticed throughout the experiment. Conversely the cell mortality was significantly higher in infected fish compared to control. The modulation of immunological parameters might reveal important clues on how innate immunity might protect fish from bacterial infections.

Overexpression of Bud5p can suppress mutations in the Gsp1p guanine nucleotide exchange factor Prp20p in Saccharomyces cerevisiae.

The gene product Prp20p, which is located in the nucleus, serves as the nucleotide exchange factor (GEF) for the small nuclear G protein Gsp1p in Saccharomyces cerevisiae, and catalyses the replacement of Gsp1-bound GDP by GTP. These proteins are involved in numerous cellular processes, including nucleocytoplasmic trafficking of macromolecules, cell cycle progression, DNA replication and maintenance of chromosome structure/stability. It is believed that in order to complete a full GDP/GTP cycle, Gsp1p has to shuttle between the nucleus and the cytoplasm, where its GTPase Activating Protein (GAP) Rna1p is located. Here, we report on the ability of Bud5p, the exchange factor for Rsr1p, to suppress conditional prp20 mutants when an extra copy of GSP1 is present. This suppression by BUD5 can be reversed by simultaneous overexpression of RNA1, and is not Rsr1p-dependent, nor allele-specific. We also show that Bud5p can physically interact with Gsplp, both in vitro and in vivo. These,findings raise the possibility that Bud5p could act as a cytoplasmic exchange factor for Gsp1p and, therefore, that a complete GDP/GTP cycle could take place in the cytoplasm.

What shadows reveal about object structure.

In a scene observed from a fixed viewpoint, the set of shadow boundaries in an image changes as a point light source (nearby or at infinity) assumes different locations. We show that for any finite set of point light sources illuminating an object viewed under either orthographic or perspective projection, there is an equivalence class of object shapes having the same set of shadows. Members of this equivalence class differ by a four-parameter family of projective transformations, and the shadows of a transformed object are identical when the same transformation is applied to the light source locations. Under orthographic projection, this family is the generalized bas-relief (GBR) transformation, and we show that the GBR transformation is the only family of transformations of an object's shape for which the complete set of imaged shadows is identical. Finally, we show that given multiple images under differing and unknown light source directions, it is possible to reconstruct both an object's surface and the light source locations up to this family of transformations from the shadows alone.

Molecular cloning of CaYRB1, the Candida albicans RanBP1/YRB1 homologue.

The yeast Ran binding protein 1 (Yrb1p) is a small protein of 23 kDa that is highly conserved among eukaryotes. It stimulates the GTPase activity of Gsp1p in the presence of the GTPase activating protein Rna1p. In addition to its role in nucleocytoplasmic transport of macromolecules, YRB1/RanBP1 could be involved in the regulation of microtubules structure and dynamics. Since microtubules are tightly associated with morphological changes, we have been interested to study the role and function of YRB1 in the pathogenic fungus Candida albicans, where there is regulated change in cellular morphology. The gene product of CaYRB1 encodes a 212 amino acid protein displaying 73% homology to the S. cerevisiae homologue. The bacterially expressed gene product has an apparent molecular weight of 35.7 kDa. We show that it can complement a S. cerevisiae yrb1 null mutant and that its mRNA does not appear to be regulated in response to conditions inducing morphological changes in C. albicans.

A localized GTPase exchange factor, Bud5, determines the orientation of division axes in yeast.

