Major upper gastrointestinal bleeding and the use of calcium channel blockers.
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Aspirin↗
Biomedical subjects
A Laszlo
Publications and source records attributed to A Laszlo.
Heat resistance in mammalian cells: lessons and challenges.
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Animals↗
Global surveillance for antituberculosis-drug resistance, 1994-1997. World Health Organization-International Union against Tuberculosis and Lung Disease Working Group on Anti-Tuberculosis Drug Resistance Surveillance.
BACKGROUND: Drug-resistant tuberculosis threatens efforts to control the disease. This report describes the prevalence of resistance to four first-line drugs in 35 countries participating in the World Health Organization-International Union against Tuberculosis and Lung Disease Global Project on Anti-Tuberculosis Drug Resistance Surveillance between 1994 and 1997. METHODS: The data are from cross-sectional surveys and surveillance reports. Participating countries followed guidelines to ensure the use of representative samples, accurate histories of treatment, standardized laboratory methods, and common definitions. A network of reference laboratories provided quality assurance. The median number of patients studied in each country or region was 555 (range, 59 to 14,344). RESULTS: Among patients with no prior treatment, a median of 9.9 percent of Mycobacterium tuberculosis strains were resistant to at least one drug (range, 2 to 41 percent); resistance to isoniazid (7.3 percent) or streptomycin (6.5 percent) was more common than resistance to rifampin (1.8 percent) or ethambutol (1.0 percent). The prevalence of primary multidrug resistance was 1.4 percent (range, 0 to 14.4 percent). Among patients with histories of treatment for one month or more [corrected], the prevalence of resistance to any of the four drugs was 36.0 percent (range, 5.3 to 100 percent), and the prevalence of multidrug resistance was 13 percent (range, 0 to 54 percent). The overall prevalences were 12.6 percent for resistance to any of the four drugs [corrected] (range, 2.3 to 42.4 percent) and 2.2 percent for multidrug resistance (range, 0 to 22.1 percent). Particularly high prevalences of multidrug resistance were found in the former Soviet Union, Asia, the Dominican Republic, and Argentina. CONCLUSIONS: Resistance to antituberculosis drugs was found in all 35 countries and regions surveyed, suggesting that it is a global problem.
Antitubercular Agents↗
Drug-resistant tuberculosis in the Dominican Republic: results of a nationwide survey.
SETTING: The Dominican Republic. OBJECTIVE: To assess the extent of drug-resistant tuberculosis (TB) following the guidelines of the World Health Organization (WHO)/International Union Against Tuberculosis and Lung Disease (IUATLD) new global surveillance project on drug resistance in TB. METHODS: Using a multi-step proportional weighted approach, a sample of 688 sequential cases of smear positive pulmonary TB diagnosed between April 1994 and April 1995 was studied in six of the country's eight health regions. Pre-treatment sputum samples were cultured on Loewenstein-Jensen medium and drug susceptibility tests were performed using the economic variant of the proportion method. RESULTS: Of 420 cases with drug susceptibility results, resistance to one or more drugs was observed in 43.8%; resistance was found in 52.1% of 117 TB cases with a history of previous antituberculosis treatment and in 40.6% of 303 new TB cases. In five of the six health regions surveyed, > or = 41% of strains were resistant to one or more drugs. Multidrug resistance (MDR) to isoniazid and rifampicin with or without resistance to other drugs was found in 43 (10.2%) of 420 cases, including 6.6% of new TB cases. In five of the six health regions > or = 8% of strains were classified as MDR. Independent predictors of MDR-TB included being in the age group 25 to 44 years (odds ratio [OR] = 4.2, 95% confidence interval [Cl] 1.5, 11.6; P = 0.005), being aged 45 years and over (OR = 4.5, 95% CI 1.4, 14.4; P = 0.009), and having a prior history of TB (OR = 3.7, 95% CI 1.9, 7.4; P = 0.0001). CONCLUSION: The proportion of Mycobacterium tuberculosis strains resistant to one or more anti-TB drugs in the Dominican Republic is among the highest observed world-wide. The severity of the problem urgently requires the full implementation of TB control strategies endorsed by the WHO and the IUATID, which include political commitment to a National TB Program, case detection utilizing sputum-smear microscopy, directly observed treatment, regular drug supply, and standardised recording and reporting systems. Also, the sale of TB drugs in the private market should be controlled.
