Cell wall extracts from the double-mutant mnn1 mnn9 strain were used as the immunogen to obtain a monoclonal antibody (MAb), SAC A6, that recognizes a specific mannoprotein-which we have named Icwp-in the walls of cells of Saccharomyces cerevisiae. Icwp runs as a polydisperse band of over 180 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of Zymolyase extracts of cell walls, although an analysis of the secretory pattern of the mannoprotein shows that at the level of secretory vesicles, it behaves like a discrete band of 140 kDa. Immunofluorescence analysis with the MAb showed that Icwp lies at the inner layer of the cell wall, being accessible to the antibody only after the outer layer of mannoproteins is disturbed by treatment with tunicamycin. The screening of a gt11 expression library enabled us to identify the open reading frame (ORF) coding for Icwp. ICWP (EMBL accession number YLR391w, frame ؉3) codes for 238 amino acids, of which over 40% are serine or threonine, and contains a putative N-glycosylation site and a putative glycosylphosphatidylinositol attachment signal. Both disruption and overexpression of the ORF caused increased sensitivities to calcofluor white and Congo red, while the disruption caused an increased sensitivity to Zymolyase digestion, suggesting for Icwp a structural role in association with glucan.The cell wall of Saccharomyces cerevisiae is made up of three components, namely, glucans, mannoproteins, and chitin, and represents some 20% of the dry weight of the cell. It consists of a layered structure, with an internal layer made up of ␤-1,3 and ␤-1,6 glucans, small amounts of chitin and mannoproteins, and an outer layer of mannoproteins (13, 23). The inner layer is responsible for the shape and mechanical strength of the wall (19,24,50), while the outer mannoprotein layer determines the surface properties of the cell, such as hydrophobicity, electrical charge, flocculence, and sexual agglutinability, as well as limiting the porosity of the cell wall (8-10, 50).The mannoproteins can be divided into three groups according to the methods used for their extraction from the cell wall: sodium dodecyl sulfate (SDS)-extractable mannoproteins (44), glucanase-extractable mannoproteins, which can be released only after glucanase digestion of the glucan layer (31,44,47), and mannoproteins extractable by reducing agents (35). The glucanase-extractable mannoproteins identified so far have two common characteristics: one is a high serine/threonine content (up to 50% of the C-terminal half of the protein), and the other is the presence of a putative glycosylphosphatidylinositol (GPI) attachment site (23,46). Several of the proteins characterized so far, such as the sexual ␣-agglutinin (27, 29, 30), the anchor subunit of the a-agglutinin (38), and the flocculin encoded by the FLO1 gene, play a role on the surface of the cell (42). In these proteins, the highly O-glycosylated Cterminal half may endow them with a rod-like structure (22) that facilitates the exposure of their ...

In this report, we present the identification of the main polypeptides that are extracted from purified cell walls of aSaccharomyces cerevisiae mnn1 mnn9 strain by reducing agents. Treatment of the purified cell walls of this strain with β-mercaptoethanol releases several mannoproteins, of which three, with apparent sizes of 120, 45, and 40 kDa, are the most abundant. Analysis of the amino-terminal sequences revealed that the 120-kDa mannoprotein is Bar1p, the protease involved in the so-called barrier activity in yeast cells, and that the 45- and 40-kDa mannoproteins are the Kex2-unprocessed and Kex2-processed forms of the gene product of open reading frame (ORF) YJL158c, an ORF that belongs to the PIR (protein with internal repeats) family of genes, composed thus far of PIR1, PIR2/HSP150, andPIR3. Accordingly we have named this gene PIR4, and Pir4 denotes the 40-kDa Kex2-processed form of the mannoprotein. We have characterized Pir4 and have shown the feasibility of using it as a fusion partner for the targeting of recombinant proteins to the cell wall.

