Mycobacterium-containing phagosomes are accessible to early endosomes and reflect a transitional state in normal phagosome biogenesis.

The success of Mycobacterium as a pathogen hinges on its ability to modulate its intracellular environment. Mycobacterium avium reside in vacuoles with limited proteolytic activity, maintain cathepsin D in an immature form and remain accessible to internalized transferrin. Artificial acidification of isolated phagosomes facilitated processing of cathepsin D, demonstrating that pH alone limits proteolysis in these vacuoles. Moreover, analysis of IgG-bead phagosomes at early time points during their formation indicates that these phagosomes also acquire LAMP 1 and cathepsin D prior to the accumulation of proton-ATPases, and are transiently accessible to sorting endosomes. This suggests that the anomolous distribution of endosomal proteins in M. avium-containing vacuoles results from their arrested differentiation in an early transitional stage through which all phagosomes pass.     

Structural analysis and proteolytic activation of Enterococcus faecalis cytolysin, a novel lantibiotic

Clinical isolates of Enterococcus faecalis more commonly produce a cytolysin than do commensal isolates. Epidemiologic evidence and animal-model studies have established a role for the cytolysin in the pathogenesis of enterococcal disease. The cytolysin consists of two structural subunits, CylL_L and CylL_s, that are activated by a third component, CylA. Genetic and biochemical characterization of CylA indicate that it is a serine protease, and that activation putatively results from cleavage of one or both cytolysin subunits. Genetic evidence also suggests that the cytolysin subunits are related to the rapidly growing class of bacteriocins termed lantibiotics. However, unlike lantibiotics, the cytolysin is lytic for eukaryotic as well as prokaryotic cells, and it consists of two structural subunits. This report describes the purification and characterization of the cytolysin subunits and detection of lanthionine-type post-translational modifications within their structures. Furthermore, the cleavage specificity of the CylA activator is reported and it is shown that proteolytic activation of both subunits is essential for activity.     

Transient transfection of mammalian cells with DNA of the plant-pathogenic Ti-plasmid and expression of marker and resident sequences

Summary The large Ti-plasmid from Agrobacterium tumefaciens strain C58 has been used for transfection experiments with mammalian cells. In DNA from Tupaia baby fibroblasts Ti-plasmid sequences could be identified by filter hybridization as long as four weeks after transfection including two cell passages. The hybridization signals decreased rapidly after addition of the Ti-plasmid DNA-coprecipitate to the cells. The signals were often not detected any more after the first day, but were visible one week after transfection. Nuclei prepared from Ti-plasmid-transfected cells hybridized to pTi-specific RNA. With the chloramphenicol acetyl transferase-gene as marker no discrimination in DNA uptake was found between the Ti-plasmid and much smaller plasmids. According to the number of nuclei with homology to pTi-sequences it is assumed that about 0.2% of the cells carry Ti-plasmid DNA in the nucleus. Analysis of RNA isolated from cells transfected with cloned segments of the Ti-plasmid revealed that the TDNA region of the Ti-plasmid was predominantly transcribed.Abbreviations CAT Chloramphenicol Acetyl Transferase - NPT Neomycin Phosphotransferase - SDS Sodium Dodecylsulfate - TK Thymidine Kinase     

Combined effects of hypertension and conditioning on coronary vascular reserve in rats

To evaluate the combined effects of cardiac overload imposed by hypertension and chronic swim training on coronary vascularity, female rats were made hypertensive by unilateral renal artery stenoses and were exercised in an 8- to 10-wk swimming program. Maximal coronary flow was assessed in isolated retrograde buffer-perfused hearts under conditions of minimal coronary resistance (15 microM adenosine or anoxia). Sedentary normotensive animals, sedentary hypertensive animals, and normotensive animals exposed to a swimming program were also studied. Swimming was associated with an 18% increase in heart weight and with increases in both absolute (ml/min) and relative (ml X g-1 X min-1) maximal coronary flow. Hypertension was associated with a 32% increase in heart weight but with a decrease in absolute and relative coronary flow compared with controls. The combined stimuli resulted in a 63% myocardial hypertrophy and a 19% increase in absolute flow. Relative coronary flow (g tissue-1) was similar in hearts from hypertensive sedentary animals and hypertensive swimmers. These data indicate that the coronary vascular deficit that accompanies the cardiac hypertrophy of hypertension is not worsened by the superimposition of an exercise load that exaggerates the hypertrophy.     