GTPases are widespread in directing cytoskeletal rearrangements and affecting cellular organization. How they do so is not well understood. Yeast cells divide by budding, which occurs in two spatially programmed patterns, axial or bipolar [1-3]. Cytoskeletal polarization to form a bud is governed by the Ras-like GTPase, Bud1/Rsr1, in response to cortical landmarks. Bud1 is uniformly distributed on the plasma membrane, so presumably its regulators, Bud5 GTPase exchange factor and Bud2 GTPase activating protein, impart spatial specificity to Bud1 action [4]. We examined the localizations of Bud5 and Bud2. Both Bud1 regulators associate with cortical landmarks designating former division sites. In haploids, Bud5 forms double rings that encircle the mother-bud neck and split upon cytokinesis so that each progeny cell inherits Bud5 at the axial division remnant. Recruitment of Bud5 into these structures depends on known axial landmark components. In cells undergoing bipolar budding, Bud5 associates with multiple sites, in response to the bipolar landmarks. Like Bud5, Bud2 associates with the axial division remnant, but rather than being inherited, Bud2 transiently associates with the remnant in late G1, before condensing into a patch at the incipient bud site. The relative timing of Bud5 and Bud2 localizations suggests that both regulators contribute to the spatially specific control of Bud1 GTPase.

Characterization of CaGSP1, the Candida albicans RAN/GSP1 homologue.

Gsp1p is a small nuclear-located GTP binding protein from the yeast Saccharomyces cerevisiae. It is highly conserved among eucaryotic cells and is involved in numerous cellular processes, including nucleocytoplasmic trafficking of macromolecules. To learn more about the GSP1 structure/function, we have characterized its Candida albicans homologue. CaGsp1p is 214 amino acids long and displays 91% identity to the ScGsp1p. There is functional complementation in S. cerevisiae, and its mRNA is constitutively expressed in the diploid C. albicans grown under various physiological conditions. Disruption of both alleles was not possible, suggesting that it could be an essential gene, but heterozygous mutants exhibited genomic instability.

Characterization of binding of Candida albicans to small intestinal mucin and its role in adherence to mucosal epithelial cells.

In order to approximate and adhere to mucosal epithelial cells, Candida must traverse the overlying mucus layer. Interactions of Candida species with mucin and human buccal epithelial cells (BECs) were thus investigated in vitro. Binding of the Candida species to purified small intestinal mucin showed a close correlation with their hierarchy of virulence. Significant differences (P < 0.05) were found among three categories of Candida species adhering highly (C. dubliniensis, C. tropicalis, and C. albicans), moderately (C. parapsilosis and C. lusitaniae) or weakly (C. krusei and C. glabrata) to mucin. Adherence of C. albicans to BECs was quantitatively inhibited by graded concentrations of mucin. However, inhibition of adherence was reversed by pretreatment of mucin with pronase or C. albicans secretory aspartyl proteinase Sap2p but not with sodium periodate. Saturable concentration- and time-dependent binding of mucin to C. albicans was abrogated by pronase or Sap2p treatment of mucin but was unaffected by beta-mercaptoethanol, sodium periodate, neuraminidase, lectins, or potentially inhibitory sugars. Probing of membrane blots of the mucin with C. albicans revealed binding of the yeast to the 66-kDa cleavage product of the 118-kDa C-terminal glycopeptide of mucin. Although no evidence was found for the participation of C. albicans cell surface mannoproteins in specific receptor-ligand binding to mucin, inhibition of binding by p-nitrophenol (1 mM) and tetramethylurea (0.36 M) revealed that hydrophobic interactions are involved in adherence of C. albicans to mucin. These results suggest that C. albicans may both adhere to and enzymatically degrade mucins by the action of Saps, and that both properties may act to modulate Candida populations in the oral cavity and gastrointestinal tract.

Isolation and characterization of the Candida albicans SEC4 gene.

The SEC4 gene product is a major component of the protein secretion machinery. More specifically, it is believed to play a pivotal role in targeting and fusion of secretory vesicles to the plasma membrane. Its recently described implication with the Saccharomyces cerevisiae Rho3p, which is required for directing growing points during bud formation, has prompted us to investigate the role and function of Sec4p in the morphological changes of the yeast pathogen Candida albicans. We have therefore cloned the C. albicans SEC4 gene. It encodes a 210 amino acids long protein sharing up to 75% homology to the S. cerevisiae homolog, when conserved changes are allowed. Its RNA is constitutively expressed in C. albicans grown under various physiological conditions. We also show that it can functionally complement a S. cerevisiae sec4 thermosensitive mutant.

