The ubiquitin-mediated proteolytic system: involvement of molecular chaperones, degradation of oncoproteins, and activation of transcriptional regulators.
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Animals↗
Biomedical subjects
A Laszlo
Publications and source records attributed to A Laszlo.
Alcohol consumption and the risk of major upper gastrointestinal bleeding.
OBJECTIVE: To examine the association between current alcohol consumption and major upper gastrointestinal bleeding. METHODS: In a case-control study in the United States, Sweden, and Hungary, 1004 incident cases with upper gastrointestinal bleeding without predisposing factors were compared with 2446 controls. Relative risks for categories of alcohol consumption (based on the number of drinks currently consumed/wk) were estimated using logistic regression; the potential confounding effects of cigarettes, nonsteroidal anti-inflammatory drugs, and other factors were controlled simultaneously. RESULTS: Compared with drinkers of < one drink/wk, the relative risks among other current drinkers ranged from 0.8 for 1-6 drinks/wk to 6.3 for > or = 35 drinks; the trend was statistically significant (p < 0.001). A significantly increased relative risk was seen for the heaviest consumption category within various subgroups: gastric and duodenal hemorrhage; males and females; age < 60 yr and > or = 60 yr; and those who consumed beer, wine, liquor, or a combination of beverages. CONCLUSIONS: These findings provide evidence that consumption of alcohol increases the risk of major gastric and duodenal bleeding in nonpredisposed individuals.
Adult↗
Development of an enzyme-linked immunosorbent assay (ELISA) combined with a streptavidin-biotin and enzyme amplification method to detect anti-2,3-di-o-acyltrehalose (DAT) antibodies in patients with tuberculosis.
IgG antibodies against the 2,3-di-o-acyltrehalose glycolipid of Mycobacterium tuberculosis were determined in a set of 49 sera from patients with pulmonary tuberculosis and 65 negative control subjects. We compared a conventional ELISA method using a beta-galactosidase anti-human IgG conjugate developed with ONPG, with an amplification ELISA system constituted of an anti-human IgG biotinylated conjugate, a streptavidin-alkaline phosphatase complex, and NADP as a substrate. The resulting NAD was measured by using a redox enzymatic recycling system of alcohol dehydrogenase, diaphorase and iodonitrotetrazolium as chromogen. With specificity set at 92.31% in both methods, we obtained a sensitivity of 42.86% in the conventional method and a sensitivity of 61.22% in the amplified method. We conclude that by using a more sensitive method we can detect cases that otherwise could be identified as false negatives.
Antibodies, Bacterial↗
Screening of synthetic trehalose 6,6'-diesters and trehalose 6-monoesters as potential immunoreactants for the serodiagnosis of tuberculosis.
The absence of serological cross-reactivity between trehalose 2,3-diester (DAT, formerly SL-IV) and synthetic trehalose 6,6'-diesters and trehalose 6-monoesters was established by ELISA testing using polyclonal immune sera raised in rabbits sensitized with "DAT". From the screening of fifteen synthetic trehalose 6,6'- and 6-esters, "mirror" pseudo cord factor no. 1, "mirror" amides no. 5 and 6, cord factor analogues 7 and 8 and trehalose 6-monoesters 10 and 11 were selected for future, more extensive serological analysis. Paired comparisons of analogues among these fifteen substances showed that serodiagnostic discrimination power was more a function of the carbon chain length of their substituent groups--as well as of their position--than of the "mirror" constitution of the molecules. More exhaustive testing of these seven compounds is needed to select the synthetic product most efficient in the ELISA serodiagnosis of tuberculosis.
Animals↗
Drug-resistant tuberculosis: laboratory issues. World Health Organization recommendations.