The gene xynC encoding xylanase C from Bacillus sp. BP-23 was cloned and expressed in Escherichia coli. The nucleotide sequence of a 3538 bp DNA fragment containing xynC gene was determined, revealing an open reading frame of 3258 bp that encodes a protein of 120567 Da. A comparison of the deduced amino acid sequence of xylanase C with known /I-glycanase sequences showed that the encoded enzyme is a modular protein containing three different domains. The central region of the enzyme is the catalytic domain, which shows high homology t o family 10 xylanases. A domain homologous t o family IX cellulose-binding domains is located in the C-terminal region of xylanase C, whilst the N-terminal region of the enzyme shows homology t o thermostabilizing domains found in several thermophilic enzymes. Xylanase C showed an activity profile similar t o that of enzymes from mesophilic microorganisms. Maximum activity was found at 45OC, and the enzyme was only stable at 55 O C or lower temperatures. Xylotetraose, xylotriose, xylobiose and xylose were the main products from birchwood xylan hydrolysis, whilst the enzyme showed increasing activity on xylo-oligosaccharides of increasing length, indicating that the cloned enzyme is an endoxylanase. A deletion derivative of xylanase C, lacking the region homologous t o thermostabilizing domains, was constructed. The truncated enzyme showed a lower optimum temperature for activity than the full-length enzyme, 35 O C instead of 4 5 OC, and a reduced thermal stability that resulted in a complete inactivation of the enzyme after 2 h incubation at 55 OC.

Background: The study of the immune system has been approached using two separate paths, the biological immune system and the behavioral immune system. Recently, Gangestad and Grebe proposed a unique integrated compensatory immune system, where both systems work together and one of them could compensate for the other when necessary. However, few studies have confirmed the existence of this integrated compensatory immune system. Our study represents an attempt to explore the existence of this unique immune system, investigating if the behavioral immune system variables increase when the biological immune system weakens with age. Material and Methods. The cross-sectional design study was made up of a final sample of 1108 participants (45.2% men and 54.2 women) aged 18–64 years. The younger group (18–21 years) was made up of students, whilst the older groups (22 to 64 years) were composed by their relatives and acquaintances, following the snow ball process. The participants completed the Perceived Vulnerability to Disease Questionnaire that assesses perceived infectability and germ aversion. Correlations, analyses of variance (ANOVAs), and independent group comparisons were performed. These analyses showed the relationships between the variables studied, the effects of age and gender in perceived infectability and germ aversion, and the differences that perceived infectability and germ aversion presented in different age-groups separated by gender. Results: A pattern emerged where germ aversion increases as both men and women get older, but perceived infectability decreases up to the age of 50, and then it increases in women from that age onward. Gender differences are only significant in younger participants, with women having higher scores than men in both variables. Conclusion: The results partially support the existence of a unique integrated compensatory biological/behavioral immune system.

In this work we have studied the disulphide-bound group of cell wall mannoproteins of Yarrowia lipolytica and Candida albicans.

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Xylanase A from Bacillus sp. BP7, an enzyme with potential applications in biotechnology, was used to test Pir4, a disulfide bound cell wall protein, as a fusion partner for the expression of recombinant proteins in standard or glycosylation-deficient mnn9 strains of Saccharomyces cerevisiae. Five different constructions were carried out, inserting in-frame the coding sequence of xynA gene in that of PIR4, with or without the loss of specific regions of PIR4. Targeting of the xylanase fusion protein to the cell wall was achieved in two of the five constructions, while secretion to the growth medium was the fate of the gene product of one of the constructions. In all three cases localization of the xylanase fusion proteins was confirmed both by Western blot and detection with Pir-specific antibodies and by xylanase activity determination. The cell wall-targeted fusion proteins could be extracted by reducing agents, showing that the inclusion of a recombinant protein of moderate size does not affect the way Pir4 is attached to the cell wall. Also, the construction that leads to the secretion of the fusion protein permitted us to identify a region of Pir4 responsible for cell wall retention. In summary, we have developed a Pir4-based system that allows selective targeting of an active recombinant enzyme to the cell wall or the growth medium. This system may be of general application for the expression of heterologous proteins in S. cerevisiae for surface display and secretion.

Cloning and sequencing of RCS1, Saccharomyces cerevisiae gene whose product seems to be involved in timing the budding event of the cell cycle, is described. A haploid strain in which the 3'-terminal region of the chromosomal copy of the gene has been disrupted produces cells that are, on average, twice the size of cells of the parental strain. The critical size for budding in the mutant is similarly increased, and the disruption mutation is dominant in a diploid heterozygous for the RCS1 gene. Spores from this diploid have a reduced ability to germinate, the effect being more pronounced in the spores carrying the disrupted copy of RCS1. However, disrupted cells recover from alpha-factor treatment equally as well as wild-type cells.