Signal transduction through small GTPases--a tale of two GAPs.
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Animals↗
Biomedical subjects
A Hall
Publications and source records attributed to A Hall.
Eye development. Function for Ras in sight.
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Animals↗
A longitudinal study of the matrix changes induced in the intervertebral disc by surgical damage to the annulus fibrosus.
A 5 x 5-mm anterolateral incision was made in the annulus fibrosus (AF) of lumbar discs of 16 sheep; four animals of similar age not operated on were used as controls. The experimental animals were sacrificed 2, 4, 6, 8, 12, and 18 months postoperatively (PO), and the incised and adjacent lumbar discs were collected. Discs were dissected into four zones: AF (zones 1 and 2) and nucleus pulposus (NP) (zones 3 and 4) corresponding to the half of the AF in which the cut was made and its opposite half, and the complementary halves of the NP. Each zone was analyzed for moisture, proteoglycan (PG), collagen, and noncollagenous protein (NCP) content. The PG extractability, aggregation, and hydrodynamic size were also examined. The NP of injured discs showed a significant loss of PGs and collagen 8 months PO, but NCP levels increased. In the incised discs, PG aggregation initially declined but recovered to within control values 6-8 months PO. The NP of discs adjacent to the incised disc also showed time-dependent changes in matrix components that included loss of collagen and PG; however, the AF matrix remained essentially uneffected. Double immunodiffusion studies indicated that a sizeable proportion of the NCPs present in the injured discs (but not the adjacent lumbar discs) were derived from serum.
Animals↗
Predictors of osteopenia in premenopausal women with anorexia nervosa.
The study retrospectively evaluated risk factors for osteopenia in anorexia nervosa (AN) patients. Sixty-nine outpatient, female anorexia nervosa patients (age range 20-40 years, mean 27.5) at varying stages of recovery, and 31 controls had lumbar spine trabecular bone density assessed with single energy computed tomography (CT) scans. An investigator-based, semistructured interview assessed weight and menstrual histories from age 10. Current exercise and dietary calcium levels were categorized and lifetime durations in categories were estimated for each subject. Bone density was significantly lower in the patient group (mean 120 mg/cm3) than in the controls (mean 148 mg/cm3, P less than 0.001). Bone density correlated significantly with duration of illness, amenorrhea, and weight histories but not with measures of dietary calcium or exercise histories. The most important predictor of bone loss on stepwise multiple regression analysis was duration of AN (23% of variance, P less than 0.001), and duration of an inadequate calcium intake in adolescence explained a further 5% of the variance (P = 0.052). There was no evidence that regular exercise and adequate dietary calcium prevented bone loss related to prolonged periods of low weight and amenorrhea in these subjects.
Adult↗
Prechronic inhalation toxicity studies of isobutyl nitrite.
Isobutyl nitrite (IBN) is a volatile liquid that has become increasingly popular as an inhaled recreational drug. To investigate short-term toxic effects and establish exposure parameters for chronic inhalation studies, F344/N rats and B6C3F1 mice were exposed to IBN vapors on a 6 hr/day + t90, 5 days/week schedule. Twelve exposures were administered at concentrations of 0, 100, 200, 400, 600, and 800 ppm IBN. This exposure series resulted in mortality in rats exposed to greater than or equal to 600 ppm and mice exposed to 800 ppm. Animals exposed at the lower concentrations developed hyperplasia of the bronchiolar and nasal turbinate epithelium (rats and mice) and lymphocytic atrophy in the spleen and thymus (mice). Longer term, 13-week, subchronic exposures were conducted at concentrations of 0, 10, 25, 75, 150, and 300 ppm IBN. Exposure to 300 ppm IBN reduced the body weight gains in both sexes of rats and in female mice. IBN-related clinical pathology changes included reduced RBC counts accompanied by moderate increases in mean corpuscular volume and reticulocyte counts, increased WBC counts, and mildly increased methemoglobin concentration. Bone marrow hyperplasia was observed in all groups of IBN-exposed rats, while in mice only females at greater than or equal to 150 ppm IBN displayed this change. Excessive splenic pulp hematopoiesis was noted in mice at all IBN exposure levels.(ABSTRACT TRUNCATED AT 250 WORDS)
Administration, Inhalation↗
Increased sensitivity of a Chinese hamster ovary cell line to alkylating agents after overexpression of the human metallothionein II-A gene.