There is a suggestion that drug resistance rates decreased in developing countries over the period 1962-85, while recent data suggest that resistance may be increasing. The initial decrease in resistance appears to be associated with well-functioning National Tuberculosis Control Programmes (NTP), while the recently observed increase may be due either to understaffed, resource-poor programmes or to the effect of the HIV epidemic, or to both. It is possible that the HIV epidemic may overwhelm the NTP, resulting in decreased programme efficiency and ultimately increased drug resistance. Resistance surveillance appears to be a good measure of programme efficiency. For research purposes, primary drug resistance surveys should be done on a sample of relevant patients which includes and distinguishes between HIV-positive and HIV-negative patients. At this time, there is not enough information to warrant a recommendation regarding HIV testing of TB patients for surveillance purposes. In order for resistance surveys to be relevant from the public health perspective, one must know the proportion of patients presenting for treatment having previously received treatment. The meaningful denominator for drug resistance surveys from the programme evaluation perspective should be the number of patients presenting for treatment. For initial drug resistance surveys the measurement should be the number of people never having received prior TB treatment with resistant bacilli, divided by the number of new patients presenting for treatment. For acquired resistance, one should look at all patients who begin treatment with susceptible bacilli who become resistant 6 months later.
Antitubercular Agents↗
A random sample survey of initial drug resistance among tuberculosis cases in Latin America.
A random sample survey of initial drug resistance among cases of tuberculosis in Latin America was carried out during the second half of the 1980s and the early 1990s. A total of 948 cultures of Mycobacterium tuberculosis isolated from patients presumed never before to have been treated for tuberculosis were collected from 30 randomly selected clusters in Latin America and tested for resistance to isoniazid, streptomycin, rifampicin, ethambutol, and thioacetazone. Initial drug resistance, although unevenly distributed, was detected in all the clusters tested and characterized one out of every six tuberculosis cases. Both single and multiple resistance to streptomycin and to isoniazid were the most prevalent forms throughout the region but were not sufficiently frequent to jeopardize significantly the outcome of short-course chemotherapy. However, localized pockets of high drug resistance occurred throughout the region and are cause for concern, especially in the case of rifampicin.
Antitubercular Agents↗
Effect of amino acid analogs on the development of thermotolerance and on thermotolerant cells.
Exposure of HA-1 Chinese hamster fibroblasts to amino acid analogs has been shown to have a heat-sensitizing effect as well as inducing the heat shock response (Li and Laszlo, 1985a). In this study, we have examined the effect of amino acid analogs on the development of thermotolerance after a brief heat shock or exposure to sodium arsenite and the effect of amino acid analogs on cells that are already thermotolerant. Exposure of HA-1 cells to amino acid analogs inhibited the development of thermotolerance following a mild heat shock or treatment with sodium arsenite. However, cells that were already thermotolerant were resistant to the sensitizing action of amino acid analogs. The refractoriness of thermotolerant cells to amino acid analog treatment developed in parallel with thermotolerance. The uptake of the arginine analog, canavanine, and its incorporation into proteins was not altered in the thermotolerant cells. Furthermore, another biological consequence of exposure to amino acid analogs, sensitization to ionizing radiation, also was not altered in the thermotolerant cells. The inhibition of the development of thermotolerance by amino acid analogs and the refractoriness of thermotolerant cells to the heat-sensitizing action of amino acid analogs lend further support the role of heat-shock proteins in the phenomenon of thermotolerance.
Amino Acids↗
Nonsteroidal anti-inflammatory drug use in relation to major upper gastrointestinal bleeding.
In a study in the United States, Sweden, and Hungary, 335 cases of gastric bleeding without predisposing factors were compared with 670 control subjects, and 239 cases of duodenal bleeding were compared with 489 control subjects. For aspirin taken at least every other day during the week before the onset of bleeding (regular use), the relative risk of gastric bleeding was 4.4 (95% confidence interval [CI], 2.9 to 6.7); for occasional use, it was 3.3 (95% CI, 2.1 to 5.0). For ibuprofen, the corresponding estimates were 1.0 (95% CI, 0.4 to 2.6) and 1.1 (95% CI, 0.5 to 2.4). For naproxen, the estimate for regular use was 4.0 (95% CI, 1.5 to 11). The estimates for any use of piroxicam (crude estimate), indomethacin, and diclofenac during the week before onset were 18 (95% CI, 4.1 to 83), 1.6 (95% CI, 0.4 to 5.9), and 0.9 (95% CI, 0.2 to 4.2), respectively. The corresponding relative risks of duodenal bleeding were 7.1 (95% CI, 4.2 to 12) and 2.2 (95% CI, 1.3 to 3.7) for the regular and occasional use of aspirin, 2.4 (95% CI, 0.5 to 11) and 0.8 (95% CI, 0.3 to 2.0) for ibuprofen, 12 (95% CI, 2.8 to 54) and 9.9 (95% CI, 2.3 to 44) for naproxen, 17 (95% CI, 3.6 to 79) for any use of piroxicam (crude estimate), and 1.7 (95% CI, 0.2 to 14) for any use of indomethacin. There was a significant trend in the risk of gastric bleeding with increasing dose of regular aspirin use (p = 0.002). The relative risk estimates for the regular use of 325 mg or less were significantly elevated for both gastric and duodenal bleeding at 3.1 and 6.4, respectively.