Adult↗
Ubiquitin-dependent degradation of certain protein substrates in vitro requires the molecular chaperone Hsc70.
Degradation of a protein via the ubiquitin system involves two discrete steps, signaling by covalent conjugation of multiple moieties of ubiquitin and degradation of the tagged substrate. Conjugation is catalyzed via a three-step mechanism that involves three distinct enzymes that act successively: E1, E2, and E3. The first two enzymes catalyze activation of ubiquitin and transfer of the activated moiety to E3, respectively. E3, to which the substrate is specifically bound, catalyzes formation of a polyubiquitin chain that is anchored to the targeted protein. The polyubiquitin-tagged protein is degraded by the 26 S proteasome, and free and reutilizable ubiquitin is released. In addition to the three conjugating enzymes, targeting of certain proteins requires association with ancillary proteins and/or post-translational modification(s). Using a specific antibody to deplete cell extract from the molecular chaperone Hsc70, we demonstrate that this protein is required for the degradation of actin, alpha-crystallin, glyceraldehyde-3-phosphate dehydrogenase, alpha-lactalbumin, and histone H2A. In contrast, the degradation of bovine serum albumin, lysozyme, and oxidized RNase A is Hsc70-independent. Mechanistic analysis revealed that the chaperone is required for the conjugation reaction; however, it does not substitute for E3. Involvement of the chaperone in the proteolytic process requires complex formation with the substrate. Formation of this complex appears to be essential in the proteolytic process. In addition, the proper function of the chaperone in the proteolytic process requires the presence of K+, which allows rapid cycles of dissociation and association of the complex. The chaperone may act by binding to the substrate and unfolding it to expose a ubiquitin ligase-binding site. In addition, it can also act directly on the ubiquitination machinery.
Actins↗
Genetic identification of Mycobacterium bovis BCG by restriction fragment length polymorphism analysis of the direct-repeat region.
Restriction fragment length polymorphism (RFLP) analysis was performed on the direct repeat (DR) regions of 14 strains of Mycobacterium bovis BCG. With AluI-digested DNA, BCG Japanese, Russian, and Mexican had differing RFLP patterns but 11 strains, including Pasteur, Glaxo, and Tice, had an identical pattern not detected in over 60 strains of the M. tuberculosis complex. DR analysis can aid in confirming the identification of clinical BCG isolates.
DNA, Bacterial↗
Analysis of genetic polymorphism in the phospholipase region of Mycobacterium tuberculosis.
mtp40 was originally identified as a short genomic region that was found in strains of Mycobacterium tuberculosis but not in Mycobacterium bovis. Subsequent studies have revealed that the sequence is part of the mpcA gene, which encodes a phospholipase C. To investigate further the distribution of the mtp40 sequence, we analyzed strains of the M. tuberculosis complex by PCR and were able to amplify the mtp40 sequence in 90 of 94 strains of M. tuberculosis and in 2 strains of Mycobacterium microti but not in M. bovis or M. bovis BCG. Based on this, we developed a dot blot assay using genomic DNA which allows M. bovis to be distinguished from the majority of M. tuberculosis strains. We also probed Southern blots of 140 clinical isolates of M. tuberculosis to determine the frequency of strains lacking mtp40. This revealed an unexpected polymorphism in the phospholipase region. Two fragments were detected in 57% of samples. The expected fragment of 0.75 kbp corresponds to the region of mpcA containing mtp40. A 2.1-kbp fragment was observed to belong to a recently discovered second phospholipase gene, mpcB. In addition, some strains appeared to lack both genes, while others showed only the presence of mpcA. A few strains had additional bands, suggesting the existence of other homologs to the two phospholipase genes. We also detected the insertion of IS6110 in the mpcA coding region of one strain. The absence of these genes in some clinical isolates raises questions about their function during infection and in the development of tuberculosis disease in humans.
Genes, Bacterial↗
Hsp70 and Hsc70 are preferentially expressed in differentiated epithelial cells in normal human endometrium and ectocervix.
