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Biomedical subjects

G M Rodriguez

Publications and source records attributed to G M Rodriguez.

12 recordsLinked to original sources
PubMed2001

The Mycobacterium tuberculosis IdeR is a dual functional regulator that controls transcription of genes involved in iron acquisition, iron storage and survival in macrophages.

In this work, we characterize genes in Mycobacterium tuberculosis that are regulated by IdeR (iron-dependent regulator), an iron-responsive DNA-binding protein of the DtxR family that has been shown to regulate iron acquisition in Mycobacterium smegmatis. To identify some of the genes that constitute the IdeR regulon, we searched the M. tuberculosis genome for promoter regions containing the consensus IdeR/DxR binding sequence. Genes preceded by IdeR boxes included a set encoding proteins necessary for iron acquisition, such as the biosynthesis of siderophores (mbtA, mbtB, mbtI), aromatic amino acids (pheA, hisE, hisB-like) and others annotated to be involved in the synthesis of iron-storage proteins (bfrA, bfrB). Some putative IdeR-regulated genes identified in this search encoded proteins predicted to be engaged in the biosynthesis of lipopolysaccharide (LPS)-like molecules (rv3402c), lipids (acpP) and peptidoglycan (murB). We analysed four promoter regions containing putative IdeR boxes, mbtA-mbtB, mbI, rv3402c and bfrA-bfd, for interaction with IdeR and for iron-dependent expression. Gel retardation experiments and DNase footprinting analyses with purified IdeR showed that IdeR binds to these IdeR boxes in vitro. Analysis of the promoters by primer extension indicated that the IdeR boxes are located near the -10 position of each promoter, suggesting that IdeR acts as a transcriptional repressor by blocking RNA polymerase binding. Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) coupled to molecular beacons, we showed that mRNA levels of mbtA, mbtB, mbtI, rv3402c and bfd are induced 14- to 49-fold in cultures of M. tuberculosis starved for iron, whereas mRNA levels of bfrA decreased about threefold. We present evidence that IdeR not only acts as a transcriptional repressor but also functions as an activator of bfrA. Three of the IdeR- and iron-repressed genes, mbtB, mbtI and rv3402c, were induced during M. tuberculosis infection of human THP-1 macrophages.

Bacterial Proteins↗
PubMed1999

Identification and characterization of two divergently transcribed iron regulated genes in Mycobacterium tuberculosis.

SETTING: Low iron availability in the host induces the expression of iron acquisition systems and virulence genes in many pathogens. IdeR is a mycobacterial iron dependent regulator that controls the iron starvation and oxidative stress responses in Mycobacterium smegmatis. It is important to determine the role of IdeR and its regulon in M. tuberculosis, as identification of iron regulated genes can aid in the design of new drugs and generation of attenuated strains. OBJECTIVE: A potential IdeR binding site was found in the M. tuberculosis genome flanked by two divergently oriented open reading frames, irg1 and irg2. The aim of this study was to determine whether irg1 and irg2 were iron and IdeR regulated genes. DESIGN: Interaction of IdeR with the putative binding sequence was examined by gel shift and footprinting assays. Transcriptional fusions of irg1 and irg2 to IacZ were used to study the effect of iron levels on the expression of these genes. RESULTS: IdeR binds to the predicted binding site, which overlaps with the irg1 promoter. irg1 and irg2 expression was decreased by iron in M. tuberculosis and in wild type M. smegmatis, but not in a M. smegmatis ideR mutant. CONCLUSION: Two M. tuberculosis iron/IdeR regulated genes were identified. irg1 is predicted to be the M. tuberculosis hisE gene, which is involved in histidine biosynthesis. It is directly upstream of the M. tuberculosis hisG. irg2 encodes a putative membrane protein that is a member of the PPE family.

Bacterial Proteins↗
PubMed1998

Extra and intracellular expression of Mycobacterium tuberculosis genes.

