Sequential fractionation and two-dimensional gel analysis unravels the complexity of the dimorphic fungus Candida albicans cell wall proteome

The cell wall proteins of Candida albicans play a key role in morphogenesis and pathogenesis and might be potential target sites for new specific antifungal drugs. However, these proteins are difficult to analyze because of their high heterogeneity, interconnections with wall polysaccharides (mannan, glucan, and chitin), low abundance, low solubility, and hydrophobic nature. Here we report a subproteomic approach for the study of the cell wall proteins (CWPs) from C. albicans yeast and hyphal forms. Most of the mannoproteins present in this compartment were extracted by cell wall fractionation according to the type of interactions that they establish with other structural components. CWPs were solubilized from isolated cell walls by hot SDS and dithiothreitol treatment followed by extraction either by mild alkali conditions or by enzymatic treatment with glucanases and chitinases. These highly enriched cell wall fractions were analyzed by two-dimensional PAGE, showing that a large number of proteins are involved in cell wall construction and that the wall remodeling that occurs during germ tube formation is related to changes in the composition of CWPs. We suggest that the CWP-chitin linkage is an important retention mechanism of CWPs in C. albicans mycelial forms. This article also highlights the usefulness of the combination of sequential fractionation and two-dimensional PAGE followed by Western blotting using specific antibodies against known CWPs in the characterization of incorporation mechanisms of such CWPs into the cell wall and of their interactions with other wall components. Mass spectrometry analyses have allowed the identification of several cell surface proteins classically associated with both the cell wall and other compartments. The physiological significance of the dual location of these moonlighting proteins is also discussed. This approach is therefore a powerful tool for obtaining a comprehensive and integrated view of the cell wall proteome.     

Evolutionary maintenance of stigma-height dimorphism in Narcissus papyraceus (Amaryllidaceae)

Stigma-height dimorphism is a sexual polymorphism in which plant populations are composed of two floral morphs that differ significantly in style length but not anther position. The morphs exhibit approach and reverse herkogamy, floral designs that in most species typically occur as monomorphic conditions. We investigated the floral biology of stigma-height dimorphism in the Mediterranean geophyte Narcissus papyraceus (Amaryllidaceae) in an effort to understand the evolutionary forces maintaining stylar polymorphism. Our survey of 66 populations in Spain, Portugal, and Morocco indicated that 56% were dimorphic with the long-styled morph at an average frequency of 0.79. The remaining 44% of populations sampled were monomorphic for the long-styled morph. In dimorphic populations there was a significant positive relation between population size and the frequency of the short-styled morph. Controlled pollinations demonstrated that N. papyraceus is self-sterile with no significant differences in female fertility between intra- and intermorph crosses. Prior self-pollination reduced seed set in flowers that were subsequently cross-pollinated. Estimates of mating patterns using allozyme markers in eight populations indicated that N. papyraceus is largely outcrossing (mean t(m) = 0.81) with no significant differences between monomorphic and dimorphic populations or style morphs. Stigma-height dimorphism in N. papyraceus is maintained in populations by insect-mediated cross-pollination with biased morph ratios and stylar monomorphism likely resulting from the combined influence of the inheritance of the polymorphism, morph-specific differences in assortative mating and founder effects.     

Upgraded expression of 5S rRNA preludes the production of fibroin by spider glands

We have developed the large ampullate glands of the orb-web spider Nephila clavipes as a model system in which to study the production of a tissue-specific secretory protein. Through simple manipulations, the glands' fibroin production can be practically abolished and subsequently elicited into high levels of synthesis through a mechanical stimulus applied to the organism. The tissue specific responses evoked by the stimulus can be monitored through time-course studies. The latter have revealed an orchestrated series of tissue and time specific macromolecular syntheses, which optimize the glandular tissues with components of the protein synthesis machinery. This work shows the upgraded accumulation of 5S rRNA in the glands as response to the stimulus within the earliest of the prelude events. Further enquiries on this accumulation must be conducted at the level of differential gene expressions, a chore we have initiated. A DNA fragment containing a single copy 5S rRNA gene has been isolated, cloned, sequenced, and transcribed in a cell-free system. We enclose a discussion on the similarity between the genomic organization of this gene to that of a 5S rRNA gene of Bombyx mori. Our studies have revealed a considerable number of similarities in the silk production strategies of Nephila clavipes and the silkworm Bombyx mori, some of them rather unusual.     