Two highly related 70K heat shock proteins, encoded by the hsc70 and hsp70 genes, are located in the nucleocytoplasmic compartment of mammalian cells. In contrast to recent cell lines, which express Hsp70 only when stressed, many human cell lines constitutively express Hsp70. The degree to which this reflects constitutive expression of Hsp70 in normal human tissues has not been extensively examined. In this study, we show by immunoblotting that human Hsp70 is constitutively expressed in the ovary, cervix, and endometrium and, by immunohistochemical analysis using Hsp70- and Hsc70-specific antibodies, that Hsp70 and Hsc70 are expressed in distinctive and predominantly overlapping patterns in the cervix and endometrium. In these two tissues, the highest levels of both proteins are seen in differentiated, non-proliferating epithelial cells, which is surprising in light of previous studies suggesting growth stimulation of hsp70 gene expression. These observations suggest the possibility that in certain human tissues, basal expression of the hsp70 and hsc70 genes is co-regulated.
Blotting, Western↗
Quality assurance programme for drug susceptibility testing of Mycobacterium tuberculosis in the WHO/IUATLD Supranational Laboratory Network: first round of proficiency testing.
SETTING: Quality assurance of the WHO/IUATLD global tuberculosis drug resistance surveillance programme. OBJECTIVE: To perform a proficiency test of drug susceptibility procedures within the WHO/IUATLD network of supranational reference laboratories (SRL). DESIGN: Identical culture panels consisting of 20 clinical isolates of Mycobacterium tuberculosis containing both drug susceptible and drug resistant cultures were tested by the 16 laboratories of the network for resistance to streptomycin, isoniazid, rifampicin and ethambutol. The drug susceptibility testing procedures included the proportion, absolute concentration and resistance ratio methods as well as their variants, including the radiometric BACTEC 460 method. RESULTS: The first round of proficiency testing has shown that the specificity of drug susceptibility testing within the SRL network was significantly higher than its sensitivity. The testing of isoniazid and rifampicin shows a high degree of agreement between the labs, but discordant results can be obtained with streptomycin and ethambutol. CONCLUSION: Drug susceptibility procedures for the testing of isoniazid and rifampicin, the two anti tuberculosis drugs which define multidrug-resistant tuberculosis, are highly reliable within the SRL network. Procedures for drug susceptibility testing of streptomycin and ethambutol are still in need of standardization.
Antitubercular Agents↗
Association between an early humoral response to Mycobacterium tuberculosis antigens and later development of tuberculosis in human immunodeficiency virus-infected individuals.
OBJECTIVE: To evaluate the association between an early humoral response to Mycobacterium tuberculosis antigens and the later development of tuberculosis (TB) disease in human immunodeficiency virus (HIV)-infected individuals. METHODS: Using an ELISA test, IgG antibodies against 4 M. tuberculosis antigens--purified protein derivative (PPD); 2,3 diacyl trehalose (DAT); a lipooligosaccharide (LOS) and a trehalose dicarboxylic acid bis N,N-dioctadecylamide (BDA.TDA)--were measured in sera from 25 HIV-infected tuberculous patients and 52 HIV-infected persons without TB. RESULTS: With the DAT and LOS antigens, a positive result in sera obtained in the 12 months preceding the onset of TB was significantly associated with later development of TB. Using the BDA.TDA antigen, the same association was observed in sera collected during the 6 months before the diagnosis of TB. No significant association was found with the PPD antigen. CONCLUSIONS: These results suggest that specific antibody markers may be useful to evaluate the risk of active TB in HIV-infected individuals, and a helpful indicator for preventive treatment.
AIDS-Related Opportunistic Infections↗
Characterization of an hsp70 related clone encoding a 33 kDa protein with homology to a protein which associates with polysomes.
Characterization of an hsp70 related Chinese hamster cDNA clone revealed that it encodes a 33 kDa protein with homology to a mouse ribosomal component p40 which associates with polysomes (Auth and Brawerman (1992) Proc. Natl. Acad. Sci. USA 89, 4368). The predicted amino acid sequence of this cDNA which differs by only two residues from p40, shares significant similarity with various members of the hsp70 family. Sequence analysis revealed a 29% identity between the N-terminal region (residues 1 to 120) of the 33 kDa protein and the ATP binding domain of the Chinese hamster hsc70, but no significant sequence similarity was found between the remaining C-terminal regions of the 33 kDa polypeptide and the hsc70 protein. The N-terminal region of the 33 kDa protein lacks the typical consensus motif for ATP binding, suggesting that the N-terminal domain of the 33 kDa protein may not be involved in ATP binding. In the light of the fact that this 33 kDa protein is a ribosomal component, we speculate that the N-terminal domain may interact with structures containing nucleotides such as RNA.
Amino Acid Sequence↗
Detection of single-base mutations by a competitive mobility shift assay.