The small GTPase Gsp1p binds to the repeat domain of the nucleoporin Nsp1p.

The small GTPase Gsp1p of Saccharomyces cerevisiae and its homologue Ran play essential roles in several nuclear processes, such as cell-cycle progression, nuclear organization and nucleocytoplasmic traffic of RNA and proteins. Gsp1p/Ran is an abundant nuclear protein that interacts with different cytoplasmic and nuclear factors. Several of the previously identified Ran-binding proteins located at the nuclear-pore complex carry a specific Ran-binding domain. So far, direct interactions between the GTPase and other proteins have been mostly characterized in higher eukaryotes. Here we report that the yeast protein Gsp1p can directly bind to the nucleoporin Nsp1p in vitro. Nsp1p does not contain a Ran-binding domain and therefore represents a distinct type of nucleoporin that associates with Gsp1p. We demonstrate that the middle domain of Nsp1p is sufficient to mediate this interaction. Importantly, we show that a conserved cluster of positively charged amino acid residues of Gsp1p located at positions 142-144 is essential for the binding reaction. Thus we have identified Nsp1p as a new candidate protein located at the nuclear pore complex of the yeast S. cerevisiae that interacts directly with Gsp1p. We further demonstrate that both Gsp1p and Nsp1p are components of larger protein complexes in vivo, supporting the idea that the association between both proteins takes place in growing cells.

Expression of a reporter gene interrupted by the Candida albicans group I intron is inhibited by base analogs.

We previously reported the identification of an intron (CaLSU) in the 25S ribosomal RNA of some Candida albicans yeast strains. CaLSU was shown to self-splice and has the potential to adopt a secondary structure typical of group I introns. The presence of CaLSU inC. albicans strains correlates with a high degree of susceptibility to base analog antifungal agents, 5-fluorocytosine (5-FC) or 5-fluorouracil (5-FU). Cell death, resulting from addition of base analogs to growing cultures, precluded demonstration of a causal relationship between CaLSU presence and susceptibility to base analogs. In the present study, CaLSU was inserted in a non-essential lacZ reporter gene and expression was examined in Saccharomyces cerevisiae. Different mutations affecting in vitro self-splicing also had similar effects on reporter gene expression in vivo. This indicates that in vivo removal of CaLSU from the reporter gene occurs through the typical self-splicing mechanism of group I introns. Base analogs inhibited expression of the reporter gene product in a concentration-dependent manner upon their addition to the cultures. This supports a model in which disruption of intron secondary structure, consecutive to the incorporation of nucleotide analogs, is a major factor determining the susceptibility of C.albicans cells to base analogs.

Evidence for degradation of gastrointestinal mucin by Candida albicans secretory aspartyl proteinase.

A zone of extracellular digestion of the mucin layer around Candida albicans blastoconidia was observed by transmission electron microscopy in the jejunum of mice inoculated intragastrically (G. T. Cole, K. R. Seshan, L. M. Pope, and R. J. Yancey, J. Med. Vet. Mycol. 26:173-185, 1988). This observation prompted the hypothesis that a putative mucinolytic enzyme(s) may contribute to the virulence of C. albicans by facilitating penetration of the mucus barrier and subsequent adherence to and invasion of epithelial cells. Mucinolytic activity was observed as zones of clearing around colonies of C. albicans LAM-1 grown on agarose containing yeast nitrogen base, glucose, and hog gastric mucin. In addition, concentrated culture filtrate obtained after growth for 24 h in yeast nitrogen base, supplemented with glucose and mucin as the sole nitrogen source, contained proteolytic activity against biotin-labelled mucin which was inhibited by pepstatin A. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the culture filtrate revealed two components of 42 and 45 kDa, with pIs of 4.1 and 5.3, respectively. A zymogram showed that mucin was degraded only by the 42-kDa component, which was also recognized by immunoblotting with an anti-secretory aspartyl proteinase (anti-Sap) 2p monoclonal antibody. The N-terminal sequence of the first 20 amino acids matched that reported for Sap2p. These results demonstrate that Sap2p is responsible for proteolysis of mucin by C. albicans in vitro and may be involved as a virulence factor in the breakdown of mucus and penetration of the mucin barrier by C. albicans.