The elevation of intracellular levels of metallothionein has been associated with the development of resistance to the cytotoxic effects of some alkylating agents. In order to study the mechanisms underlying metallothionein associated drug resistance we transfected the alkylating agent sensitive CHO cell line MMC-1 (Robson et al., 1985) with an episomally replicating plasmid coding for the human metallothionein II-A gene. Two transfectants were isolated which carried about 10 copies of the plasmid. The basal expression of metallothionein was increased from undetectable levels in the recipient cell line MMC-1 to between 22 and 26 micrograms metallothionein per 10(7) cells in the transfectants. Treatment with cadmium salts increased metallothionein levels a further 1.7-fold. Cytotoxicity assays revealed that MTII-A transfection increased the sensitivity of the parental MMC-1 cells to N-methyl-N'-nitro-N-nitrosoguanidine and N-nitroso-N-methylurea. These results suggest that metallothionein does not act as a simple scavenger of alkylating agents, but interacts with unknown factor(s) in host cells.
Alkylating Agents↗
Glutathione S-transferase activity and isoenzyme distribution in ovarian tumour biopsies taken before or after cytotoxic chemotherapy.
A study involving the measurement of glutathione S-transferase activities and isoenzyme distributions in human ovarian tumours has been carried out. These tumours have been obtained either at initial debulking surgery, prior to cytotoxic chemotherapy, or at second look laparotomy following chemotherapy. The response rates of these two groups to chemotherapy differ markedly, with patients who have relapsed following initial chemotherapy showing a reduction in response rates to subsequent chemotherapy. Analysis of these data show no statistically significant differences between the glutathione S-transferase activity or isoenzyme distribution in these two groups of patients. Significant differences were observed in the glutathione-S-transferase activities (GST) between tumours and normal ovaries. GST activities in pre-chemotherapy tumours (n = 33, P = 0.01) and post-chemotherapy tumours (n = 20, P = 0.001) where significantly higher than the GST activity in normal ovaries (n = 15). One feature was the expression of the basic isoenzyme which is expressed more in normal ovaries than in tumours. No differences in these parameters were observed in normal peritoneal tissue taken from patients before or after chemotherapy. These data do not support the hypothesis that changes in glutathione S-transferase enzyme activity or isoenzyme expression are major determinants of response to chemotherapy in ovarian tumours.
Adult↗
Intracellular localization of the P21rho proteins.
The three mammalian ras proteins associated specifically with the plasma membrane and this is essential for their biological activity. Two signals encoded within the extreme COOH terminus of the proteins specify this cellular localization; a CAAX box in combination with either a polybasic domain (p21K-rasB) or a palmitoylation site (p21Ha-ras and p21N-ras). All members of the ras-like and rho-like subfamilies of the ras superfamily of small GTP-binding proteins also have CAAX boxes with potential second site sequences resembling either p21K-rasB or P21N-ras/Ha-ras. However it is not at all clear that they are each located at the plasma membrane, and in fact one of the ras-like proteins, rap1, has been localized to the Golgi (Beranger et al., 1991). None of the mammalian rho-like subfamily has yet been localized. Three forms (A, B, and C) of p21rho, the prototype of this family are known; the COOH termini of p21rhoA and p21rhoC resemble p21K-rasB with a polybasic domain, whereas p21rhoB resembles p21N-ras/Ha-ras with two cysteine residues as potential palmitoylation sites. Despite this similarity to the p21ras proteins, rho proteins have been purified from both particulate and cytosolic fractions of a variety of tissues. In order to localize definitively the three rho proteins we have used an epitope tagging approach coupled to microinjection of living cells. We show that a small fraction of all three proteins is localized to the plasma membrane but the majority of p21rhoA and p21rhoC is cytosolic whereas p21rhoB is associated with early endosomes and a pre-lysosomal compartment. Along with the results obtained with chimeric molecules using heterologous proteins attached to rho COOH termini, this suggests that the p21rho proteins cycle on and off the plasma membrane and this may have important implications for their biological function.
Amino Acid Sequence↗
Ras-related GTPases and the cytoskeleton.