We have developed an assay for the rapid screening of point mutations in specific genes. Our assay is based upon competitive hybridization of differentially labeled wild-type and mutant oligonucleotide probes to a PCR-generated DNA template and a subsequent analysis of the mobility of the probe-template hybrids. The assay is referred to as a competitive mobility shift assay. Generation of a hybridization stringency gradient allows perfect-matched hybrids to be formed to a greater extent at a slightly higher stringency than the corresponding mismatched hybrids. The stringency gradient is achieved by carrying out the hybridizations at a steadily decreasing temperature (from 95 to 20 degrees C) in a thermal cycler. This step allows the assay to be competitive while avoiding the need to establish precise hybridization conditions for each gene-specific probe, a major disadvantage associated with reverse oligonucleotide hybridization. The assay is rapid and sensitive and can selectively detect mutant DNA in the presence of a large (up to one million-fold) excess of wild-type DNA.
DNA Mutational Analysis↗
Uncoupling of M-phase kinase activation from the completion of S-phase by heat shock.
Chronic exposure of asynchronous HeLa cell cultures to 41.5 degrees C leads to an accumulation of cells in the S-phase, spontaneous premature chromosome condensation, and loss of clonogenicity (M.A. Mackey, S. L. Anolik, and J. L. Roti Roti. Cancer Res., 52: 1101-1106, 1992). In this report, we show that increases in histone H1 kinase activity during 41.5 degrees C exposure occur coincidentally with the appearance of premature chromosome condensation. Furthermore, this kinase activity is shown to be associated with M-phase kinase complexes containing cyclin B1. These increases in the activity of M-phase kinase were found to occur concomitantly with an elevation in cyclin B1 mRNA and an accumulation of cyclin B1 protein. Because cyclin B1 transcription begins in the S-phase, it is probable that the heat-induced delay in the S-phase allows the accumulation of abnormally high cyclin B1 levels. Elevated cyclin B1 levels could then account for the observed abrogation of the cell cycle checkpoint, which usually assures that mitosis does not proceed until DNA replication is complete. This involvement of M-phase kinase in heat-induced cytotoxicity demonstrates the importance of the coordinate regulation of the processes of DNA replication and entry into mitosis.
Cell Cycle↗
Amplification and altered expression of the hsc70/U14 snoRNA gene in a heat resistant Chinese hamster cell line.
We have recently demonstrated that the heat resistant phenotype of the HR-1 variant isolated from HA-1 Chinese hamster fibroblasts after a series of heat shocks is associated with the increased expression of Hsc70, the constitutive form of Hsp70 (Laszlo and Li 1985). Here, we report the cloning and characterization of the Chinese hamster hsc70 gene and its organization and expression in wild type HA-1 and permanently heat resistant HR-1 cells. DNA sequencing revealed that the structure and nucleotide sequence of the hamster hsc70 gene is highly homologous to the human and rat genes coding for Hsc70. Three of the eight introns of the hamster hsc70 gene encode U14 small nucleolar RNAs, as has been demonstrated in other species. Although putative transcriptional elements, including a TATA box, two inverted CAT boxes, and two sets of heat shock elements (HSEs) are completely conserved in the human and hamster hsc70 genes, the regulation of expression of the hamster hsc70 gene is different from that reported for its human counterpart in that the mRNA coding for Hsc70 increases at least 10-fold after a mild heat shock in Chinese hamster cells while no induction of Hsc70 by heat shock has been reported in human cell lines. In situ hybridization revealed a complex chromosomal rearrangement in HR-1 cells which results in the 4- to 5-fold amplification of the hsc70 gene as indicated by genomic Southern blots. In association with this amplification of the hsc70 gene, the levels of Hsc70 mRNA and U14 snoRNA are increased in the HR-1 cells under both normal growing conditions and after heat shock. Thus, the elevated expression of both Hsc70 and U14 snoRNA might play a role in the heat resistant phenotype of the HR-1 cells. This is the first report of the amplification of a heat shock gene and the possible induction of gene amplification by heat shock.
Amino Acid Sequence↗
Tuberculosis bacteriology laboratory services and incremental protocols for developing countries.
Tuberculosis causes more deaths worldwide than any other single infectious disease. Most new cases occur in developing countries, where the emergence of HIV/AIDS-associated multidrug-resistant Mycobacterium tuberculosis could disrupt the effective delivery of chemotherapy. Diagnosis, one of the cornerstones of the modern national tuberculosis control programs in developing countries, is based on sputum smear microscopy, which can be delivered effectively only through a national laboratory network. Tuberculosis diagnostic services can be delivered in lock step with the expanding laboratory network in an incremental manner, beginning with the simplest procedure and ending with the most sophisticated ones.