Determination of prednisolone and the most important associated compounds in ocular and cutaneous pharmaceutical preparations by micellar electrokinetic capillary chromatography

A micellar electrokinetic capillary chromatographic method to separate prednisolone, prednisolone acetate, naphazoline, Zn-bacitracin, sulfacetamide and phenylefrine is described. The separation was carried out by using a fused-silica capillary (57 cmx75 micrometer I.D.) at 25 degrees C and 30 kV, using a 5 mM phosphate-5 mM borate buffer adjusted to pH 8.2, 50 mM sodium dodecyl sulfate (SDS) and 10% methanol-water (v/v) as background electrolyte. Under these conditions, the run time was 8 min and the limits of quantification were about 1.0 mg/l for every component. The method was applied to pharmaceutical preparations and the results provided recoveries close to 100% and the method gave good results when compared with a reference multivariate calibration spectrophotometric method.     

Regulatory mechanisms for modulation of signaling through the cell integrity Slt2-mediated pathway in Saccharomyces cerevisiae

Signal transduction mediated by the mitogen-activated protein kinase (MAPK) Slt2 pathway is essential to maintain the cell wall integrity in Saccharomyces cerevisiae. Stimulation of MAPK pathways results in activation by phosphorylation of conserved threonine and tyrosine residues of MAPKs. We have used an antibody that specifically recognizes dually phosphorylated Slt2 to gain insight into the activation and modulation of signaling through the cell integrity pathway. We show that caffeine and vanadate activate this pathway in the absence of osmotic stabilization. The lack of the putative cell surface sensor Mid2 prevents vanadate- but not caffeine-induced Slt2 phosphorylation. Disruption of the Rho1-GTPase-activating protein genes SAC7 and BEM2 leads to constitutive Slt2 activation, indicating their involvement as negative regulators of the pathway. MAPK kinases also seem to participate in signaling regulation, Mkk1 playing a greater role than Mkk2 in signal transmission to Slt2. Additionally, one of the phosphatases involved in Slt2 dephosphorylation is likely to be the dual specificity phosphatase Msg5, since overexpression of MSG5 in a sac7Delta mutant eliminates the high Slt2 phosphorylation, and disruption of MSG5 in wild type cells results in increased phospho-Slt2 levels. These data present the first evidence for a negative regulation of the cell integrity pathway.     

Variation in tomato host response to Bemisia tabaci (Hemiptera: Aleyrodidae) in relation to acyl sugar content and presence of the nematode and potato aphid resistance gene Mi

Two commercial cultivars of tomato, Alta and Peto 95, the accession line number LA716 of Lycopersicon pennellii and lines 94GH-006 and 94GH-033 (backcrosses between Peto 95 and LA716), with different leaf acyl sugar contents were screened for resistance to Bemisia argentifolii Bellows & Perring (corresponding to the Spanish B-biotype of Bemisia tabaci (Gennadius)), in greenhouse- and field-no-choice experiments. There was no oviposition on LA716 (with the highest acyl sugar content) while the greatest fecundity and fertility values were observed on the cultivar Alta (no acyl sugar content). However, no clear relationship was found between the low acyl sugar content in the other tomato cultivars tested and whitefly reproduction. Thus, resistance to B. tabaci did not appear to correlate with acyl sugar content below a threshold level of 37.8 microg cm-2 leaf. In a greenhouse choice-assay, B. tabaci exhibited reduced host preference and reproduction on the commercial tomato cultivars Motelle, VFN8 and Ronita all of which carry the Mi gene resistance to Meloidogyne nematodes and the aphid Macrosiphum euphorbiae (Thomas), than on the Mi-lacking cultivars Moneymaker, Rio Fuego and Roma. When data of Mi-bearing plants were pooled, the mean values for daily infestation and pupal production of B. tabaci were significantly lower than those of Mi-lacking plants. This reflected a level of antixenosis- and antibiosis-based resistance in commercial tomato and indicated that Mi, or another closely linked gene, might be implicated in a partial resistance which was not associated either with the presence of glandular trichomes or their exudates. These findings support the general hypothesis for the existence of similarities among the resistance mechanisms to whiteflies, aphids and nematodes in commercial tomato plants.     

