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R A Carchman

Publications and source records attributed to R A Carchman.

At least 37 records · Page 2Linked to original sources
PubMed1987

Human T lymphocyte mitogenesis in response to the B oligomer of pertussis toxin is associated with an early elevation in cytosolic calcium concentrations.

Pertussis toxin was found to serve as a mitogen in the human T lymphocyte, an effect which could be mimicked by its resolved binding component, the B oligomer. The mechanism of action of this component appeared to involve a rapid and sustained elevation of cytosolic calcium levels, as monitored by fura-2 fluorescence. The source of mobilized calcium was predominantly extracellular, suggesting that the binding of the B oligomer to the T cell plasma membrane in some way elicited calcium channel activation. Notably, the influx of calcium was not observed with cholera toxin, an AB toxin lacking mitogenic effects on the human T lymphocyte.

Benzofurans↗
PubMed1987

A correlation between phorbol diester-induced protein phosphorylation and superoxide anion generation in HL-60 cells during granulocytic maturation.

As HL-60 cells matured along the granulocytic pathway, phorbol diester-induced superoxide anion production was compared to phorbol diester-induced protein phosphorylation using an in vitro phosphorylation technique. Maturation was induced by 0, 2, 4, or 6 days incubation with dimethyl sulfoxide (Me2SO). In 0 day Me2SO HL-60 cells, phorbol 12-myristate 13-acetate induced phosphorylation of protein pp29 (Mr = 28,600) and to a lesser extent protein pp76 (Mr = 76,300). With increased time of Me2SO incubation, phorbol 12-myristate 13-acetate induced phosphorylation of pp212 (Mr = 211,800), pp134 (Mr = 134,200), and pp76, whereas the phosphorylation of pp29 did not change appreciably. In close agreement with this increase in protein phosphorylation was the observed increase in phorbol diester-induced superoxide anion formation. Morphological characterization of cells during Me2SO-induced differentiation reveals that these increases in phorbol diester responses are probably attributable to the proportional rise in metamyelocytes, band, and segmented neutrophils. A variety of phorbol diesters increased superoxide anion generation in HL-60 cells differentiated into granulocyte-like cells by 6-day incubation with Me2SO. The structure-activity relationship of these phorbol diester derivatives for protein phosphorylation was strongly correlated to their ability to increase superoxide anion generation. Thus, we propose that phorbol diester-induced phosphorylation of pp212, pp134, and pp76, but not pp29 may play a role in mediating the functional response of phorbol diester-induced superoxide anion generation in HL-60 cells differentiated into mature granulocyte-like cells.

Cell Differentiation↗
PubMed1987

The evaluation of biological interactions using response surface methodology.

Response surface methodology was employed in the statistical analysis of the combination exposures of genotoxic agents, bischloroethylnitrosourea with cis-diamminedichloroplatinum (II) and cis-diamminedichloroplatinum (II) with X rays. The measured endpoint in each case was sister chromatid exchanges in V79 Chinese hamster cells. The combination experiments employed a factorial design in which cells were treated, in various concentration combinations, with two agents simultaneously. Bis-chloroethylnitrosourea and cis-diamminedichloroplatinum (II) each exhibited curvilinear concentration-related increases in sister chromatid exchanges. X rays exhibited a dose-dependent increase in sister chromatid exchanges. For the cis-diamminedichloroplatinum (II)/X ray combinations, response surface methodology indicates a less-than-additive interaction, suggested by the non-parallel concentration-response curves of one agent at varying concentrations of the other, and a slight dose-dependent increase in sister chromatid exchanges due to X rays alone. Both cis-diamminedichloroplatinum and bis-chloroethylnitrosourea exhibited concentration-related increases in sister chromatid exchanges, cis-diamminedichloroplatinum (II) being 8-10 times (dependent on what level of effect was compared) more potent than bis-chloroethylnitrosourea. For the cis-diamminedichloroplatinum (II)/bis-chloroethylnitrosourea combinations, an increasingly less-than-additive interaction was detected. The analysis of these combinations demonstrates the strength of response surface methodology, a collection of mathematical and statistical techniques for detecting, analyzing and describing the biological effects resulting from exposures to multiple cytotoxic agents. The descriptive ability of these procedures is shown to be useful in that it leads to the suggestion of hypotheses regarding mechanisms of action.

Animals↗
PubMed1987

Evaluation of the interaction of three genotoxic agents in eliciting sister-chromatid exchanges using response surface methodology.

