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Biomedical subjects

J Mitchell

Publications and source records attributed to J Mitchell.

At least 19 recordsLinked to original sources
PubMed1992

Defective guanyl nucleotide-binding protein beta gamma subunits in a forskolin-resistant mutant of the Y1 adrenocortical cell line.

Forskolin-resistant mutants derived from Y1 adrenocortical cells display decreased responsiveness both to receptor and postreceptor stimulators of adenylyl cyclase and decreased amounts of the alpha subunits of the GTP-binding proteins (G proteins) that mediate stimulation (Gs) and inhibition (Gi) of adenylyl cyclase--namely, Gs alpha and Gi alpha-2. This phenotype is suggestive of a mutation that affects the processing or plasma membrane incorporation of G protein alpha subunits. Since the membrane attachment of heterotrimeric G proteins has been ascribed in part to the beta gamma subunits, we examined the quantity and functional activity of beta gamma subunits in wild-type Y1 and forskolin-resistant Forsk-10r-9 and Forsk-10r-3 cells. We now show that two assays previously used to examine the activity of purified beta gamma subunits--namely, to support either rhodopsin-catalyzed guanyl nucleotide exchange on Gt alpha or pertussis toxin-catalyzed ADP-ribosylation of Gt alpha--can be used with detergent extracts of cells. In both assays the beta gamma activity in Forsk-10r-9 and Forsk-10r-3 extracts was decreased by 53-76% compared with wild-type Y1 extracts. When normalized for immunoreactive beta subunit, the beta gamma activity in the Forsk-10r-9 samples was decreased by 55-57% compared with the wild-type Y1 samples. These results suggest that a mutation of one of the G protein beta or gamma subunits may result in the multiple defects of adenylyl cyclase activity and apparent loss of G protein alpha subunits seen in the forskolin-resistant mutant cells. The frequency with which these spontaneous mutations arise in the Y1 cell line suggests that they may contribute more generally to genetic abnormalities in signal transduction.

Adenosine Diphosphate Ribose↗
PubMed1992

Expression of insulinlike growth factor (IGF) and IGF-binding protein genes in human lung tumor cell lines.

BACKGROUND: The presence of multiple, low-molecular-weight, insulinlike growth factor (IGF)-binding proteins in lung tumor cell-conditioned medium and lung cancer patient serum has been recently reported. PURPOSE: To begin to elucidate the genetic basis for these observations, the present study examines the expression by lung tumor cell lines of three IGF-binding protein genes, namely, IGFBP-1, IGFBP-2, and IGFBP-3. Since IGF-binding proteins are thought to modulate the biologic action of the IGFs, the relationship between the expression of IGF-binding protein genes and the genes encoding IGF-I and IGF-II also has been investigated. METHODS: Gene expression was studied in four small-cell lung cancer (SCLC) and three non-small-cell lung cancer (NSCLC) cell lines using Northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR) for IGFBP-1. RESULTS: IGFBP-1 gene expression was detected by Northern blot analysis in one NSCLC cell line only. However, RT-PCR revealed that the IGFBP-1 gene was expressed in all four SCLC cell lines and in two of the three NSCLC lines. Northern blot analysis of IGFBP-2 gene expression demonstrated that all lung tumor cell lines expressed this gene. A low level of IGFBP-3 gene expression was detected in one SCLC cell line and in all three NSCLC cell lines. All lung tumor cell lines expressed the IGF-II gene as determined by Northern blot analysis. In marked contrast, none of the lines showed evidence of IGF-I gene expression using this method. However, RT-PCR revealed a low level of IGF-I gene expression in one SCLC and one NSCLC cell line only. CONCLUSIONS: These observations indicate 1) that IGF-binding proteins secreted by lung tumors are encoded by at least three different genes; 2) that there may be a close association between IGF-II and IGFBP-2 gene expression, such that, where there is production of IGF-II, IGFBP-2 is the principal BP; and 3) that the IGF-II gene is more widely expressed than the IGF-I gene in human lung tumor cell lines.

Base Sequence↗
PubMed1992

Genetic and environmental determinants of factor VII coagulant activity in ethnic groups at differing risk of coronary heart disease.