Development of a method to detect secretory mucinolytic activity from Candida albicans.

Ultrastructural examinations of sites where Candida albicans invaded the bowel wall after oral intragastric inoculation of infant mice suggested that blastoconidia are capable of progressive extracellular digestion of the intestinal mucus barrier. Microplate assay methods, based on biotin or digoxigenin-labelling systems, were therefore devised for quantitation of protease and glycosidase activities against the glycoprotein mucin. Labelled mucin was adsorbed on microplate wells, incubated with sample to be assayed for enzyme activity, and the remaining labelled mucin was quantitated by spectrophotometry. Proteolytic activity against mucin was demonstrated using concentrated culture filtrate of C. albicans strain LAM-1, grown in yeast nitrogen base medium containing mucin as sole nitrogen source. The activity was inhibited by boiling for 10 min or by incubation with the aspartyl proteinase inhibitor pepstatin A.

A gene from Saccharomyces cerevisiae which codes for a protein with significant homology to the bacterial 3-phosphoserine aminotransferase.

During the sequencing of the gene GSP2 from Saccharomyces cerevisiae, we have encountered an adjacent open reading frame having strong homology to the 3-phosphoserine aminotransferase (E.C.2.6.1.52) from other organisms. In this report, we present the sequence for this yeast SERC, and evidence that its deletion from the yeast genome leads to serine dependency. The sequence has been deposited in the GenBank data library under Accession Number L20917.

Prp20, the Saccharomyces cerevisiae homolog of the regulator of chromosome condensation, RCC1, interacts with double-stranded DNA through a multi-component complex containing GTP-binding proteins.

Prp20, a homolog to the mammalian negative regulator of chromosome condensation, RCC1, is retained on double-stranded (ds) DNA-cellulose when extracts are prepared from asynchronously growing wild-type yeast strains. Conversely, neither Prp20 from ts mutant cell extracts nor wt yeast Prp20 produced in Escherichia coli, bind to dsDNA-cellulose. In vitro reconstitution assays using E. coli-expressed Prp20 and inactivated ts mutant extracts of prp20-1 reveal that the Prp20 protein requires the assistance of other proteins in the cell extract to promote its binding to dsDNA. Immunoprecipitations and sizing-column-chromatography indicate that the Prp20 protein binds to the dsDNA column through a multicomponent complex composed of six to seven proteins, which has a collective molecular mass greater than 150,000 Da. At least three of the members of this Prp20 complex will bind GTP in vitro. Moreover, the Prp20 complex is shown to specifically lose its ability to bind dsDNA during the DNA replication phase of the cell cycle. This loss of dsDNA binding during the S phase of the cell cycle does not affect the proper organization of the nucleoplasm and appears to be reversed before the cell enters mitosis.

Action of spontaneously produced beta interferon in differentiation of embryonal carcinoma cells through an autoinduction mechanism.

In the current study, we have addressed the role of interferons (IFNs) in controlling the differentiation of pluripotent P19 embryonal carcinoma (EC) cells. Blocking IFN activity in the culture medium of differentiating cells with antibodies leads to a strong decrease in the degree of differentiation. The antibodies are active for a relatively short time. During this time, IFN-beta mRNA can be detected in the differentiating cells, as can increases of IFN stimulation response element-binding activity and NF-KB. The timing of IFN action also coincides with the accumulation of cytoplasmic double-stranded RNA (dsRNA) and with a drop in dsRNA unwindase-modificase activity. A model for the involvement of autoinduction of IFN by intracellular dsRNA in the control of differentiation in this system is presented.