Chinese hamster cells (V79) were treated with ethylnitrosourea (ENU), bischloroethylnitrosourea (BCNU), and cis-diamminedichloroplatinum(II) (DDP) alone and in combination. Sister chromatid exchanges (SCEs) were quantitated as measures of genotoxicity of the three agents. The combination experiment employed a factorial design in which cells were treated, in various concentration combinations, with all agents simultaneously. Response surface methodology using a polynomial model in treatment variables to approximate the mean the distribution of SCE events was employed for analysis of the interactions of the three genotoxic agents. Due to unequal variances of the number of SCEs in the various treatment groups, a weighted least-squares analysis was used to estimate the parameters of the dose-response relationship. The single-agent results suggest that the DDP concentration-response curve has a much steeper slope than the ENU and BCNU curves, and is concave downward as compared to the relatively linear concentration-response curves of ENU and BCNU. The combination results suggest that ENU and DDP are involved in a negative interaction. The BCNU/DDP interaction, the ENU/BCNU interaction, and the three-factor interaction are not statistically significant. The analysis of these data demonstrates the usefulness of a statistical procedure for evaluating the biological effects resulting from exposure to multiple cytotoxic agents. The methodology can be used with many other types of endpoints and is not limited by the number of treatment agents.

Animals↗
PubMed1986

Estimation and analysis of the concentration-response surfaces associated with multiple-agent combinations.

Chinese hamster cells (V79) were treated with ethylnitrosourea (ENU) and cis-diamminedichloroplatinum(II) (DDP) alone and in combination. Sister chromatid exchanges (SCEs) were quantified as measures of genotoxicity of the two agents. The combination experiment employed a factorial design in which cells were treated, in various concentration combinations, with both agents simultaneously. Response surface methodology, using a polynomial model based on a negative binomial distribution of SCE events, was employed for analysis of the interactions of the two genotoxic agents. The negative binomial distribution, a generalization of the Poisson distribution, is required since SCEs are discrete variables which, under the conditions of these experiments, have a distribution which exhibits extra-Poisson variability. The model of the ENU/DDP combinations indicated an increasingly less-than-additive effect resulting from increasing concentrations of each agent in the combination. The analysis of these experiments demonstrates the usefulness of a powerful statistical procedure for evaluating the biological effects resulting from exposure to multiple cytotoxic agents. The methodology can be used with many other types of endpoints and is not limited by the number of treatment agents.

Cells, Cultured↗
PubMed1986

The kinetics of in vivo sister chromatid exchange induction in mouse bone marrow cells by ethylnitrosourea and methylnitrosourea.

Administration of ethylnitrosourea (ENU) (20, 25, 40, 50, and 60 mg/kg body weight) or methylnitrosourea (MNU) (25, 40, 50, 60, 75, and 80 mg/kg body weight) to male CD-1 mice 2 hr after sc implantation of a 5-bromo-2'-deoxyuridine (BrdUrd) pellet (55 mg) resulted in a dose-dependent increase in sister chromatid exchanges (SCE) in bone marrow cells. Treatment with ENU (50 mg/kg body weight) at several time points prior to BrdUrd implantation resulted in a multiphasic curve of SCE induction indicating at least two events that result in SCEs. Treatment with ENU at the time of BrdUrd implantation and post-BrdUrd reflected a similar mechanism apparent in the pre-BrdUrd curve. Treatment with MNU (50 mg/kg body weight) pre- and post-BrdUrd resulted in a linear monophasic curve of SCE induction in both the pre- and post-BrdUrd time periods. The overall MNU time-course curve resembled an inverted V function suggesting the mechanisms of SCE induction for ENU and MNU are different. These observations suggest that at least one explanation for the differences in the time courses for ENU and MNU SCE induction may result from a more persistent lesion being induced by ENU. In addition, these results indicate that in vivo SCE protocols which utilize a single acute chemical exposure at or near the time of BrdUrd labeling may not be useful for judging the relative activities of genotoxic agents.

Animals↗
PubMed1986

Application of response-surface methodology to detect interactions of genotoxic agents in cultured mammalian cells.

Response-surface methodology (RSM) techniques provide a useful statistical approach for the design and analysis of experiments involving multiple variables. Although it has been used for some time in the areas of chemical engineering and agriculture, RSM has only recently been applied to the solution of biological problems. Here we have utilized RSM to investigate the interaction of two direct-acting, monofunctional alkylating agents [ethyl methanesulfonate (EMS) and ethylnitrosourea (ENU)] in Chinese hamster V79 cells with respect to the in vitro induction of sister chromatid exchanges (SCEs). A factorial design was employed in which the cells were exposed to the agents singly and in simultaneous combinations for 4 h. The cells were collected for SCE determination 30 h after treatment. The analysis revealed concentration-dependent increases in SCEs for both of the agents, with ENU being the more effective on an equimolar basis. In addition, single- and multiple-agent interactions were detected. The most important finding was that over the treatment range studied, a significant negative interaction occurs between EMS and ENU with regard to SCE induction. It is suggested that RSM not only may be useful in determining the statistical relevance of experimental variables but also may generate hypotheses the evaluation of which could provide additional insights into the underlying mechanisms involved.