Trichomonas vaginalis: chromatin and mitotic spindle during mitosis

The mitotic phases and the changes that the chromatin and mitotic microtubules undergo during mitosis in the sexually transmitted parasite Trichomonas vaginalis are described. Parasites arrested in the gap 2 phase of the cell cycle by nutrient starvation were induced to mitosis by addition of fresh whole medium. [(3)H] Thymidine labeling of trichomonad parasites for 24 h showed that parasites have at least four synchronic duplications after mitosis induction. Fixed or live and acridine orange (AO)-stained trichomonads analyzed at different times during mitosis by epifluorescence microscopy showed that mitosis took about 45 min and is divided into five stages: prophase, metaphase, early and late anaphase, early and late telophase, and cytokinesis. The AO-stained nucleus of live trichomonads showed green (DNA) and orange (RNA) fluorescence, and the nucleic acid nature was confirmed by DNase and RNase treatment, respectively. The chromatin appeared partially condensed during interphase. At metaphase, it appeared as six condensed chromosomes, as recently reported, which decondensed at anaphase and migrated to the nuclear poles at telophase. In addition, small bundles of microtubules (as hemispindles) were detected only in metaphase with the polyclonal antibody anti-Entamoeba histolytica alpha-tubulin. This antibody showed that the hemispindle and an atractophore-like structure seem to duplicate and polarize during metaphase. In conclusion, T. vaginalis mitosis involves five mitotic phases in which the chromatin undergoes different degrees of condensation, from chromosomes to decondensed chromatin, and two hemispindles that are observed only in the metaphase stage.     

Y-chromosomal diversity in Europe is clinal and influenced primarily by geography, rather than by language

Clinal patterns of autosomal genetic diversity within Europe have been interpreted in previous studies in terms of a Neolithic demic diffusion model for the spread of agriculture; in contrast, studies using mtDNA have traced many founding lineages to the Paleolithic and have not shown strongly clinal variation. We have used 11 human Y-chromosomal biallelic polymorphisms, defining 10 haplogroups, to analyze a sample of 3,616 Y chromosomes belonging to 47 European and circum-European populations. Patterns of geographic differentiation are highly nonrandom, and, when they are assessed using spatial autocorrelation analysis, they show significant clines for five of six haplogroups analyzed. Clines for two haplogroups, representing 45% of the chromosomes, are continentwide and consistent with the demic diffusion hypothesis. Clines for three other haplogroups each have different foci and are more regionally restricted and are likely to reflect distinct population movements, including one from north of the Black Sea. Principal-components analysis suggests that populations are related primarily on the basis of geography, rather than on the basis of linguistic affinity. This is confirmed in Mantel tests, which show a strong and highly significant partial correlation between genetics and geography but a low, nonsignificant partial correlation between genetics and language. Genetic-barrier analysis also indicates the primacy of geography in the shaping of patterns of variation. These patterns retain a strong signal of expansion from the Near East but also suggest that the demographic history of Europe has been complex and influenced by other major population movements, as well as by linguistic and geographic heterogeneities and the effects of drift.