It has been shown previously that individuals possessing the Gln353 allele of factor VII have significantly lower factor VIIc levels. In this population based study of Europeans, Afro-Caribbeans and Gujarati Indians, the Gln353 allele was associated with lower factor VIIc in all groups, carriers having factor VIIc levels 20-25% below the group mean. Although the Afro-Caribbeans had the lowest factor VIIc levels, the frequency of the Gln353 allele was not different from the European sample. However, in the Gujaratis, the frequency of the Gln353 allele was significantly higher than in the Europeans (0.25 compared to 0.09, P less than 0.001). Factor VIIc is known to be positively correlated with plasma triglyceride levels, although the Gujaratis, having the highest mean triglyceride levels, did not have the highest mean factor VIIc levels. On examination of the relationship between triglycerides and factor VIIc in the Gujaratis there was a correlation (r = 0.23, P = 0.13) in individuals homozygous for the factor VII Arg353 allele, but no correlation (r = 0.001, P = 0.5) among Gln353 carriers. This striking difference suggests that the effect of triglycerides on factor VIIc is genotype specific and thus provides an example of gene-environment interaction. The high frequency of the Gln353 allele, with its associated lack of relationship between triglyceride and factor VIIc levels, may explain the lower than expected factor VIIc levels in the Gujaratis.

Africa↗
PubMed1992

The significance of platelets with increased RNA content (reticulated platelets). A measure of the rate of thrombopoiesis.

Direct flow cytometric measurement of nucleic acid content in individual platelets is possible using the fluorescent dye Thiazole Orange (Becton-Dickinson, San Jose, CA). When applied to studies of thrombocytopenic patients, platelets with elevated nucleic acid content ("reticulated platelets") can be identified and quantitated. Labeling of these platelets is saturable and is abolished by treatment with RNAse. It has been suggested that, similar to the erythrocyte reticulocyte response to anemia, the number of these platelets appearing in the circulation may provide an estimate of the rate of thrombopoiesis. The authors studied 229 thrombocytopenic patients, measuring both reticulated platelets and platelet-associated immunoglobulin. The results show that for the subset of patients with normal levels of platelet-associated immunoglobulin, the average absolute number of reticulated platelets is independent of platelet count and remains in the normal range. For those with elevated levels of platelet-associated immunoglobulin, the absolute number of reticulated platelets increases in patients who are moderately thrombocytopenic (60 to 100 x 10(9)/L) but decreases to normal or subnormal levels as thrombocytopenia worsens. The latter finding has been duplicated in studies of mice made thrombocytopenic by injection of anti-platelet antiserum. These results are consistent with the hypothesis that reticulated platelets are subject to peripheral destruction at the same rate as mature platelets, and that in the severely thrombocytopenic patient their level may decrease despite an appropriate marrow thrombopoietic response.

Animals↗
PubMed1992

Induction of ornithine decarboxylase in specific subpopulations of murine epidermal cells following multiple exposures to 12-O-tetradecanoylphorbol-13-acetate, mezerein and ethyl phenylpropriolate.

Single applications of 12-O-tetradecanoylphorbol-13-acetate (TPA), mezerein or ethyl phenylpropriolate (EPP) to mouse skin at appropriate doses cause similar degrees of hyperplasia and comparable levels of induction of epidermal ornithine decarboxylase (ODC) activity. Multiple (n = 5) treatments with these agents, in contrast, resulted in large differences in induced ODC activity (TPA much greater than mezerein greater than EPP) with no differences in the degree of hyperplasia or [3H]thymidine pulse-labeling among the multiple treatment groups. To attempt to explain the cellular basis for the greater ODC-inducing ability of TPA relative to mezerein and EPP in chronic exposure protocols, immunocytochemical and flow cytometric analyses were performed. Immunocytochemistry using an ODC-specific polyclonal antibody revealed substantially different pattern of ODC-positive cells in chronically exposed epidermis than observed with single exposures. TPA treatment resulted in very pronounced immunostaining of the perifollicular cells, with little evidence of specific staining in the interfollicular epidermis mezerein treatment yielded staining in both interfollicular and some perifollicular areas, while EPP treatment produced the least amount of specifically stained cells, all of which were in the interfollicular epidermis. Flow cytometric analysis of keratinocytes isolated from chronically treated skin identified three distinct subpopulations that bound varying amounts of ODC antibody. Chronic treatment of CD-1 murine epidermis with TPA appeared to cause the expansion of an intermediate sized cell subpopulation that was not apparent with EPP or mezerein. Our results suggest that chronic treatment of murine epidermis with the potent complete tumor promoter TPA leads to the selective expansion of a keratinocyte subpopulation that is hyperinducible for ODC and may be identical to the cells in the perifollicular region previously identified. These observations also suggest that the weaker tumor promoters mezerein and EPP are less capable of causing expansion of this specific subpopulation, which may be an important target cell population for neoplastic transformation in mouse epidermis.

Alkynes↗
PubMed1992

Autism and developmental abnormalities in children with perinatal cocaine exposure.