Animals↗
PubMed1986

Effect of in vitro differentiation on phorbol diester receptor number in human promyelocytic leukemia (HL-60) cells.

Phorbol diester (PDE) tumor promoters affect cells by interacting with specific receptors on the plasma membrane. Cells are known to regulate their receptor populations in response to many external and internal stimuli, including differentiation. The human promyelocytic leukemia cell line HL-60, which can be induced by dimethyl sulfoxide to differentiate into mature granulocytes, was used as a model to examine whether PDE receptors are regulated as cells differentiate in vitro. PDE binding was measured using [20-3H]phorbol 12,13-dibutyrate ([3H]PDBu). Specific binding of [3H]PDBu to the undifferentiated cells was rapid, reversible, and time and concentration related. One class of noncooperative binding sites was found with approximately 3.3 X 10(5) cells, having a Kd of 27.5 nM. [3H]PDBu could be displaced from the binding sites by a series of biologically active PDEs, but not by inactive ones. The characteristics of [3H]PDBu binding to the differentiated HL-60 cells were almost identical to those of the undifferentiated cells, except that there was an increase in the number of binding sites to 9.1 X 10(5) cells. Production of reactive oxygen metabolites by the cells, as monitored by chemiluminescence (CL) in response to PDEs, was examined before and after dimethyl sulfoxide differentiation, to determine if the change in receptor density was accompanied by a change in cell function. Only the differentiated HL-60 cells produced a quantifiable CL response when exposed to PDEs. The potency of the PDEs in causing CL generation was the same as that for displacing [3H]PDBu. The correlation between CL generation and affinity for the binding site suggests that the PDE binding sites mediate this effect of the PDEs and, therefore, are receptors. These studies indicate that HL-60 cells regulate their PDE receptors as they differentiate in vitro, with minimal extracellular influence. This may reflect a portion of an internal mechanism to enhance or change response to an endogenous ligand for the PDE receptor as cells mature.

Caenorhabditis elegans Proteins↗
PubMed1985

Reversibility of phorbol diester-induced macrophage spreading.

Phorbol 12-myristate 13-acetate and phorbol 12, 13-dibutyrate induced spreading of mouse macrophages with 50% effective concentrations of 3 nM and 35 nM, respectively. Macrophages treated with 100 or 1000 nM phorbol 12, 13- dibutyrate showed a time related decrease in spreading after washout. Spreading induced by 1, 10, or 100 nM phorbol 12-myristate 13-acetate was irreversible; however, washed phorbol 12,13-dibutyrate-treated cells respread after a second exposure to this compound. Washout of 3[H]phorbol diesters corroborated these observations in that 5% of 3H-phorbol 12-myristate 13-acetate and only 0.1% 3[H]phorbol, 12,13-dibutyrate remained associated with washed cells. Since phorbol 12-myristate 13 acetate is much more lipophilic than phorbol 12,13-dibutyrate, the reversibility of phorbol diester-induced macrophage spreading may depend upon the lipophilicity of the derivative utilized.

Animals↗
PubMed1985

Alterations in calcium metabolism in phorbol ester-treated mouse peritoneal macrophages.

Phorbol 12-myristate 13-acetate (PMA)-treated macrophages exhibited a two-fold increase in the rate of 45Ca++ efflux and over a three-fold increase in the size of the exchangeable calcium pool, resulting in almost a seven-fold increase in the slow phase of calcium efflux. The calcium antagonist 8-(N,N-diethylamino) octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) by itself did not affect calcium efflux in macrophages; but abolished the PMA-induced increase in the rate of calcium efflux. The divalent cationphore A23187 increased the rate constant of the fast phase of calcium efflux two-fold when applied alone or when applied with PMA. These effects might be linked to ionophore enhancement and TMB-8 inhibition of PMA-induced macrophage chemotaxis and spreading (previously reported in Cell Calcium 3:503-514 and Cancer Research 43:3385-3391). No change in calcium efflux was observed if cells were exposed to PMA only during the efflux experiment suggesting that a prolonged exposure to PMA is required to elicit changes in calcium flux. Increased 45Ca++ remained in treated cells at each time point perhaps reflecting the PMA-induced increase in exchangeable calcium.