Aged↗
Putative determinants of the cellular response to hyperthermia.
Recently, it has been demonstrated that two different thermal resistant states found in Chinese hamster cells, one transient, associated with thermotolerance, and the other permanent, associated with the increased expression of the cognate member of the hsp 70 family, are characterized by faster recovery from heat-induced perturbations in several cellular processes (Laszlo 1992b). These processes include total cellular protein and RNA synthesis, the localization of hsp70, the organization of vimentin, and the protein composition of the nucleus. In the present study, the recovery from heat-induced perturbations in cellular physiology was extended further to two more types of Chinese hamster cells: permanently heat resistant cells in which thermoresistance is associated with the overexpression of hsp27 and heat-sensitive cell lines. When the heat-resistant hsp27 transfected cell lines were compared with the control wild-type cell line, the recovery of protein synthesis from heat-induced inhibition was similar in the normal and hsp27 transfected cells, while the recovery from heat-induced inhibition of total RNA synthesis and the recovery from heat-induced increased association of hsp70 with nuclei were both more rapid in the hsp27 transfected cell lines. In the permanently heat-sensitive cell lines, the kinetics of recovery from heat-induced inhibition of protein synthesis did not correlate with the heat sensitive state. However, delays in the recovery from heat-induced alterations in total cellular RNA synthesis and from heat-induced excess nuclear association of hsp70 were associated with the heat-sensitive state. Overall, these results suggest that the kinetics of recovery from heat-induced alterations in total cellular RNA synthesis and the localization of hsp 70 are putative candidates for being determinants of the cellular response to hyperthermia, and thus have the potential to form the basis of predictive assays for use in conjunction with clinical hyperthermia.
Adaptation, Physiological↗
Rapid broth macrodilution method for determination of MICs for Mycobacterium avium isolates.
A multicenter study was done to investigate the accuracy and reproducibility of a method for determining the MICs of antimicrobial agents against the Mycobacterium avium complex in 7H12 broth with the BACTEC system. In phase I, with eight drugs and 10 strains, intralaboratory reproducibility was 95.7 to 100%, allowing a 1-dilution difference upon repeat testing. The results of phase II testing with 41 additional strains were consistent with those obtained in phase I, with good interlaboratory reproducibility. The radiometric method was validated by sampling and plating of the same broth cultures and determining, by the number of CFU per milliliter, the lowest drug concentration that inhibited more than 99% of the initial bacterial population. Three test concentrations of each drug and the tentative interpretation of results are proposed. Radiometric MIC determination has the potential to become the method of choice for clinical microbiology laboratories and evaluation of new agents for the treatment of M. avium infections, both pulmonary and disseminated.
Anti-Bacterial Agents↗
A stress-inducible 40 kDa protein (hsp40): purification by modified two-dimensional gel electrophoresis and co-localization with hsc70(p73) in heat-shocked HeLa cells.