Incorporation of the available data on rac in neutrophils, CDC42 in yeast, and rho in fibroblasts suggests a general model for the function of rho-like GTPase (Figure 1). Conversion of an inactive cytoplasmic rho-related p21GDP/GDI complex to active p21. GTP occurs by inhibition of GAP and/or stimulation of exchange factors in response to cell signals. p21.GTP is then able to interact with its target at the plasma membrane. This could result in a conformational change in the target, enabling it to bind cytosolic protein(s). Alternatively, p21.GTP could be actively involved in transporting cytosolic protein(s) to the target. A GAP protein, perhaps intrinsic to the complex, would stimulate GTP hydrolysis allowing p21.GDP to dissociate. Solubilization of p21GDP by interaction with GDI would complete a cycle. What about the nature of the final complex? The rac-regulated NADPH oxidase complex in neutrophils is currently the best understood and most amenable to further biochemical analysis. Two plasma-membrane bound subunits encode the catalytic function necessary for producing superoxide, but the two cytosolic proteins, p47 and p67, are essential for activity. Why the complexity? Production of superoxide is tightly coordinated with phagocytosis, a membrane process driven by rearrangement of cortical actin. This is not unrelated to the membrane ruffling and macropinocytosis that we observe in fibroblasts microinjected with p21rac. It is tempting to speculate, therefore, that in neutrophils rac is involved not only in promoting the assembly of the NADPH oxidase but also in the coordinate reorganization of cortical actin leading to phagocytosis. For CDC42 controlled bud assembly in yeast, the components of the plasma-membrane complex are not so clear. By analogy with rac in neutrophils, it seems likely that CDC42 is involved in promoting the assembly of cytosolic components at the bud site on the plasma membrane. These putative cytosolic proteins have not yet been identified, but BEM1 and ABP1 are two possible candidates. The biochemical basis for the stimulation of adhesion plaques and actin stress fibers by p21rho in fibroblasts is also unclear. However, components of the adhesion plaque such as vinculin and talin are known to be cytosolic when not complexed with integrin receptors, and rho could be involved in regulating their assembly into the adhesion plaque. Several things are still difficult to incorporate into this model. First the target for CDC42, the bud site, although not yet structurally defined requires the activity of another small GTPase, BUD1. Similarly, in activated neutrophils, the NADPH oxidase is found in a complex with rap1, the mammalian homologue of BUD1 (BoKoch et al., 1989). It seems likely, therefore, that the target is not simply a plasma-membrane protein but may be a complex of proteins whose formation is under the control of the rap1/BUD1 GTPase. The other black box in this model is the actin connection: activation of bud assembly by CDC42 is followed by actin polymerization, activation of NADPH oxidase in neutrophils occurs concomitantly with phagocytosis, a cortical actin-dependent process, and p21rho in fibroblasts couples the formation of adhesion plaques to actin stress fibers. One possible link between the GTPase-driven assembly of a plasma-membrane complex and actin polymerization could involve the SH3 domain. Interestingly, both p47 and p67 and yeast ABP1 and BEM1 have SH3 domain. If rho-like GTPases recognize plasma-membrane targets already associated with cortical actin, then this could promote an interaction with a subset of SH3-containing proteins. The result of this would be a GTPase-regulated aggregation of a group of proteins at a single site in the plasma membrane. It is not too difficult to imagine biological processes where such a spatial integration of different biochemical activities would be essential: coupling the assembly of bud components to the formation of actin fibers in yeast; or the activation of NADPH oxidase to phagocytosis in neutrophils; or the assembly of adhesion plaques and the formation of actin stress fibers in fibroblasts are just three examples that have emerged so far. In conclusion, although rho-like GTPases clearly have distinct roles in different mammalian cell types and in yeast, their underlying mechanism of action appears to be strikingly similar. Whether this will remain so when there are some biochemical data to back up these initial observations, time will tell.
Actins↗
Distinct patterns of actin organization regulated by the small GTP-binding proteins Rac and Rho.
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3T3 Cells↗
A monoclonal antibody for distinction of invasive and noninvasive clinical isolates of Entamoeba histolytica.
