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Biomedical subjects

A C Miller

Publications and source records attributed to A C Miller.

At least 55 records · Page 3Linked to original sources
PubMed1994

Calpain activation in apoptosis.

Programmed cell death is an active process wherein the cell initiates a sequence of events culminating in the fragmentation of its DNA, nuclear collapse, and disintegration of the cell into small, membrane-bound apoptotic bodies. Examination of the death program in various models has shown common themes, including a rise in cytoplasmic calcium, cytoskeletal changes, and redistribution of membrane lipids. The calcium-dependent neutral protease calpain has putative roles in cytoskeletal and membrane changes in other cellular processes; this fact led us to test the role of calpain in a well-known model of apoptotic cell death, that of thymocytes after treatment with dexamethasone. Assays for calcium-dependent proteolysis in thymocyte extracts reveal a rise in activity with a peak at about 1 hr of incubation with dexamethasone, falling to background at approximately 2 hr. Western blots indicate autolytic cleavage of the proenzyme precursor to the calpain I isozyme, providing additional evidence for calpain activation. We have also found that apoptosis in thymocytes, whether induced by dexamethasone or by low-level irradiation, is blocked by specific inhibitors of calpain. Apoptosis of metamyelocytes incubated with cycloheximide is also blocked by calpain inhibitors. These studies suggest a required role for calpain in both "induction" and "release" models of apoptotic cell death.

Animals↗
PubMed1994

Decreased content of the IL1 alpha processing enzyme calpain in murine bone marrow-derived macrophages after treatment with the benzene metabolite hydroquinone.

Benzene is an important industrial chemical known to produce hematotoxicity in mice and humans. Hydroquinone, a major metabolite of benzene, inhibits conversion of the precursor form of IL1 alpha (pre-IL1 alpha) to IL1 alpha in murine bone marrow-derived macrophages in vitro, and a similar effect can be demonstrated in vivo after treatment of mice with benzene. The protease which converts pre-IL1 alpha to IL1 alpha is calpain. We examined decreases in calpain content in bone marrow-derived macrophages as a possible mechanism underlying hydroquinone-induced decreases in pre-IL1 alpha conversion. Hydroquinone, at concentrations which were not overtly cytotoxic, decreased total calpain activity in macrophages by 10-30%. Using immunoblot analysis macrophage calpain II levels were shown to be decreased by approximately 50% after treatment with hydroquinone. Under the same conditions, no changes were observed in calpain I content using immunoblot analysis. These data show that decreased calpain II content represents a potential mechanism of hydroquinone-induced inhibition of pre-IL1 alpha processing, and may contribute to benzene-induced alterations in bone marrow stromal cell function and myelotoxicity.

Animals↗
PubMed1994

Calpain-induced downregulation of activated protein kinase C-alpha affects lung epithelial cell morphology.

A few minutes after mouse lung epithelial cell lines were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), the cells rounded up and pulled away from their neighbors. Several hours later, the cells flattened out to resume their original morphology. To begin to characterize the enzymology underlying these changes, the subcellular distribution and intracellular content of the TPA receptor, protein kinase C (PKC), and its putative endogenous regulator, the Ca(2+)-dependent protease, calpain, were investigated. Of eight PKC isozymes examined in several tumorigenic and nontumorigenic cell lines, all cells contained PKC-alpha, PKC-delta, and PKC-zeta. TPA rapidly (5 min) translocated PKC-alpha from the cytosol to the particulate fraction; PKC-alpha concentrations then decreased with continued TPA exposure. PKC-zeta levels and intracellular location were not affected. An inhibitor of PKC activity, GF 109203X, prevented the initial morphological change. The calpain II isozyme was also found in all cell lines, and its cellular content increased as a result of TPA treatment. Calpain inhibitor I did not affect the initial shape change but prevented subsequent flattening of the cells. We therefore conclude that PKC activation is required for the TPA-induced alterations in lung cell morphology and that calpain mediates the return to a flattened epithelial appearance.

Animals↗
PubMed1993

Guidelines for the management of spontaneous pneumothorax. Standards of Care Committee, British Thoracic Society.

Simple flow diagrams, with explanatory notes, have been devised to assist in the immediate and subsequent management of patients presenting to casualty departments with spontaneous pneumothorax. They cover decision making about which patients require admission; whether a drainage procedure is necessary; if so, the appropriate method, including technical details; inpatient treatment; referral of inpatients to a respiratory specialist; and follow up arrangements. The guidelines, designed for incorporation into casualty and ward handbooks, have already proved valuable in several district general hospitals, and can be used as a basis for audit.