To understand how Mycobacterium tuberculosis survives and grows in an infected host, we are studying the mycobacterial transcriptional machinery and its response to stresses encountered in vitro and in vivo. Much has been learned about sigma factors and other transcriptional regulators concerning their roles in controlling mycobacterial gene expression. It has recently been shown that sigma A is the essential housekeeping sigma factor and the alternative sigma factor sigma B, not essential for growth in a laboratory setting, is required for a robust protective response to various environmental stresses. We are also studying the mechanism by which the R522H mutation in sigma A prevents the transcription of certain genes, including some that are believed necessary for virulence. Also under investigation is the mycobacterial iron acquisition apparatus and its regulation, as metabolism of this essential element plays a key role in microbial pathogenesis. We have identified and characterized the major mycobacterial iron regulator IdeR that blocks the synthesis of the iron uptake machinery and have identified target genes in M. smegmatis and M. tuberculosis that are directly repressed by IdeR. Recent studies have examined the control of M. tuberculosis gene expression in vivo. Among these new approaches are an in vivo expression technology system to identify M. tuberculosis genes that are induced in macrophages and mice and a novel RT-PCR method that allows an accurate comparison between the levels of specific mRNAs in M. tuberculosis grown in vitro with those found in bacteria growing in human macrophages.

Animals↗
PubMed1996

Compliance with AZT treatment regimen of HIV-seropositive injection drug users: a neglected issue.

Although treatment with zidovudine (AZT) is now recommended for asymptomatic and symptomatic HIV-infected persons with CD4+ cell counts of 0.20 to 0.50 x 10(9)/L and under, data gathered from a small convenience sample of current and former injection drug users with AIDS in the New York City metropolitan region suggest that noncompliance with HIV/AIDS-related therapeutic regimen may be common in this population. This paper enumerates the reasons for noncompliance offered by these informants, reviews the general literature on treatment compliance to identify additional potential reasons for non-adherence to AZT treatment regimen, and outlines some suggestions for future research into this important issue that may prompt changes in the antiviral delivery system.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Lysosomal hydrolases are present in melanosomes and are elevated in melanizing cells.

Melanosomes, the subcellular site of melanin synthesis and deposition, may be related to the endolysosomal lineage of organelles. To determine if melanosomes contain lysosomal hydrolases, we examined the subcellular distribution of five of these enzymes in melanocytes cultured from C57BL/6J mice. Analyses of Percoll gradient density centrifugations demonstrated that beta-hexosaminidase, beta-galactosidase, beta-glucuronidase, and cathepsins B and L all co-sedimented with tyrosinase-rich densely sedimenting melanosomes. The melanosomal distribution of these enzymes was confirmed in studies of melanocytes cultured from albino mice and of melanocytes rendered amelanotic by transfection with the v-rasHa oncogene (which lack dense, melanized melanosomes). In these cells, only a less dense peak of activity for each hydrolase was present. The level of each hydrolase was elevated in black cells when compared with albino cells. Metabolic labeling studies confirmed that the increase in beta-glucuronidase in black versus albino cells resulted mainly from increased synthesis of this enzyme. The data suggest that melanosomes represent specialized lysosomes present within melanocytes, that they contain a broad array of lysosomal hydrolases, and that the levels of these hydrolases are elevated in cells actively engaged in pigment production.

Animals↗
PubMed1995

Destructive proteolysis by cysteine proteases in antigen presentation of ovalbumin.

Most native antigens require digestion by acidic proteases in order to be recognized in the context of major histocompatibility complex class II by T helper cells (Th). We have studied the roles of three different acidic proteases, cathepsin D, cathepsin B and cathepsin L, in the processing of ovalbumin (OVA) for presentation in the context of I-Ad. We report that digestion of OVA in vitro with the aspartyl protease cathepsin D generates the epitope OVA322-336, which is recognized by I-Ad-restricted OVA-specific Th in the presence of paraformaldehyde-fixed antigen-presenting cells (APC). In contrast, digestion of OVA with the cysteine proteases cathepsin B and L not only failed to generate an epitope, but also destroyed OVA322-336. In the presence of fixed APC expressing I-Ad. OVA322-336 was protected from destructive proteolysis by cathepsin L. These results illustrate the dependence of epitope selection on the intracellular proteolytic environment in APC, and suggest that mechanisms must exist for protection of epitopes from destructive proteolysis in the processing compartments.

Amino Acid Sequence↗
PubMed1995

Seroprevalence and risk factors associated with HTLV-I/II infection in injection drug users in northern New Jersey.

