beta-Glucanases from Candida albicans: purification, characterization and the nature of their attachment to cell wall components

beta-Glucanase activities were found associated with Candida albicans and their culture fluids. Mild acid treatment of the organisms led to rapid inactivation of beta-glucanase activities, the degree of loss increasing with the age of the cultures; the results suggested an extracytoplasmic location of the cell-associated enzymes. Most of the beta-glucanase activities were associated with the cell walls in organisms phenotypically resistant to amphotericin B methyl ester (AME). Two proteins (I and II) exhibiting beta-glucanase activity were isolated and purified by conventional procedures from cell-free extracts, cell-wall autolysates and culture fluids of C. albicans sensitive and phenotypically resistant to AME. The purified enzymes appeared homogeneous on isoelectric focusing, gel electrophoresis and ultracentrifugation, with molecular weights of 150000 (I) and 49000 (II). Both enzymes hydrolysed cell walls purified from AME-sensitive and phenotypically resistant organisms, but showed different substrate specificities and patterns of activity. Enzyme II hydrolysed (1 leads to 3)-beta-glycans by an endolytic mechanism releasing laminaritetraose as the initial product. Glucose was the only product released by enzyme I. The properties of th individual enzymes were unaffected by their localization or the age of the culture of the organisms. The loosening of the polysaccharide packing by ultrasonic treatment of cell walls purified from AME-resistant organisms increased the beta-glucanase activities bound to the walls, but did not solubilize them. Autolysis of cell walls released 58 to 66% of their beta-glucanase activity in 20 h, but no further release was attained on prolonged incubation. The amount of beta-glucanase activity released by autolysis was increased by a variety of pretreatments. Diethyl pyrocarbonate inhibited beta-glucanase activity and prevented autolysis. Evidence is presented indicating that interactions with lipids, polysaccharides and other cell wall proteins may be involved in the control of the activity of the cell wall-associated beta-glucanases in organisms phenotypically resistant to AME.     

Phenotypic resistance to amphotericin B in Candida albicans: relationship to glucan metabolism

The phenotypic resistance to amphotericin methyl ester (AME) of stationary phase cultures of Candida albicans was decreased by alkaline pH values and by treatment with 2-mercaptoethanol or glucanase preparations, and was increased by acid pH values, increased aeration, treatment with N-ethylmaleimide, or the presence of inhibitors of protein synthesis such as trichodermin. The effects of such treatments on endogenous glucanase activity and on the incorporation of glucose residues into the 'glucan fraction' of the organism were studied. The changes in the endogenous levels of lytic activities on laminarin [as a measure of the total (1 leads to 3)-beta-D-glucanase] and on p-nitrophenyl-beta-D-glucoside [reflecting the exo-(1 leads to 3)-beta-D-glucanase] were followed in C. albicans cells under a variety of conditions. Treatments which increased AME sensitivity stimulated both total and exo-(1 leads to 3)-beta-D-glucanase activities, while treatments which promoted resistance decreased the levels of both (1 leads to 3)-beta-D-glucanases. Changes in the 'glucan fraction' were followed by incubating suspensions of organisms in the presence of trace amounts of [U-14C]glucose. The rate of incorporation of radioactivity fell during the first 2-3 d of stationary phase culture and then rose to high values by 7-8 d; AME resistance increased throughout this period. The rate of incorporation was markedly stimulated by prior treatment of the organisms with 2-mercaptoethanol or glucanase and inhibited by trichodermin or treatment with N-ethylmaleimide. The addition in the concentration range 0.3-3 mM of the glucose analogues beta-D-allose, 3-O-methyl-D-glucose, 2-deoxy-D-glucose or 5-thio-D-glucose to cultures 24 h after inoculation prevented any further increase in AME resistance for the next 2-3 d and resulted in a decrease in the level of resistance established at the time of addition. Radioactivity from 14C- or 3H-labelled analogues added, 24 h after inoculation, to stationary phase cultures was incorporated into the 'glucan fraction' of the organisms. The incorporation of glucose residues into the 'glucan fraction' is controlled by the activity of glucanases in producing glucose acceptor sites. The results reported confirm that there is a correlation between glucan metabolism, glucanase activity and resistance to AME, in that any factor leading to increased glucanase action also results in decreased resistance and vice versa, while incorporation of certain glucose analogues into the 'glucan fraction' delays the further increase in resistance.     

