Raul Mancilla

New insights into protein O-mannosylation in actinomycetes

Glycosylation is a common post-translational modification of surface exposed proteins and lipids present in all kingdoms of life. Information derived from bacterial genome sequencing, together with proteomic and genomic analysis has allowed the identification of the enzymatic glycosylation machinery. Among prokaryotes, O-mannosylation of proteins has been found in the actinomycetes and resembles protein O-mannosylation in fungi and higher eukaryotes. In this review we summarize the main features of the biosynthetic pathway of O-mannosylation in prokaryotes with special emphasis on the actinomycetes, as well as the biological role of the glycosylated target proteins.

High-Level Expression of NRAMP1 in Peripheral Blood Cells and Tuberculous Granulomas from Mycobacterium bovis-Infected Bovines

ABSTRACT By Western blotting, we demonstrate high-level expression of NRAMP1 proteins in peripheral blood cells and granulomas ofMycobacterium bovis-infected bovines. Immunohistochemistry of granulomatous lesions showed heavily labeled epithelioid macrophages and Langhans cells. These data suggest that M. bovisinfection enhances NRAMP1 expression and that active tuberculosis can occur despite this response.

Binding and Activation of Human Plasminogen by Mycobacterium tuberculosis

ABSTRACT The first evidence of the interaction of Mycobacterium tuberculosis with the plasminogen system is herein reported. By FACScan analysis and affinity blotting, lysine-dependent binding of plasminogen to M. tuberculosis was demonstrated. The binding molecules were 30-, 60-, and 66-kDa proteins present in cell wall and soluble protein extracts. The activation of plasminogen, which occurred only in presence of fibrin and was not inhibited by the host serpin, α2-antiplasmin, was also demonstrated.

Mammary 5′deiodinase (5′D) during the breeding cycle of the rat: indirect evidence that 5′D type I is specific to the alveolar epithelium

The present study analyses the activity of 5′deiodinases type I and II in mammary gland during the breeding and estrous cycle of the rat, and includes indirect evidence that 5′D-I is present only in the alveolar epithelium. Data show that the mammary gland exhibits 5′D-II activity throughout the developmental period and its activity varies along the allometric growth of the gland. 5′D-I is detected during the differentiation stages of the alveolar epithelium (puberty, late pregnancy) and its activity rises significantly (10-fold) 24 h after delivery. Data also show that 5′D-I activity is not present in the fat pads of the gland. These findings suggest that during its differentiation and functional stages, the mammary gland requires an elevated and compartmentalized production of T3.

Rapid Screening Test for Tuberculosis Using a 38-kDa Antigen From Mycobacterium tuberculosis

A screening test for the diagnosis of tuberculosis by immunodot (Idt) is described, using an antigen of Mycobacterium tuberculosis, namely, a 38‐kDa glycoprotein which has shown great specificity in previous serologic analyses. The test was used to examine 28 sera from patients with lung tuberculosis. Of these, 85% were positive by micro‐ELISA and by the Idt test herein described. Control sera from healthy subjects (n=20) gave negative results for ELISA and for Idt, which indicates that the screening test is highly specific. The test is easy to handle and requires no equipment and is therefore particularly useful for field studies. J. Clin. Lab. Anal. 12:126–129, 1998.

Characterization of the raphe nuclei of the reptile Ctenosaura pectinata

The brain stem of the lizard Ctenosaura pectinata was studied in 10 microns thick sections following the Nissl and eosin-hematoxilin techniques. Furthermore, the distribution of serotonin-containing neuronal somata in this encephalic region was determined by means of an indirect immunofluorescence technique using a specific antibody to serotonin. Two of the cellular groups of the brain stem were identified as the superior and inferior raphe nuclei, which show serotonergic cells of variable size (between 17 and 30 microns). The results obtained in the present study together with information coming from other authors, suggest that serotonergic neuronal systems placed at brain stem level of vertebrates are phylogenetically ancient.