Cocaine in all forms is the number one illicit drug of choice among pregnant women. Records of 70 children with cocaine exposure in utero who were referred for developmental evaluation at a large inner-city hospital were reviewed in an effort to determine whether a specific pattern of abnormalities could be discerned. Patients received physical examinations, neurological screenings, and behavioral and developmental assessments based on the Gesell Developmental Inventory, and the Denver Developmental Screening Test. Documentation of specified drug use was obtained by history. Mean age (SEM) at referral was 19.2 (1.7) months. All mothers used cocaine in one of its forms, although polydrug use was common. Growth parameters were low (median = 15th percentile). Significant neurodevelopmental abnormalities were observed, including language delay in 94% of the children and an extremely high frequency of autism (11.4%). The high rate of autistic disorders not known to occur in children exposed to alcohol or opiates alone suggests specific cocaine effects.

Adolescent↗
PubMed1992

Newborn screening for sickle cell and other hemoglobinopathies: a Canadian pilot study.

We estimated incidence of HbS disease in Quebec. It is approximately 9 cases per 100,000 births (equivalent to the incidence of the hyperphenylalanemias). Accordingly, we performed a voluntary pilot study in 9 self-identified ethnic groups; 3528 families were counselled about the relevance of newborn screening for hemoglobinopathies; and 2779 cord blood samples were collected (participation rate, 78.7%) and analyzed for Hemoglobin S and other hemoglobin variants by cellulose acetate electrophoresis. There were 95 (3.42%) positive tests on the initial (cord blood) samples, of which only 40 could be confirmed because of low participation in follow-up. We identified 8 false-positive tests; 7 had been classified initially as alpha-thalassemia trait and one as HbC heterozygosity on the first test. The relative frequency of hemoglobinopathy genes (confirmed) was: 52.5% HbS; 22.5% alpha-thalassemia; 22.5% other mutation; all but one patient with sickle cell disease were heterozygotes; the majority (71%) of HbS genes were accounted for by the 7% of screened newborns who were Black; a further 24% of the HbS genes were accounted for by 7% with Central American ancestry. Record linkage of the findings in heterozygotes for use later in life is an unsolved problem. Seventy five first-degree relatives of the 48 probands were screened in follow-up studies (64% of parents participated); 5 couples at risk for having a future child with a hemoglobinopathy were identified. Attitudes toward follow-up varied among the ethnic groups. The single family with an affected newborn (sickle cell anemia) was counselled effectively; the infant received penicillin prophylaxis.(ABSTRACT TRUNCATED AT 250 WORDS)

Anemia, Sickle Cell↗
PubMed1991

Phosphorylation by calcium calmodulin-dependent protein kinase II and protein kinase C modulates the activity of nitric oxide synthase.

Nitric oxide synthase purified from rat brain, which is Ca2+ and calmodulin dependent, was phosphorylated by calcium calmodulin-dependent protein kinase II as well as protein kinase C. Phosphorylation by calcium calmodulin-dependent protein kinase II resulted in a marked decrease in enzyme activity (33% of control) without changing the co-factor requirements, whereas a moderate increase in enzyme activity (140% of control) was observed after phosphorylation by protein kinase C. These findings indicate that brain nitric oxide synthase activity may be regulated not only by Ca2+/calmodulin and several co-factors, but also by phosphorylation.

Adenosine Triphosphate↗
PubMed1991

Spin trap protection from tumor necrosis factor cytotoxicity.

Tumor necrosis factor (TNF) facilitates superoxide production, and spin traps may detoxify superoxide by acting as superoxide dismutase mimics. We investigated the ability of a stable nitroxide spin trap, TEMPOL, to protect TNF-sensitive cells from exogenously added TNF. WEHI or L929 cells were incubated with TNF (500 units/ml) for 18 hr either simultaneously with 0 to 8 mM TEMPOL or with the TEMPOL added at varying intervals after TNF exposure. A dose-dependent increase in survival was noted in the TEMPOL-treated cells, with 92 +/- 2% survival of WEHIs treated with 4 mM TEMPOL compared to 26 +/- 1% survival for non-TEMPOL-exposed cells (P2 less than 0.01). Significant increases in survival could be accomplished with as late as 15-hr delayed addition of the compound. The mechanism of protection does not seem to involve newly synthesized protein, and Northern blot analysis revealed that TEMPOL does not induce the genes for MnSOD or Cu-ZnSOD. The ability of TEMPOL to protect against TNF injury, even when exposure is delayed, may prove useful in conditions thought to be associated with free radical-lymphokine interactions such as ischemia-reperfusion, oxygen toxicity, or sepsis.

Animals↗
PubMed1991

Changes to synaptic ultrastructure in field CA1 of the rat hippocampus following intracerebroventricular injection of kainic acid.