Approximately 10% of the world population is infected with Entamoeba histolytica, but only 10% of the carriers develop symptomatic amebiasis. This discrepancy could be explained by the genotypic differences between the morphologically indistinguishable invasive and noninvasive strains of E. histolytica currently identified by zymodeme analysis, a technique that is unsuitable for routine diagnostic laboratories. Here we report the production of a monoclonal antibody against E. histolytica and its use in an immunofluorescence assay to identify invasive isolates cultured from stool samples of infected patients in several regions where amebiasis is endemic: Bangladesh, Colombia, and Mexico. After testing a total of 88 E. histolytica isolates, the correlation between zymodeme characterization and the immunofluorescence assay with the invasive isolate-specific monoclonal antibody was 100%. The epitope detected by the invasive isolate-specific monoclonal antibody resides in a previously undescribed internal protein with molecular masses of 84 and 81 kDa in axenic and polyxenic E. histolytica strains, respectively.
Animals↗
Small GTP-binding proteins--a new family of biologic regulators.
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GTP-Binding Proteins↗
Cystatin C based peptidyl diazomethanes as cysteine proteinase inhibitors: influence of the peptidyl chain length.
The peptidyl diazomethanes Cbz-Gly-CHN2, Boc-Val-Gly-CHN2, H-Leu-Val-Gly-CHN2, Cbz-Leu-Val-Gly-CHN2 and Cbz-Arg-Leu-Val-Gly-CHN2, with peptidyl portions modelled after the proposed cysteine proteinase interacting N-terminal segment of human cystatin C, were synthesized. Their efficiency as cysteine proteinase inhibitors was tested against papain, human cathepsin B and bovine cathepsin B. All, except Cbz-Gly-CHN2, were found to be irreversible inhibitors of the tested enzymes. Each addition of an amino acid residue to their peptidyl portions resulted in an increased inhibition rate of all three enzymes. These data suggest that the arginyl residue of the tetrapeptidyl diazomethane, and also the corresponding arginyl residue in native cystatin C, interact with a S4 substrate pocket subsite of both papain and cathepsin B. The most efficient inhibitor, Cbz-Arg-Leu-Val-Gly-CHN2, inhibited papain and cathepsin B with rate constants of the same order of magnitude as those for L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guanidin o)butane (E-64). The high water-solubility of Cbz-Arg-Leu-Val-Gly-CHN2 allowing it to be dissolved to molar concentrations without use of non-physiological additives, makes it suitable for in vitro and in vivo cysteine proteinase inhibition studies.
Amino Acid Sequence↗
Further diagnostic use of an invasive-specific monoclonal antibody against Entamoeba histolytica.
Genotypic differences between invasive and non-invasive E. histolytica could explain the 1:10 ratio of symptomatic/asymptomatic infection worldwide. Currently, zymodeme analysis is used to differentiate invasive from non-invasive E. histolytica strains but the technique is cumbersome and expensive. In accordance with the WHO research priorities for amebiasis we report here the further use of an invasive-specific monoclonal antibody against E. histolytica in immunofluorescence, to identify isolates cultured from stool samples of patients from three geographically distant endemic regions: Bangladesh, Colombia and Mexico. We tested 107 E. histolytica isolates and the correlation between zymodeme characterization and the immunofluorescence assay was 100%.
Animals↗
Synthesis in E. coli of a polypeptide with human leukocyte interferon activity. 1980.
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Cloning, Molecular↗
Evaluating measures to control intestinal parasitic infections.