Humans↗
PubMed1993

Increased radioresistance of EJras-transformed human osteosarcoma cells and its modulation by lovastatin, an inhibitor of p21ras isoprenylation.

Alterations in ras oncogene expression have been associated with increased cellular resistance to ionizing radiation. As an extension of studies with murine cell models, we have now explored the radioresponses of human osteosarcoma (HOS) sub-clones that differ in their EJras expression. Quantitative analysis revealed a tight correlation between the amounts of ras-encoded mRNA and p21 produced, and the degree of cell radioresistance. Interestingly, treatment of the ras-transformed cells with lovastatin, an inhibitor of p21ras post-translational processing via the mevalonate pathway, markedly decreased their radioresistance. Under the experimental conditions used, lovastatin prevented the membrane association, but not the biosynthesis, of p21. The decline in radiation resistance following lovastatin treatment could not be attributed to perturbation of cholesterol metabolism or to non-specific cell-cycle effects. In agreement, lovastatin did not alter the radiation responses of control HOS cells that do not express EJras, or those with an activated met oncogene. The results indicate that elevation in ras gene expression can lead to increased radioresistance of human tumor cells. It appears, however, that p21ras membrane localization is critical for maintenance of the radioresistant phenotype, thus providing a target for pharmacological intervention.

Animals↗
PubMed1993

Serine/threonine protein kinases and calcium-dependent protease in senescent IMR-90 fibroblasts.

Three enzymes relevant to signal transduction were compared in replicating, quiescent and senescent human diploid fibroblasts (HDF). These were Ca(2+)-dependent thiol protease (calpain), cAMP-dependent protein kinase (Pk-A), and calcium/phospholipid-dependent protein kinase C (Pk-C). The amounts of these enzymes in quiescent HDF were slightly greater or the same as in replicating HDF. In contrast, senescent HDF exhibited higher Pk-C, Pk-A and proteolytic activities than did either replicating or quiescent cells. While the elevated protein kinase activities could be accounted for by the larger size of senescent cells relative to younger cells, the increased calpain activity exceeded this size differential. Immunoblotting studies with antisera to both Pk-C and calpain demonstrated increased enzyme concentrations in parallel with the increased activities. Photolabeling cell extracts with an analog of cAMP, 8-N3-[32P]cAMP, provides an estimate of Pk-A concentration. By this criterion, senescent HDF have more Pk-A molecules than do young cells that are either replicating or quiescent. Only the type I isozyme of Pk-A (Pk-A-I) was observed in any of these cells. Photolabeling with 8-N3-[32P]cAMP demonstrated more degradative fragments of the Pk-A regulatory subunit (RI) in senescent cells also. This is a logical consequence of the increased calpain activity in senescent cells, since RI is a substrate for calpain. These results imply that senescent cells do not fail to enter S phase owing to inadequate concentrations of Pk-A or Pk-C. Rather, the increased quantities of these enzymes in senescent cells may reflect aberrations elsewhere along signal transduction pathways that coordinate cell size with cell proliferation.

Calcium↗
PubMed1993

Differences in radiation-induced micronuclei yields of human cells: influence of ras gene expression and protein localization.

Expression of ras has been correlated with increased intrinsic resistance to ionizing radiation. In this study we show that increased EJras expression in human cells is associated with a decrease in the frequency of radiation-induced micronuclei. The experimental system consisted of human osteosarcoma-derived cell lines which quantitatively vary in their EJras expression. There was a dose-dependent relationship between radiation dose and micronuclei formation in all cell lines tested. Human osteosarcoma cells, in which the ras level was undetectable, had the highest frequency of micronuclei production at all radiation doses tested. At 4 Gy the most radioresistant cells exhibited a 41.5 +/- 5% decrease in the production of micronuclei concomitant with high ras expression in comparison with the relatively radiosensitive parental cell line. Cells expressing a low amount of EJras demonstrated a 23 +/- 3% decrease in micronuclei induction compared with parental cells. Treatment of cells with lovastatin, an inhibitor of ras-encoded p21ras post-translational processing via the mevalonate pathway, markedly decreased the yield of micronuclei formation in cells transfected with ras; the drug had no effect on radiation-induced micronuclei formation in parental cells. The use of the in vitro micronuclei assay has provided a convenient way to visualize differences in the genotoxic damage induced by ionizing radiation in cells which express different amount of EJras. The results indicate that elevation of ras expression in human cells can lead to a decrease in the number of radiation-induced micronuclei formed and that this relationship is dependent on membrane association of ras-encoded p21.

