Protein mannosyltransferases (Pmt proteins) initiate O glycosylation of secreted proteins in fungi. We have characterized PMT6, which encodes the second Pmt protein of the fungal pathogen Candida albicans. The residues of Pmt6p are 21 and 42% identical to those of C. albicans Pmt1p and S. cerevisiae Pmt6p, respectively. Mutants lacking one or two PMT6 alleles grow normally and contain normal Pmt enzymatic activities in cell extracts but show phenotypes including a partial block of hyphal formation (dimorphism) and a supersensitivity to hygromycin B. The morphogenetic defect can be suppressed by overproduction of known components of signaling pathways, including Cek1p, Cph1p, Tpk2p, and Efg1p, suggesting a specific Pmt6p target protein upstream of these components. Mutants lacking both PMT1 and PMT6 are viable and show pmt1 mutant phenotypes and an additional sensitivity to the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid). The lack of Pmt6p significantly reduces adherence to endothelial cells and overall virulence in a mouse model of systemic infection. The results suggest that Pmt6p regulates a more narrow subclass of proteins in C. albicans than Pmt1p, including secreted proteins responsible for morphogenesis and antifungal sensitivities.Secreted proteins in fungi can get modified by the attachment of short glycosyl chains consisting of one to seven mannoses to serine or threonine residues (reviewed in reference 38). The first mannosylation step in O glycosylation occurs in the endoplasmic reticulum, presumably cotranslationally, and is mediated by protein mannosyltransferases (Pmt proteins). In the yeast Saccharomyces cerevisiae seven PMT genes are known (20,24,28,37); their paralogous gene products, by their degree of homology, can be grouped in at least two subclasses consisting of either the Pmt1 and Pmt5 proteins or the Pmt2, Pmt3, and Pmt6 proteins (10). We recently isolated and characterized the PMT1 gene of the important human fungal pathogen Candida albicans (41). Pmt homologues in Drosophila melanogaster (25) and humans (21) have also been described, and Pmt homologues deduced from "expressed sequence tags" occur in nematodes, plants, and mammals, although their enzymatic functions as Pmt proteins have not yet been demonstrated. In C. albicans, O-glycosyl chains initiated by Pmt proteins are extended further by mannosyltransferases including Mnt1p (3).Although the molecular details of target protein recognition by Pmt proteins are unknown, it appears that Pmt proteins can have a preference for certain glycosylation targets. Thus, the lack of Pmt1 and Pmt2 proteins in S. cerevisiae mutants affects O glycosylation of a set of secreted proteins overlapping with, but different from, the set affected in mutants lacking Pmt4p (16), and certain cell wall proteins are affected differently by mutations in PMT genes (28). Recently, the Axl2p protein, involved in axial budding, was recognized as a specific target of mannosylation by Pmt4p (29). Unknown O-glycosylated proteins are needed for gener...

Due to the high permeability of endothelial cell layers derived from macrovascular vessels, precise determination of their barrier function towards ion movement requires refined experimental techniques. We thus cultured bovine aortic endothelial cells (BAEC) directly on thin gold-film electrodes and measured the electrochemical impedance to study their passive electrical properties in general and during beta-adrenergic stimulation. Impedance spectra (10-2.10(6) Hz) of confluent cell monolayers revealed that the electrical characteristics of the cells can be modelled by a simple resistor-capacitor parallel network. Under control conditions the overall resistance of confluent BAEC monolayers was 3.6+/-0.6 Omega.cm2 (n=30) and the capacitance was 0. 6+/-0.1 microF/cm2. Both quantities are discussed with respect to morphological characteristics of these cells. Stimulation of BAECs with the synthetic beta-adrenoceptor agonist isoproterenol leads to a concentration-dependent, highly specific increase of the cell layer resistance characterized by a concentration for half-maximal response (EC50) of 0.3+/-0.1 microM. The cell layer capacitance, however, remained unaffected. Using impedance measurements at a single frequency, we analysed the response of BAECs to treatment with isoproterenol in comparison with several chemically unrelated compounds known to stimulate the adenosine 3',5'-cyclic monophosphate (cAMP)-dependent signal transduction cascade. These studies confirmed that the enhancement of the cell layer resistance after beta-adrenergic stimulation is mediated by an increase in intracellular cAMP.