Intestinal parasitic infections are among the most common infections of humans in developing countries, but the resources available for their control are severely limited. Careful evaluation of control measures is essential to ensure that they are cost-effective. The evaluation of the effects of control on intestinal helminths and intestinal protozoa requires an understanding of the different epidemiological patterns of these two groups of parasites. The transmission dynamics and morbidity associated with the major helminth infections (Ascaris lumbricoides, Trichuris trichiura and the hookworms) are dependent on the size of the worm burdens. Thus the important parameter for evaluating the impact of control on morbidity and transmission is the intensity of infection, which can be assessed by determining the mean density of parasite eggs in faecal specimens. Estimation of intensity is subject to systematic errors, however, due to the complex pattern of worm burden distributions. The frequency distribution of burdens is highly overdispersed, and individuals exhibit predisposition to particular levels of infection. Furthermore, mean intensity is age-dependent, in a species-specific manner, and is clustered spatially and within families. These complex patterns imply that the estimation of intensity is exceptionally sensitive to the size and demographic structure of the population sample selected for assessment. They also have the effect that prevalence estimates, the most commonly used measures of infection in communities, can seriously mislead. Paradoxically, prevalence is least useful where infection is most common because the relationship between prevalence and intensity is most markedly non-linear when the prevalence is high. Thus in areas where control is most needed, evaluation using prevalence might suggest that control had failed while evaluation by intensity would, correctly, show the measure of success. With the major protozoan infections (Entamoeba histolytica, Giardia duodenalis and Cryptosporidium parvum) an estimate of intensity is of little value and the central parameter for evaluation is prevalence. Prevalence does exhibit age and spatial heterogeneity, which may be species-specific, so there remains a need to ensure a consistent sample structure, although this is less critical than for the helminths. The major constraint on evaluating the control of protozoan infections is the need to identify pathogenic species and, in some cases, pathogenic strains. Harmless commensal protozoans are ubiquitous and often morphologically very similar to pathogens, but their control is both unnecessary and impracticable. Species such a E. histolytica appear to exist as strains with differing pathogenicity, thus control will be cost-effective only if the focus is on pathogenic strains. Effective diagnosis is therefore central to the evaluation of the control of protozoan infections.(ABSTRACT TRUNCATED AT 400 WORDS)
Adolescent↗
Identification of the human platelet GTPase activating protein for the CDC42Hs protein.
The CDC42Hs protein appears to be an isoform of the ras-related GTP-binding protein G25K and is an apparent human homolog of the Saccharomyces cerevisiae cell-division-cycle protein, CDC42Sc. In this study, we report the identification of a GTPase-activating protein (GAP) for CDC42Hs from human platelets (designated from here on as CDC42Hs-GAP). The CDC42Hs-GAP activity was solubilized from platelet membranes, recovered through successive chromatography steps (the final step being Mono-Q chromatography), and purified approximately 3500-fold. The CDC42Hs-GAP activity appeared to correspond to a polypeptide with an apparent Mr of approximately 25,000. The GTPase activities of the purified human platelet CDC42Hs, the Escherichia coli-recombinant CDC42Hs, and the Spodoptera frugiperda-recombinant GTP-binding proteins are all stimulated by the CDC42Hs-GAP to identical extents, which indicates that the recombinant CDC42Hs proteins are as effective as the native human platelet protein in coupling to the GAP. However, a mutant form of the E. coli-recombinant CDC42Hs which contains a valine residue at position 12 (CDC42HsVal-12) has a significantly reduced intrinsic GTPase activity (relative to the wild type CDC42HsGly-12) which is not stimulated by the CDC42Hs-GAP. The CDC42Hs-GAP also does not stimulate the GTPase activities of the ras or rap GTP-binding proteins; however, it is capable of a weak stimulation of the GTPase activity of mammalian rho. Based on the apparent similarities in the molecular size of the CDC42Hs- and rho-GAPs (i.e. 25-30 kDa), and the cross-reactivity of rho with the CDC42Hs-GAP, it seems likely that the CDC42Hs- and rho-GAPs will constitute a specific subclass of the ras-related GAP superfamily.
Blood Platelets↗
Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1.
Professional phagocytes, such as neutrophils and monocytes, have an NADPH oxidase that generates superoxide and other reduced oxygen species important in killing microorganisms. Several components of the oxidase complex have been identified as targets of genetic defects causing chronic granulomatous disease. The complex consists of an electron transport chain that has as its substrate cytosolic NADPH and which discharges superoxide into the cavity of the intracellular phagocytic vacuole. The only electron transport component identified so far is a low-potential cytochrome b, apparently the only membrane component required. At least three cytosolic factors are also necessary, two of which, p67phOx and p47phOx, have been identified by their absence in patients with chronic granulomatous disease. A third component, sigma 1, is required for stimulation of oxidase activity in a cell-free system. The active components of purified sigma 1 are two proteins that associate as heterodimers, and here we report that these are the small GTP-binding protein p21rac1 and the GDP-dissociation inhibitor rhoGDI.
Amino Acid Sequence↗