Cell Line↗
PubMed1993

Posttranscriptional down-regulation of ras oncogene expression by inhibitors of cellular glutathione.

Alterations in intracellular glutathione (GSH) content are known to affect intrinsic responses to ionizing radiation. More recently, it became apparent that radiation responses may depend also on the expression of specific oncogenes, including ras. These findings, suggesting a possible link between GSH and ras, led us to examine the effect of various GSH modulators on ras expression. Treatment of c-Ha-ras-transformed NIH 3T3 cells with L-buthionine S'R'-sulfoximine, dimethylfumarate, or N',N'-1,3-bis(trans-4-hydroxycyclohexyl)-N'-nitrosourea resulted in dose- and time-dependent reduction in ras mRNA steady-state levels followed by a decrease in ras-encoded p21 protein production. The effect on ras correlated with the extent of GSH decline, was common to different members of the ras family, and was independent of the mode of oncogene activation or cell phenotype. Indeed, similar drug effects were observed with murine cells in which overexpression of the c-Ha-ras proto-oncogene was due to transcriptional activation (PR4, nontumorigenic) or gene amplification (NIH 136, tumorigenic) and with malignant cells expressing a mutated Ha-ras (RS504). Moreover, N-ras, EJras, and Ki-ras in human tumor cells were similarly affected. Molecular analysis revealed a significant decrease in ras mRNA half-life in cells subjected to GSH inhibition, an effect that required de novo protein synthesis, but there was no change in the rate of gene transcription. These results indicate that pharmacological manipulation of cellular GSH content can down-regulate ras expression at the posttranscriptional level by destabilizing ras transcripts. The potential clinical implications are discussed.

3T3 Cells↗
PubMed1992

Inhibition of glutathione reductase activity by a carbamoylating nitrosourea: effect on cellular radiosensitivity.

Nitrosoureas inactivate cellular glutathione reductase. N,'N'1,3-bis(trans-4-hydroxycyclohexyl)-N'-nitrosoureas (BCyNU), a nitrosourea reported to selectively inhibit glutathione reductase (GR) activity, was examined to determine if it could be used as a means to inhibit cellular levels of this enzyme in radiobiology studies. Confirmation of drug-induced inhibition of GR activity was demonstrated using a cell-free model system employing purified GR. Cellular studies with Chinese hamster V79A03 showed that BCyNU decreased cellular glutathione content concomitant with an inhibition of specific GR activity. Under relatively nontoxic conditions, cellular exposure to BCyNU (25 microM, 0.25 h) either before or after radiation treatment, increased cellular radiosensitivity with the optimum time for drug addition being immediately following radiation. At a BCyNU dosage which produced less than or equal to 5% cell toxicity, a marked decrease in radioresistance was characterized as a reduction in both Dq (24 +/- 1.5%) and Do (8 +/- 0.5%) concomitant with a 25 +/- 2% decrease in cellular glutathione reductase (GR) activity. At cytotoxic drug dosages (25 microM, 1 h; cell survival 79 +/- 7%), a marked radiosensitization manifested by a 1.25 +/- .07-fold reduction in the Dq was observed concomitant with a 49 +/- 4% decrease in GR activity. Using cells enriched in different stages of the cell cycle, BCyNU caused cell-age dependent cytotoxicity with preferential killing of cells in the radioresistant late-S-phase, a likely explanation for its radiosensitizing capabilities at high drug dosages. Data obtained at nontoxic drug dosages suggest that GR-inactivation may be an important component of cellular response to free-radical induced damage.

Animals↗
PubMed1992

Stress, appraisal, and coping in mothers of disabled and nondisabled children.

The cognitive-behavioral model of stress and coping provided the framework for this study of mothers of physically disabled children (n = 69) and mothers of nondisabled children (n = 63). The first aim was to test for differences between groups in responses to stressful parenting events. As hypothesized, mothers of disabled children reported higher levels of depressive symptomatology. Differences in psychological distress and health status approached significance. The second aim was to explore the mediating influences of coping strategies and cognitive appraisals. As predicted, emotion-focused coping was related to increased psychological distress in mothers of disabled children whereas problem-focused coping was associated with decreased distress. Both relationships were significant even after controlling for differences in type of parenting stressor. Hierarchical regressions showed that 58 and 25% of the variance in psychological distress was explained by the independent variables; however, the predictors were different for the two groups of mothers. Factors that may explain the differential effectiveness of coping and appraisal, and clinical implications of this study are discussed.