Several lines of evidence suggest that endothelial dysfunction and damage present early steps in the pathophysiology of vascular complications in diabetes mellitus [1±3]. Several studies using cultured endothelial cells clearly show that incubation of these cells with high concentrations of glucose leads to severe changes in the proliferation, the adhesive and synthetic properties [1]. Consequently, regulation of vascular relaxation by endothelium becomes disturbed in diabetes [2,3]. In addition to glucose, high concentrations of proinsulin have been shown to promote the synthesis of the plasminogen activator inhibitor type-1 (PAI-1) [4]. This observation indicates that endothelial function can be directly influenced not only by glucose, but also by proinsulin. Thus, high proinsulin levels may contribute to the development of vascular complications in diabetes, if secreted excessively. In line with this assumption proinsulin is increased in the late pre-insulin-dependent diabetes mellitus [5] and especially in non-insulin-dependent diabetic patients with abnormal prohormone convertase PC2 and PC3 activity [6] or with a point mutation in the Diabetologia (1998)

Migration of the fungal pathogen Candida albicans across the endothelial cell layer is considered a prerequisite for the invasion of multiple organs occurring in systemic candidiasis. We developed an experimental system in which C. albicans migrates from a luminal compartment across a monolayer of bovine aortic endothelial cells on a porous filter support to an abluminal compartment. In this system, a C. albicans wild-type strain (ATCC 10261) traverses the endothelial monolayer in a time-, glucose-, and cell concentration-dependent manner. A mutant derivative unable to grow and form hyphae (SGY-243) migrates at a reduced rate. Concomitant to transendothelial migration, the permeability of the endothelial monolayer for dextran diffusion markers is significantly increased. This increase in transendothelial exchange occurs before fungal cells are detectable in the abluminal compartment and is time, glucose, and cell concentration dependent. A mutant strain (hOG301) unable to interact with endothelial cells does not alter endothelial permeability. Thus, transendothelial migration of C. albicans is able to damage the barrier function of an endothelial monolayer. Our experimental system, which reflects key stages of transendothelial migration of C. albicans including adherence and passage across endothelial cells and the extracellular matrix, may be a useful model for comparisons of transendothelial migration characteristics of Candida strains.

No abstract

Barrier function of endothelial cells (EC) was modulated using beta-adrenergic agonists, e.g., isoproterenol (ISO) and formoterol (FOR). To get a direct comparison between EC from different vascular sources, we isolated EC from aorta (BAEC) and retina (BREC) of the same calf. For permeability studies, EC were cultured on polycarbonate filters. At confluency, transendothelial exchange of the diffusion marker fluorescein isothiocyanate-dextran was determined. Microvascular retinal EC monolayers are half as permeable as monolayers from macrovascular BAEC. When EC are stimulated with beta-adrenergic receptor (AR) agonists, monolayer permeability decreases, and the amount of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) increases in both cell types. Comparison of the half-maximum concentrations causing change in permeability (pEC50) shows direct coupling between beta-AR and adenylate cyclase. The beta 2-selective agonist FOR stimulates cAMP synthesis in BAEC with a pEC50 value of 9.37 and decreases permeability with a pEC50 value of 9.72. In BREC, the pEC50 values of ISO concerning stimulation of cAMP synthesis and the decrease of permeability are also very similar, 5.32 and 5.34, respectively. BREC are not as sensitive to beta 2-AR agonists as BAEC. The pEC50 value of FOR influence on BREC permeability is 8.77 in comparison with 9.72 for BAEC. These results could be interpreted with different affinities of the beta 2-selective AR agonist in BREC and BAEC.

No abstract

No abstract