Animals↗
PubMed1985

Application of response surface methods for evaluating the interactions of soman, atropine, and pralidioxime chloride.

Response surface methods were employed to model survival data obtained in guinea pigs following subcutaneous exposure to soman (GD; 30-84.6 micrograms/kg) with various treatment regimens (i.e., atropine/pralidioxime chloride [ATR/2-PAM] combinations, given im, 1 min post-GD). The analysis of the proportions of surviving animals in the various groups revealed that the use of individual treatment agents (i.e., ATR or 2-PAM) was effective in increasing survival. The level of GD exposure altered the nature of the ATR/2-PAM interaction. Exploration of the response surface indicates that the optimal treatment combinations (greater than 94% survival) of ATR/2-PAM change as a function of GD exposure in the following manner: GD 30 micrograms/kg-ATR/2-PAM, 217 mg/kg/0 mg/kg; GD 42.4 micrograms/kg-179/29 mg/kg; mg/kg; GD 60 micrograms/kg-148/83 mg/kg; GD 84.6 micrograms/kg-168/150 mg/kg. At higher exposures of GD (greater than 42.4 micrograms/kg), therapeutic synergy was observed with the use of the treatment combinations, as compared to optimal single agent treatments. Evaluation of the apparent toxicities of treatment combinations can also be determined in this procedure. RSM is not geometrically restricted by the number of variables under consideration. A variety of experimental designs, when used in conjunction with RSM, permit the modeling of multiple responses and provide estimates of optimal therapeutic modalities subject to constraints (e.g., behavioral toxicity).

Animals↗
PubMed1985

Assessing pyridostigmine efficacy by response surface modeling.

The therapeutic efficacy of atropine sulfate/pralidoxime chloride (ATR/2-PAM) treatment (im) therapy, and pyridostigmine bromide (PYR) pretreatment (oral) therapy were evaluated in soman-challenged guinea pigs. ATR/2-PAM efficacy was assessed as protective ratio (PR = treated soman LD50/control soman LD50); PYR efficacy was assessed both as PR and by response surface modeling (RSM) techniques. The optimal ATR/2-PAM treatment gave a PR of 3.78. PYR pretreatment (1 hr) produced a dose(log)-dependent (r = 0.96) inhibition of whole blood AChE and afforded significant (p less than 0.05) increases in PR (with doses greater than 0.12 mg/kg PYR) against soman when followed by 64 mg/kg ATR/100 mg/kg 2-PAM treatment. These PRs, however, were poorly correlated (r = 0.45) with the corresponding level of PYR-induced AChE inhibition. In contrast, RSM analysis of efficacy indicated that the optimal ATR/2-PAM dose combination varied as a function of both the soman-challenge level and the PYR pretreatment dose. Efficacy was therefore evaluated for varying PYR pretreatment doses in combination with the appropriate optimal ATR/2-PAM treatment (as determined by RSM for each soman challenge dose and PYR dose evaluated). When assessed in this manner, PYR efficacy (PYR) was found to be highly correlated (r = 0.97) with PYR-induced AChE inhibition. Since percentage of AChE inhibition was directly correlated with PYR dose (log), these results indicate that PYR pretreatment efficacy is a highly correlated, dose-dependent phenomenon, providing ATR/2-PAM treatment is optimized.

Animals↗
PubMed1984

Development of the adult respiratory distress syndrome: progressive alteration of neutrophil chemotactic and secretory processes.

Chemotaxis and lysosomal enzyme release in peripheral blood neutrophils taken from patients before, during, and after recovery from the adult respiratory distress syndrome (ARDS) were studied. This allowed for correlation of cellular function with changes in a patient's clinical status. It was found that neutrophils from 8 of 9 patients with the fully developed syndrome exhibited a profound depression of chemotaxis (63% depressed, P = 0.0001) and a fourfold elevation of basal lysosomal enzyme release relative to neutrophils from healthy controls (P = 0.0001). These findings of depression of chemotaxis and enhanced basal enzyme release were also detected in neutrophils taken from 7 of 11 patients in whom clinical risk factors (eg, sepsis, pneumonia) for the syndrome had developed. Following resolution of the adult respiratory distress syndrome, the above changes in neutrophil function resolved in the four patients studied during convalescence. Healthy neutrophils exposed to plasma samples (untreated or zymosan-activated) from control subjects and patients with ARDS could not be distinguished with respect to chemotaxis and enzyme secretion. It is concluded that patients in whom ARDS develops show profound but reversible changes in peripheral neutrophil activity which can be measured following the development of a clinical predisposition for the syndrome. Further, the presence of a humoral substance capable of promoting chemotaxis or enzyme secretion from healthy neutrophils in the untreated plasma of patients suffering from ARDS was not demonstrated. This suggests that alteration of neutrophil activity measured in patients with the fully developed syndrome may be cellular in origin.