Adaptation, Psychological↗
PubMed1991

Brain protein synthesis in rabbits following low level aluminium exposure.

1. Infant rabbits received s.c.injections 3 times weekly of low doses of aluminium (A1) maltolate (0.5-1.5 mg Al/kg body wt) or aluminium lactate (8 mg Al/kg body wt) from 5 or 10 days of age to 14 or 22 days of age. 2. Brain was used to provide a cell-free protein synthesizing system and this system exhibited increased activity in preparations from Al-exposed infants. The mRNA fraction obtained from the brain polysomal RNA also was more active following Al exposure. 3. The synthesis of immunoprecipitable calmodulin was significantly elevated.

Aluminum↗
PubMed1991

DNA damage in cultured human skin fibroblasts exposed to excimer laser radiation.

Ultraviolet excimer lasers are being considered for use in a variety of refractive and therapeutic procedures, the long-term biologic consequences of which are unknown. The effect of sublethal doses of 193-nm laser radiation on cellular DNA was examined in cultured human skin fibroblasts. In contrast to 248 nm, treatments with the 193-nm laser radiation below 70 J/m2 did not cause significant pyrimidine dimer formation in the skin cells. This was indicated by the lack of excision repair activities (unscheduled DNA synthesis assay), and further demonstrated by direct analysis of pyrimidine dimers in DNA from irradiated cells. However, a low level of unscheduled DNA synthesis could be detected following irradiation at 193 nm with 70 J/m2. Both the 193-nm and 248-nm radiation were able to induce chromosomal aberrations, as indicated by a micronucleus assay. A dose-dependent increase in micronuclei frequency was observed 48 and 72 h after laser irradiation. These results indicate that exposure of actively replicating human skin fibroblasts to sublethal doses of either 193- or 248-nm laser radiation can result in genotoxicity.

Cell Survival↗
PubMed1991

The laryngeal mask airway. An unusual complication.

The laryngeal mask airway is an important addition to the anaesthetist's armamentarium, but its use is not without the possibility for misfortune. We encountered an unusual and potentially serious complication. A patient's epiglottis became trapped between the pliable grates in the mask portion of the laryngeal mask and partially obstructed his airway. Should this problem occur and remain unnoticed, in addition to the problem of airway obstruction during the anaesthetic, the oedematous epiglottis could be severely injured upon removal of the laryngeal mask. This, in turn, could result in airway obstruction requiring emergency treatment.

Adult↗
PubMed1991

Hookworm folliculitis.

A case of persistent folliculitis in a 21-year-old man was demonstrated to be due to Ancylostoma caninum larvae. Treatment with oral thiabendazole was curative. Cutaneous larva migrans may be due to A caninum, but this presentation appears to be unique. The literature concerning etiology and pathogenesis of larva migrans is discussed with reference to this case.

Adult↗
PubMed1991

Increased radiation resistance in transformed and nontransformed cells with elevated ras proto-oncogene expression.

The cellular Ha-ras oncogene, activated by missense mutations, has been implicated in intrinsic resistance to ionizing radiation. This study shows that the overexpression of the unmutated gene (proto-oncogene) may also be involved in how the cells respond to radiation. The experimental system consisted of mouse NIH 3T3-derived cell lines which carry multiple copies of a transcriptionally activated human c-Ha-ras proto-oncogene. Both tumorigenic (RS485) and revertant nontumorigenic subclones (PR4 and 4C3) which have high levels of ras expression exhibited a marked increase in radioresistance as measured by D0 compared to control NIH 3T3 cells. Other nontransformed cells with elevated levels of ras (phenotypically revertant line 4C8-A10) also had a significantly increased resistance to radiation, further indicating an association between ras and radioresistance. The increased radioresistance of the RS485 and phenotypic revertants could not be explained by a differential expression of the myc or metallothionein I genes or by variations in cell cycle. The correlation between increased ras proto-oncogene expression and radioresistance suggests that the ras encoded p21, a plasma membrane protein, may participate in the cellular responses to ionizing radiation.

Animals↗