Data from an AIDS Demonstration Research project in Paterson, NJ, that enrolled out-of-treatment injection drug users (IDUs) were analyzed to detect demographic patterns and risk factors associated with infection with human immunodeficiency virus (HIV) and human T-lymphotropic virus types I or II (HTLV-I/II). Of 410 IDUs screened, 44.2% were HIV-positive and 19.3% were HTLV-I/II-positive. African-Americans were significantly more likely than other racial groups to be HTLV-I/II-seropositive and to be HIV-seropositive. Over one-fifth of African-Americans--but no Latinos or whites--were doubly infected with HIV and HTLV-I/II. In logistic regression analysis, African-American race, long-term injection drug use, and age were significant predictors of HTLV-I/II-seropositivity. While the associations between recent needle practices and HTLV-I/II-seropositivity fell short of significance, the trends in the data were consistent with a hypothesis that HTLV-I/II is transmitted through the sharing of injection equipment. Public health implications of the data are discussed.

Adolescent↗
PubMed1994

A comparison of male and female intravenous drug users' risk behaviors for HIV infection.

Despite the central role played by female intravenous drug users (IVDUs) in the worsening AIDS statistics of states in the northeastern United States, the relative paucity of research into the HIV risk behaviors--particularly risky needle practices--of female drug injections has left significant gaps in researchers' understanding of how and to what extent such women may differ in their risks from their better-studied male counterparts. This study, derived from a sample of 769 out-of-treatment IVDUs residing in an area (Paterson, New Jersey) characterized by high levels of AIDS and HIV infection among drug users, attempts to address this lacuna in the research literature by comparing the drug usage, AIDS knowledge, and needle and sexual behaviors of male and female IVDUs that place them at risk for HIV infection. In this sample, gender was found to be unrelated to HIV serostatus, injection frequency and injected drug of choice, and to most dimensions of knowledge about AIDS and the means of HIV transmission. Overall, it appears that the average Paterson female IVDU may be at greater risk for HIV infection as a result of involvement with a drug-using sex partner than because of especially risky needle practices, for females in this sample were significantly more likely than males to report injecting with a sex partner in the previous 6 months, and female IVDUs with one sex partner were more than twice as likely as males with one partner to report that this individual was an IVDU. Condom use was relatively rare, particularly among those with one partner. Moreover, female IVDUs were significantly more likely than males to be daily users of crack cocaine, and significantly more likely to report poorer health. However, current needle and sexual practices were found to be unrelated to HIV seropositivity among both males and females. In logistic regression analysis, only length of IV drug involvement was found to be independently associated with HIV seropositivity for both sexes. Implications of the data for future prevention efforts aimed at female IVDUs are discussed.

Adolescent↗
PubMed1992

Role of cathepsin D in antigen presentation of ovalbumin.

Modification of protein Ag by proteolysis is one of the principal steps in the presentation of Ag to Th cells. However, little is known about the enzymes participating in these events, their specificity or the characteristics of the natural fragments that they produce. Cathepsin D (CD) is an aspartyl protease identified in endosomes of APC. In this report, the role of CD in the processing of OVA has been investigated. OVA digested in vitro with purified CD was able to stimulate IL-2 secretion by three different OVA-specific I-Ad restricted Th cell hybridomas when it was presented by fixed APC. The digest of OVA was recognized in the context of I-Ad, but not by I-Ak-restricted OVA-specific Th cells. No difference was observed in the ability of OVA digested with CD to stimulate Th cells in the absence of FCS or in the presence of protease inhibitors indicating that extracellular proteases were not likely to contribute to processing of OVA. Taken together, these results suggest that CD is necessary and sufficient for the generation of an antigenic epitope from OVA. A fragment containing the epitope was isolated from the OVA digest by reverse phase HPLC. This fragment, which migrates in SDS-PAGE as a 10-kDa polypeptide, is a potent epitope. Its capacity to activate Th cells is compared to that of the tryptic peptide OVA323-339.

Animals↗
PubMed1989

Hepatitis B virus replication in hepatitis B and D coinfection.

The clinical course, changes in liver function tests and the behaviour of viral markers over the course of time have been examined in 45 patients with acute hepatitis B and 14 patients with acute hepatitis caused by B and D viruses coinfection. There were no significant differences either in the clinical course or in the liver function tests, in the two groups. The changes in serum viral markers were as follows: HBV-DNA was the first marker to disappear; this was closely followed by HBeAg, and HBsAg was the last marker to become negative, during convalescence. This pattern was not altered by Delta coinfection. When we quantified serum HBV-DNA in both groups of patients, we found that Delta virus infection led to parital inhibition of HBV replication, so that serum HBV-DNA levels were significantly lower in those patients with acute hepatitis B who were simultaneously infected with Delta virus.

Adolescent↗