Decreased Expression of T-Cell Costimulatory Molecule CD28 on CD4 and CD8 T Cells of Mexican Patients with Pulmonary Tuberculosis

Patients with tuberculosis frequently develop anergy, a state of T-cell hyporesponsiveness in which defective T-cell costimulation could be a factor. To know if the expression of T-cell costimulatory molecules was altered in tuberculosis, we analyzed the peripheral blood T-cell phenotype of 23 Mexican patients with pulmonary tuberculosis. There was severe CD4 (P < .001) and CD8 (P < .01) lymphopenia and upregulation of costimulatory molecule CD30 on CD4 and CD8 T cells (P < .05); this increase was higher in relapsing tuberculosis. The main finding was severe downregulation of the major costimulatory molecule CD28 on both CD8 and CD4 T cells (P < .001). Depletion of the CD4/CD28 subset, a hitherto undescribed finding, is relevant because CD4 T cells constitute the main arm of the cell-mediated antimycobacterial immune response.

A microsatellite study of bovine solute carrier family 11 a1 (Slc11a1) gene diversity in Mexico in relation to bovine tuberculosis

Bovine tuberculosis, caused by Mycobacterium bovis, is a disease of socio-economic and public health importance and of significance to international trade regulation. Allelic variants of several genes have been implicated in the genetic susceptibility to tuberculosis in some human populations, but little is known in cattle. We surveyed 34 European, 18 Asian, 20 Creole and 23 hybrid bovines for polymorphisms of the bovine solute carrier family 11 a1(Slc11a1) gene, formerly known as natural resistance associated macrophage protein (Nramp1), gene by typing the cattle using two microsatellite loci closely linked to this gene. The microsatellites used were 311-22, located at the 3’ untranslated region (3’ UTR) of the Slc11a1 gene, and ARO28 situated about 0.6 cM upstream of the same gene Based on allele size in base pairs (bp) we determined five 311-223 locus variants (221, 223, 225, 227 and 229 bp) and 12 ARO28 loci. There was marked diversity and a very high level of heterozygosity in most of the cattle surveyed except the Europeans bovines and especially Holsteins in relation to the 3’ UTR microsatellite locus.

Mycobacterial glycolipids di-O-acylated trehalose and tri-O-acylated trehalose downregulate inducible nitric oxide synthase and nitric oxide production in macrophages

BackgroundTuberculosis (TB) remains a serious human health problem that affects millions of people in the world. Understanding the biology of Mycobacterium tuberculosis (Mtb) is essential for tackling this devastating disease. Mtb possesses a very complex cell envelope containing a variety of lipid components that participate in the establishment of the infection. We have previously demonstrated that di-O-acylated trehalose (DAT), a non-covalently linked cell wall glycolipid, inhibits the proliferation of T lymphocytes and the production of cytokines.ResultsIn this work we show that DAT and the closely related tri-O-acylated trehalose (TAT) inhibits nitric oxide (NO) production and the inducible nitric oxide synthase (iNOS) expression in macrophages (MØ).ConclusionsThese findings show that DAT and TAT are cell-wall located virulence factors that downregulate an important effector of the immune response against mycobacteria.

Dendritic cells that phagocytose apoptotic macrophages loaded with mycobacterial antigens activate CD8 T cells via cross-presentation

While homeostatic apoptosis is immunologically silent, macrophage apoptosis during Mycobacterium tuberculosis infection can potentially induce an immune response against the mycobacteria. To examine the role of dendritic cells in this response, macrophage apoptosis was induced by incubating the macrophage with cell wall extracts of mycobacteria expressing LpqH. The apoptogenic proteins of the cell wall extracts were engulfed by the macrophage and then were translocated from the cytosol to the nuclei of the dying cells. Dendritic cells that engulfed the apoptotic macrophages acquired an immunogenic phenotype that included upregulation of MHC-I, increased expression of the costimulatory molecules, CD40, CD80, and CD86, and increased production of IL-12, IL-10, TNF-α, and TGF-β. In addition, the dendritic cells triggered a proliferative response of CD8+ T cells with IFN-γ production via cross-presentation. Taken together, these findings support a model in which phagocytosis of whole apoptotic cells carrying mycobacterial antigens promotes a potentially protective immune response.