We have previously reported that a novel 40 kDa protein is induced by heat shock and several environmental stresses in mammalian and avian cells and that the N-terminal amino acid sequence of this 40 kDa protein has homology with the bacterial DnaJ heat-shock protein. We have purified this protein (40 kDa heat-shock protein, hsp40) from HeLa cells by modified two-dimensional gel electrophoresis and generated a polyclonal antibody against hsp40. This antibody was highly specific for human hsp40 and cross-reacted weakly with rat and Chinese hamster hsp40. Indirect immunofluorescence revealed that the hsp40 in HeLa cells accumulates in the nucleus, especially in the nucleolus, during heat shock and returns to the cytoplasm during the recovery period. The kinetics of the accumulation in the nucleoli and subsequent return to the cytoplasm of hsp40 was similar to that of hsp70. In addition, hsp40 was co-localized with hsc70(p73) in heat-shocked HeLa cells as demonstrated by double immunofluorescence staining. These results suggest that hsp40 (a DnaJ homologue) and hsp70 (a DnaK homologue) may act in concert to repair (refold) denatured proteins and protein aggregates in the nuclei and nucleoli of heat-shocked HeLa cells.
Cell Nucleolus↗
Initial characterization of heat-induced excess nuclear proteins in HeLa cells.
Exposure of mammalian cells to hyperthermia is known to cause protein aggregation in the nucleus. The presence of such aggregates has been detected as the relative increase in the protein mass that is associated with nuclei isolated from heated cells. We have characterized these excess nuclear proteins from the nuclei of heated HeLa cells by two-dimensional gel electrophoresis. The abundance of cytoskeletal elements which co-purify with the nuclei did not increase with exposure to hyperthermia, indicating that these proteins are not part of the excess nuclear proteins. In contrast, several specific polypeptides become newly bound or increase in abundance in nuclei isolated from heated cells. Members of the hsp 70 family were identified as a major component of the excess nuclear proteins. Among the other excess nuclear proteins we identified ten that had apparent molecular weights of 130, 95, 75, 58, 53, 48, 46, 37, 28, and 26 kilodaltons. Since hsp 70 is mainly cytoplasmic in non-heated cells, its association with nuclei in heated cells indicates that one mechanism accounting for the heat-induced excess nuclear proteins is the movement of cytoplasmic proteins to the nucleus. We also obtained evidence that increased binding of nuclear proteins is another mechanism for this effect. No overall increase or decrease in the phosphorylation of nuclear proteins was found to be associated with such altered binding or movement from the cytoplasm to the nucleus.
Cytoskeletal Proteins↗
The relationship between hsp 70 localization and heat resistance.
Using indirect immunofluorescence we have investigated the kinetics of nuclear accumulation and removal of hsp 70 in HA-1 Chinese hamster fibroblasts exposed to elevated temperatures. The kinetics of accumulation of hsp 70 in the nuclei were found to be time/temperature dependent at all temperatures tested (42-45 degrees C). At a given temperature, the fraction of cells manifesting nuclear localization of hsp 70 increased with exposure time. For a given duration of heating, the fraction of cells manifesting nuclear localization of hsp 70 increased with the temperature. The kinetics of the nuclear accumulation of hsp 70 were similar for normal HA-1 cells, their heat-resistant variants, and transiently thermotolerant cells (triggered by prior exposure to a brief heat shock or to sodium arsenite). Upon return to 37 degrees C after heat shock, the kinetics of removal of the hsp 70 associated with the nucleus was dependent on the severity of the initial heat challenge. However, for a given heat dose, the decay of nuclear localization of hsp 70 was more rapid in thermotolerant and heat-resistant cells than in their normal counterparts. These results suggest that the increased levels of hsp 70 associated with the transient or permanently heat-resistant state may play a direct role in restoring and/or repairing heat-induced nuclear and nucleolar alterations associated with heat-induced cell killing. Furthermore, they also suggest that the heat-resistant state may involve ameliorated repair of heat-induced cellular alterations.
Animals↗
The thermoresistant state: protection from initial damage or better repair?