Acetylglucosaminidase↗
PubMed1983

The kinetics of in vivo sister chromatid exchange induction in mouse bone marrow cells by alkylating agents: cyclophosphamide.

Administration of cyclophosphamide (5, 10, 20 and 25 mg/kg body weight) to male CD-1 mice 2 hr after subcutaneous implantation of a 5-bromo-2'-deoxyuridine (BrdUrd) pellet (55 mg) resulted in a dose-dependent increase in sister chromatid exchanges (SCE) in bone marrow cells. Treatment with cyclophosphamide (15 mg/kg body weight) at the time of BrdUrd implantation and 2, 6.5, and 13 hr post-BrdUrd implantation resulted in the induction of approximately 19 SCE/cell indicating that the bone marrow SCE response was independent of the time of administration. Treatment with cyclophosphamide (15 mg/kg body weight) at 26, 19, 13, and 6 hr prior to BrdUrd implantation resulted in baseline SCE (3.3 SCE/cell) at 26 hr with an increasing number of SCE/cell with decreasing time prior to BrdUrd implantation. These results compare favorably with those obtained by Kram et al [1981] with mitomycin C (MMC) using a similar protocol. The time-dependent induction of SCE is qualitatively similar for CP and MMC, both of which are bifunctional alkylating agents metabolically activated by oxidation and reduction, respectively, and suggests that these two compounds may induce SCE by a similar mechanism.

Animals↗
PubMed1983

Calcium modulation of phorbol ester-induced alterations in murine macrophage morphology.

The phorbol ester tumor promoter phorbol-12-myristate-13-acetate (PMA) induced mouse resident peritoneal macrophage spreading in an in vitro system in a time- and dose-dependent manner; this process was modified by agents which alter intracellular calcium metabolism. After a 35-min incubation with PMA, 50% of the macrophages were spread (as classified by at least a 2-fold increase in cell surface area). Also at 35 min, the median effective concentration for PMA induction of spreading was 1.6 ng/ml. The intracellular calcium antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate inhibited PMA-induced cell spreading with a one-half maximal inhibitory concentration of 8.0 microM. The calcium ionophore A23187 enhanced PMA-induced spreading by 30% at 0.1 to 10 nM. The histological dye ruthenium red, which purportedly increases intracellular calcium by displacing membrane-bound calcium stores, enhanced PMA-induced spreading up to 75% at 1.0 pM. The cationic chelator ethyleneglycolbis(beta-aminoethylether)-N,N-tetraacetic acid (1.8 and 3.6 mM) had no effect on PMA-induced spreading. Thus, PMA-induced spreading was independent of extracellular calcium but was modulated by agents altering intracellular calcium metabolism. Microfilament formation, a proposed mechanism of cell spreading, also depends on intracellular calcium availability. The microfilament inhibitor cytochalasin B inhibited PMA-induced spreading with a one-half maximal inhibitory concentration of 1 microM. Future experiments should investigate the hypothesis that calcium availability to the cytoskeletal elements regulates the morphological effects of PMA on macrophages.

Animals↗
PubMed1983

Antagonist of phorbol ester receptor-mediated chemotaxis in mouse peritoneal macrophages.

Biologically active phorbol ester derivatives displace [3H]phorbol-12, 13-dibutyrate from thioglycollate-elicited mouse peritoneal macrophages in a time-, temperature-, and concentration-dependent manner. Scatchard analysis revealed an apparent Kd of 54.1 nM and 8.0 X 10(5) sites/cell, indicating that these macrophages possess saturable, high-affinity phorbol ester-binding sites. These derivatives also act as chemoattractants for the macrophage at equivalent concentrations. A notable exception to this pattern is phorbol-12,13-diacetate. Phorbol-12,13-diacetate inhibits specific binding of [3H]phorbol-12,13-dibutyrate (concentration required for a 50% inhibition of the maximum specific binding of [3H]phorbol-12,13-dibutyrate, 2.6 microM) and chemotaxis to phorbol-12-myristate, 13-acetate (concentration required for a 50% inhibition of the maximum chemotactic response, 0.39 microM) while exhibiting no activity as a chemoattractant at concentrations up to 10(-5) M. The data indicate that phorbol-12,13-diacetate may be an antagonist for receptor-mediated chemotaxis to phorbol-12-myristate, 13-acetate in the macrophage.

Animals↗