Regulation of sodium and water excretion by catecholamines

There is considerable evidence that the renal nerves contribute to the regulation of salt and water excretion by a direct effect on tubular reabsorption, independent of changes in renal hemodynamics. Whereas the effect of the adrenergic nervous system on sodium reabsorption appears to be established in anesthetized animals, it has been suggested that the basal activity of the renal sympathetic nerves in conscious dogs is too low to have a significant effect on sodium reabsorption by the proximal tubules. However, denervation natriuresis and diuresis have recently been demonstrated in conscious euvolemic and conscious volume-expanded rats. The effects of renal nerve stimulation on the handling of sodium and water by the proximal tubule can be mimicked by infusion of the α-adrenergic agonist norepinephrine and prevented by infusion of an α-adrenergic antagonist. This confirms that they are mediated by α-receptors. The adrenergic nervous system may have an independent role in the control of sodium excretion or may be complementary to other systems such as the renin-angiotensin-aldosterone system.     

Effects of theophylline on insulin receptors and insulin action in the adipocyte

Summary The effects of theophylline on insulin receptors and insulin action in isolated rat adipocytes were studied. Theophylline reduced insulin binding by a decrease of receptor affinity. As concentration-response curves revealed, the effect was paralleled by a reduction of the cellular ATP content. Basal as well as insulin-stimulated glucose transport (2-deoxyglucose and 3-O-methylglucose uptake) were inhibited by much smaller theophylline concentrations (0.15–0.6 mM ) than those necessary to reduce insulin binding and to lower ATP levels (1–4.8 mM), or to stimulate lipolysis (0.3-2.4 mM). Insulin fully antagonized the effect of theophylline on lipolysis but failed to reverse the inhibition of glucose transport completely. The results suggest that (a) theophylline impairs insulin action at a post-receptor level and, at higher concentrations, by a decrease of receptor binding, (b) the reduction of insulin receptor affinity probably reflects ATP depletion of the adipocyte, and (c) the xanthine inhibits glucose transport independently from its effects on lipolysis.     

Detection and characterization of two antigens specific for cell walls ofCandida albicans mycelial growth phase

Antiserum againstCandida albicans ATCC 10231 mycelium growth phase absorbed with yeast cells and intracellular material of mycelium cells showed a positive reaction with mycelium cells in immunofluorescence assay, whereas with yeast cells the reaction was negative. Mycelium and blastospore cell wall were extracted with dithiothreitol (DTT_My- and DTT_B-extract). When DTT_My was separated in gel-electrophoresis, two glycoprotein bands of 87 and 67 Kd could be detected. In immunoblot these bands showed a strong reaction with mycelium cell wall-specific antiserum, but also a weak reaction with the blastospore antiserum. Whereas pronase treatment destroyed antigenicity, mannanase treatment did not. After enzymatic digestion with endoglycosidase H, four major enzymatic digestion products were found at 37, 35, 30, and 27 Kd when protein staining was performed. The digestion products at 37 and 35 Kd could be made visible through glycoprotein staining. Antibodies of yeast-phase-immunized animals reacted only with the 37 and 30 Kd bands, whereas the digestion products at 35 and 27 Kd were also detected by mycelium cell wall-specific antiserum. This proves the existence of two mycelium cell wall-specific antigens (35 Kd and 27 Kd) that can be isolated from the DTT-mycelium cell wall extract by endoglycosidase H treatment.     

Cone-shaped HIV-1 capsids are transported through intact nuclear pores

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