Africa↗
Hsp70 translocates into a cytoplasmic aggregate during lymphocyte activation.
The percentage of T and B lymphocytes expressing a distinct cytoplasmic aggregate enriched in spectrin, ankyrin, and in several other proteins including protein kinase C greatly increases following various activation protocols. Members of the 70 kDa family of heat shock proteins (hsp70) temporarily bind to and stabilize unfolded segments of other proteins, a function apparently required for proper protein folding and assembly. Considering the multiprotein and dynamic nature of the lymphocyte aggregate, the possibility that hsp70 also might be associated with components of this structure is considered here. Double immunofluorescence analysis indicates that hsp70 is a component of the lymphocyte aggregate and is coincident with spectrin in a subpopulation of freshly isolated, untreated lymphocytes from various murine tissues and in a T-lymphocyte hybridoma. When cell lysates of lymph node T cells are immunoprecipitated using an antibody against hsp70 or spectrin and then analyzed by Western blot utilizing the alternate antibody, it was found that hsp70 and spectrin coprecipitated with one another. Moreover, this coprecipitation could be abolished by addition of ATP. This latter observation was extended to lymphoid cells using a transient permeabilization procedure, and it was shown that addition of exogenous ATP results in the dissipation of the aggregate structure itself. Finally, conditions that result in T-cell activation and aggregate formation, i.e., treatment with the phorbol ester PMA or T-cell receptor cross-linking, also lead to the repositioning of hsp70 into the aggregate from a membrane/cytosolic locale in congruence with spectrin. These data suggest that hsp70 is an active component of the aggregate and that it may function in the interactions believed to occur in this unique activation-associated organelle.
Adenosine Triphosphate↗
Effects of hypothermia, potassium , and verapamil on the action potential characteristics of canine cardiac Purkinje fibers.
BACKGROUND: Hypothermia may induce hypokalemia and increase intracellular Ca2+ by affecting serum K+ and Ca2+ fluxes across the cell membrane. These ionic alterations may significantly change the electrophysiologic characteristics of the cardiac action potential and may induce cardiac arrhythmias. The current study was undertaken to determine whether electrophysiologic changes in Purkinje fibers induced by hypothermia could be reversed by manipulating the extracellular K+ and transmembrane Ca2+ fluxes by Ca2+ channel blockade with verapamil. METHODS: A conventional microelectrode method was used to determine the effects of hypothermia (32 +/- 0.5 degrees C and 28 +/- 0.5 degrees C) and various external K+ concentrations ([K+]o) (2.3, 3.8, and 6.8 mM) on maximum diastolic potential, maximum rate of phase 0 depolarization (Vmax), and action potential duration (APD) at 50% (APD50) and at 95% (APD95) repolarization in isolated canine cardiac Purkinje fibers. To evaluate the contribution of the slow inward Ca2+ current to action potential changes in hypothermia, the experiments were repeated in the presence of the Ca(2+)-channel antagonist verapamil (1 microM). RESULTS: Variations of [K+]o induced the expected shifts in maximum diastolic potential, and hypothermia (28 degrees C) induced moderate depolarization, but only when [K+]o was > or = 3.9 mM (P < 0.05). Hypothermia decreased Vmax at all [K+]o studied (P < 0.05). Regardless of the temperature, Vmax was not affected by verapamil when [K+]o was < or = 3.9 mM, but at 6.8 mM [K+]o in hypothermia Vmax was significantly lower in the presence of verapamil. Hypothermia increased both the APD50 and the APD95. The effects of verapamil on APD were temperature and [K+]o dependent; between 37 degrees C and 28 degrees C with 2.3 mM [K+]o in the superfusate, verapamil did not affect APD. At 28 degrees C in the presence of verapamil, the APD50 and APD95 decreased only if the [K+]o was > or = 3.9 mM. CONCLUSIONS: Verapamil and K+ supplementation in hypothermia may exert an antiarrhythmic effect, primarily by reducing the dispersion fo prolonged APD.
Action Potentials↗
The ubiquitin-mediated proteolytic system: involvement of molecular chaperones, degradation of oncoproteins, and activation of transcriptional regulators.
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Animals↗