GSP1 and GSP2, genetic suppressors of the prp20-1 mutant in Saccharomyces cerevisiae: GTP-binding proteins involved in the maintenance of nuclear organization.

The temperature-sensitive mutation prp20-1 of Saccharomyces cerevisiae exhibits a pleiotropic phenotype associated with a general failure to maintain a proper organization of the nucleus. Its mammalian homolog, RCC1, is not only reported to be involved in the negative control of chromosome condensation but is also believed to assist in the coupling of DNA replication to the entry into mitosis. Recent studies on Xenopus RCC1 have strongly suggested a further role for this protein in the formation or maintenance of the DNA replication machinery. To elucidate the nature of the various components required for this PRP20 control pathway in S. cerevisiae, we undertook a search for multicopy suppressors of a prp20 thermosensitive mutant. Two genes, GSP1 and GSP2, were identified that encode almost identical polypeptides of 219 and 220 amino acids. Sequence analyses of these proteins show them to contain the ras consensus domains involved in GTP binding and metabolism. The levels of the GSP1 transcript are about 10-fold those of GSP2. As for S. cerevisiae RAS2, GSP2 expression exhibits carbon source dependency, while GSP1 expression does not. GSP1 is an essential gene, and GSP2 is not required for cell viability. We show that GSP1p is nuclear, that it can bind GTP in an in vitro assay, and finally, that a mutation in GSP1p which activates small ras-like proteins by increasing the stability of the GTP-bound form causes a dominant lethal phenotype. We believe that these two gene products may serve in regulating the activities of the multicomponent PRP20 complex.

Regulated expression of Krox-24 and other serum-responsive genes during differentiation of P19 embryonal carcinoma cells.

In order to identify genes that may play a role in the onset of the differentiation program elicited by retinoic acid, we analyzed, in P19 embryonal carcinoma cells, the expression of genes that are part of the early response of mouse fibroblasts to growth factor stimulation. In this paper, we show that a sequence-specific transcriptional activator, Krox-24, is rapidly induced, under conditions that promote differentiation of P19 cells. Expression of three other serum- and retinoic acid-stimulated genes (clones AC36, C1, and G39) was also studied. Induction of these genes occurs during the first 48 h of exposure of cells to retinoic acid, a period that precedes cell type determination. Our results suggest that different mechanisms regulate the expression of the Krox-24 gene in differentiating P19 cells. A labile repressor seems to be responsible for control of Krox-24 expression in P19 embryonal carcinoma cells. Inactivation of this repressor following retinoic acid treatment resulted in several peaks of activation of the Krox-24 gene, mediated by different mechanisms, some of which did not require de novo protein synthesis. In contrast, activation of AC36 required de novo protein synthesis, and that of C1 and G39 did not. The four genes are differentially expressed in several mouse tissues and during mouse embryonic development.

Functional substitution of mouse ribosomal protein L27' for yeast ribosomal protein L29 in yeast ribosomes.

A cDNA clone of mouse ribosomal protein L27' was shown previously to be 62% identical in amino acid residues to yeast ribosomal protein L29. The L27' cDNA was expressed in yeast to determine the ability of the mouse protein to substitute for yeast L29 in assembling a functional ribosome. In a yeast strain resistant to cycloheximide by virtue of a recessive mutation in the L29 protein, the murine cDNA did not produce a sensitive phenotype, indicating failure of the mouse L27' protein to assemble into yeast ribosomes. However, when the mouse L27' gene was expressed in cells devoid of L29 and otherwise inviable, the murine protein supported normal growth, demonstrating that mouse ribosomal protein L27' indeed was interchangeable with yeast L29. We conclude that mouse ribosomal protein L27' is assembled into ribosomes in yeast, but yeast L29 is assembled preferentially when both L29 and L27' are present in the same cell.