CD69 Does Not Affect the Extent of T Cell Priming

CD69 is rapidly upregulated on T cells upon activation. In this work we show that this is also the case for CD69 expression on dendritic cells (DC). Thus, the expression kinetics of CD69 on both cell types is reminiscent of the one of costimulatory molecules. Using mouse models of transgenic T cells, we aimed at evaluating the effect of monoclonal antibody (MAb)-based targeting and gene deficiency of CD69 expressed by either DC or T cells on the extent of antigen (Ag)-specific T cell priming, which could be the result of a putative role in costimulation as well as on DC maturation and Ag-processing and presentation. CD69 targeting or deficiency of DC did not affect their expression of costimulatory molecules nor their capacity to induce Ag-specific T cell proliferation in in vitro assays. Also, CD69 targeting or deficiency of transgenic T cells did not affect the minimal proliferative dose for different peptide agonists in vitro. In in vivo models of transgenic T cell transfer and local Ag injection, CD69 deficiency of transferred T cells did not affect the extent of the proliferative response in Ag-draining lymph nodes (LN). In agreement with these results, CD69 MAb targeting or gene deficiency of Vaccinia-virus (VACV) infected mice did not affect the endogenous formation of virus-specific CD8⁺ T cell populations at the peak of the primary immune response. Altogether our results argue against a possible role in costimulation or an effect on Ag processing and presentation for CD69.     

Primary structure of the human fgr proto-oncogene product p55c-fgr.

Normal human c-fgr cDNA clones were constructed by using normal peripheral blood mononuclear cell mRNA as a template. Nucleotide sequence analysis of two such clones revealed a 1,587-base-pair-long open reading frame which predicted the primary amino acid sequence of the c-fgr translational product. Homology of this protein with the v-fgr translational product stretched from codons 128 to 516, where 32 differences among 388 codons were observed. Sequence similarity with human c-src, c-yes, and fyn translational products began at amino acid position 76 of the predicted c-fgr protein and extended nearly to its C-terminus. In contrast, the stretch of 75 amino acids at the N-terminus demonstrated a greatly reduced degree of relatedness to these same proteins. To verify the deduced amino acid sequence, antibodies were prepared against peptides representing amino- and carboxy-terminal regions of the predicted c-fgr translational product. Both antibodies specifically recognized a 55-kilodalton protein expressed in COS-1 cells transfected with a c-fgr cDNA expression plasmid. Moreover, the same protein was immunoprecipitated from an Epstein-Barr virus-infected Burkitt's lymphoma cell line which expressed c-fgr mRNA but not in its uninfected fgr mRNA-negative counterpart. These findings identified the 55-kilodalton protein as the product of the human fgr protooncogene.     

Purification of an exo-1,3-beta-glucanase from Candida utilis.

An exo-1,3-beta-glucanase (EC 3.2.1.-) has been purified from the culture fluid of the yeast Candida utilis, and its biochemical properties have been studied. The amino acid analysis revealed a high content of acidic amino acids. The purified enzyme had 20% carbohydrate and a net negative charge showing higher affinity for laminarin than for p-nitrophenyl-beta-D-glucopyranoside and yeast cell-wall 1,3-beta-glucans. In addition, the enzyme hydrolyzed the substrates starting from the nonreducing ends, releasing glucose as the exclusive hydrolysis product. The enzyme activity was strongly inhibited by lactones and also by some heavy-metal ions.     

Assessment of membrane-bound mammal mitochondrial adenine nucleotide translocase topography by experimental antibodies

To gain insight into the immunogenicity of mitochondrial adenine nucleotide translocase (ANT), we raised antibodies against purified bovine heart ANT by induction of ascitic fluid in male Balb/c mice. We identified the antigenic determinants detected by these antibodies by (1) immunodetection of GST-ANT fusion proteins and selected partial constructs of ANT, (2) immunodetection of chemically synthesized overlapping peptides on solid support, and (3) back-titration ELISA. Results revealed a short epitope spreading of the antibodies, resulting in a small number of antigenic determinants. Thus, each antibody detects one or two major epitopes located in the putative hydrophilic loops M2 and M3. No evidence for the antigenicity of the first 133 amino acids of ANT was obtained. These well-characterized antibodies were used to study the topography of the membrane-bound ANT by back-titration ELISA with mitochondrial membranes. We demonstrated that amino acids 145-150 and 230-237 are fully accessible to the antibodies in native ANT, whereas regions 133-140 and 244-251 are not. Furthermore, we used mitochondria devoid of the outer membrane (mitoplasts) and inside-out submitochondrial particles (SMP) to establish the matrix or cytosolic orientation of loops M2 and M3. The results clearly show that these loops have a matrix orientation and thus support the six transmembrane segment model of ANT topography in the inner mitochondrial membrane.