To assess the nature and extent of ultrastructural damage due to low unilateral intracerebroventricular doses of kainic acid, treated rats were killed at survival times from 8 h to 14 weeks. Degenerative changes in field CA1 of the hippocampus included dark profiles (often presynaptic), lucent areas enveloping axonic or dendritic elements, damaged myelin sheaths, and enlarged glial profiles. The effect of kainic acid ipsilaterally was maximal at three days but also apparent up to 14 weeks. Contralateral CA1 showed similar though less extensive abnormalities. These observations suggest that, despite rapid synaptic replacement (Nadler et al., Brain Res. 191, 387-403, 1980), long-term electrophysiological abnormalities (Cornish and Wheal, Neuroscience 28, 563-571, 1989) may stem not only from inappropriate reactive synaptogenesis but also from a continuing state of neuronal degeneration.

Animals↗
PubMed1991

Flow cytometric assessment of platelet function in patients with peripheral arterial occlusive disease.

This study compared new and traditional measures of platelet function in 16 patients with severe peripheral arterial occlusive disease and 15 age-matched controls. Circulating platelets were characterized by the use of fluorescence flow cytometry to assess platelet aggregate formation and expression of the secretion-dependent alpha granule membrane protein GMP-140, by measurement of plasma beta-thromboglobulin (beta-TG), and by performance of platelet-rich plasma aggregation studies. In addition, blood samples were treated with graded concentrations of adenosine diphosphate (ADP; 0 to 10 mumols/L) to characterize by fluorescence flow cytometry the secretory and aggregatory responses to mild stimulation. No differences were detected between the two groups with regard to platelet function in unstimulated circulating blood by use of these techniques. Values (mean +/- SEM) observed were: GMP-140-positive platelets, 11% +/- 3% versus 13% +/- 2%; platelet aggregates in circulating whole blood, 4% +/- 1% versus 9% +/- 3%; plasma beta-TG, 92 +/- 12 versus 94 +/- 22 ng/ml; and ED50 (concentration of ADP required to produce half maximal aggregation), 3.8 +/- 1.1 versus 3.1 +/- 0.5 mumol/L in the patients with peripheral arterial occlusive disease and controls, respectively. Treatment with ADP caused a dose-related increase in GMP-140 expression in both groups, without significant differences in this parameter between the groups at any given concentration. However, stimulation with ADP concentrations greater than 1 mumol/L resulted in more frequent aggregate formation in the control than in the peripheral arterial occlusive disease group (25% +/- 4% versus 11% +/- 2%, respectively at 5.0 mumols/L, p = 0.002).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1991

Delusions and hallucinations of cocaine abusers and paranoid schizophrenics: a comparative study.

We compared delusions and hallucinations of 100 cocaine abusers and 100 paranoid schizophrenic subjects admitted to an East Texas state psychiatric hospital. Subjects in both groups feared that individuals or organized groups might harm them in some way, but delusions of the paranoid schizophrenic subjects were more often bizarre than those of the cocaine abuse subjects. "Cocaine bugs" (parasitosis) were more often found in the cocaine abuse subjects. Command hallucinations were found in both groups, but the commands of the schizophrenic group more often related to harming or killing others. Cocaine abusers had a greater frequency of visual hallucinations (47 to 7), distinguished by shadows, flashing lights ("snow lights"), objects moving and bugs crawling on the arm. Finally, the most distinguishing characteristics were identity delusions, possession delusions, grandiose delusions (other than identities and possessions), and delusions that their families were imposters (Capgras Syndrome) reported by paranoid schizophrenics. No such delusions were reported by the cocaine abusers.

Adolescent↗
PubMed1991

Platelet activation and aggregation during cardiopulmonary bypass.

Increases in plasma concentrations of platelet granule products such as platelet factor 4 and beta-thromboglobulin during cardiopulmonary bypass suggest that platelets are activated during extracorporeal circulation. Subsequent circulation of these activated platelets may be responsible for the ubiquitous platelet dysfunction associated with cardiopulmonary bypass. Using flow cytometry and a monoclonal antibody directed against an alpha-granule membrane protein, granule membrane protein 140 (GMP-140), which is expressed on the platelet surface membrane after activation, we directly measured the percentage of circulating activated platelets in 41 patients before, during, and after cardiopulmonary bypass. In addition, we compared the GMP-140 expression with platelet aggregation in response to adenosine diphosphate (ADP). Cardiopulmonary bypass produced a significant increase in the percentage of GMP-140-positive platelets persisting in the circulation; the percentage peaked at a mean of 29% (range 10-58%) before separation from extracorporeal circulation. A significant percentage of these activated platelets continued to circulate in the early postoperative period. Simultaneous measurement of platelet aggregation in response to ADP demonstrated an aggregation defect that had a time course distinct from platelet activation and whose magnitude did not correlate with the degree of platelet activation in individual patients. We conclude that cardiopulmonary bypass causes a complex constellation of platelet defects, which include alpha-granule release, prolonged circulation of activated, "spent" platelets, and impaired platelet aggregation.

Adenosine Diphosphate↗