The induction of and recovery from heat-induced perturbations in several cellular parameters were examined in normal, transiently thermotolerant, and permanently heat-resistant HA-1 Chinese hamster fibroblasts. The initial heat-induced perturbations in total cellular protein synthesis, RNA synthesis, vimentin-containing intermediate filaments, and nuclear protein mass were similar in the three different cell types which display various levels of thermal resistance as determined by clonogenic survival. The posthyperthermia recovery from the heat-induced perturbations in all of the cellular parameters was more rapid in both the permanently heat-resistant cells and in the transiently thermotolerant cells. This response was observed in cells in which transient thermotolerance was induced by either a mild heat shock or exposure to sodium arsenite. The development and decay of the capacity for more rapid recovery from the initial heat-induced perturbations in total cellular protein and RNA synthesis paralleled the development and decay of clonogenic thermotolerance. Overall, these results support the notion that more rapid recovery from similar levels of heat-induced perturbations in various cellular parameters are a salient feature of both the transiently and permanently heat-resistant state.
Animals↗
Evaluation of synthetic pseudo cord-factor-like glycolipids for the serodiagnosis of tuberculosis.
A number of glycolipids were evaluated in an ELISA test for their serodiagnostic usefulness in tuberculosis. One hundred and twelve (112) sera belonging to bacteriologically confirmed TB patients, patients with pathologies other than tuberculosis and healthy individuals were examined against several synthetic "mirror" pseudo cord factors (analogues of trehalose-6,6'-dimycolate or TDM) using natural cord factor and another recently described natural glycolipid (SL-IV) of Mycobacterium tuberculosis as control antigens. Analysis of the results shows that all synthetic "mirror" pseudo cord factors, except one with a short 8-carbon chain, were better recognized by the sera of tuberculosis patients than natural cord factor, with sensitivity and specificity values in the ELISA test similar to those reported for M. tuberculosis species-specific SL-IV. Of all antigens tested in this study, BDA. TDA, a bis(N,N-dioctadecylamide) of "trehalose dicarboxylic acid", [(alpha-D-glucopyranosyluronic acid) (alpha-D-glucopyranosiduranic acid)], showed the highest serodiagnostic discriminating power (93% sensitivity and specificity). We postulate that either these artificial molecules are cross-reactants of similarly structured native glycolipids of M. tuberculosis or that they bear closer resemblance to actual phagosome-lysosome-modified antigens than to native mycobacterial ones.
Cord Factors↗
Thin-layer chromatography systems for the identification of Mycobacterium tuberculosis, M. bovis BCG, M. kansasii, M. gastri and M. marinum.
Knowledge of mycobacterial glycolipid antigens and the study of their specificity have resulted in their utilization as species markers. We describe a thin-layer chromatography method which could serve as a useful adjunct for the identification of Mycobacterium tuberculosis, M. bovis BCG, M. kansasii, M. gastri and M. marinum.
Antigens, Bacterial↗
Interactive effects of volatile anesthetics, verapamil, and ryanodine on contractility and calcium homeostasis of isolated pregnant rat myometrium.
OBJECTIVE: Both volatile anesthetics and Ca2+ antagonists decrease uterine contractility. The interactive effects of anesthetics, verapamil, and ryanodine on myometrial muscular activity and on intracellular Ca2+ availability were examined. STUDY DESIGN: The effects of minimum alveolar concentrations of 0.5, 1.0, and 2.0 of halothane, enflurane, and isoflurane on electrically stimulated isometric mechanical activity of isolated longitudinal myometrium strips from 45 pregnant rats (15 to 21 days), superfused with Krebs' solution with or without 5.0 mmol/L Ca2+, 10(-7) and 10(-6) mol/L verapamil, and 10(-6) mol/L ryanodine, were compared. Analysis was performed by analysis of variance and Duncan test. RESULTS: All three anesthetics and verapamil produced dose-dependent depression of contractility. Increasing the Ca2+ concentration from 2.5 to 5.0 mmol/L in the superfusate partly reversed the depression. An additive effect of verapamil and anesthetics, especially enflurane, on contractile force was observed. Ryanodine had no effect on contractility, but it could partly counteract the depressant effect of anesthetics. CONCLUSION: Clinically used concentrations of volatile anesthetics modify Ca2+ availability and depress uterine contractility. General anesthesia, especially by enflurane, in patients being treated with calcium antagonists may represent a higher risk.
Anesthetics↗
The effects of hyperthermia on mammalian cell structure and function.
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Animals↗