L3L4ES antigen and secretagogues induce histamine release from porcine peripheral blood basophils after Ascaris suum infection.

The aim of this paper was to investigate the role of porcine basophils in protective immunity. Experimental pigs were infected with 10(3) Ascaris suum eggs daily for 21 days. Control pigs were maintained helminth-free. Circulating porcine basophils were isolated from the anticoagulated whole blood of A. suum-infected and noninfected pigs by dextran (4.5%) sedimentation of erythrocytes or by the centrifugation of dextran-isolated leukocytes through discontinuous Percoll gradients. Results showed that 2.2% of the isolated leukocytes, stained with May-Grunwald Giemsa, were basophils. Each basophil from infected pigs contained 1.30 x 10(-2) to 1.20 x 10(-1) pg of histamine. Peripheral blood basophils (PBBs) from infected swine released 49% specific histamine when induced with A. suum-derived antigen (L3L4ES), 55% with anti-immunoglobulin G, and 62% with calcium ionophore A23l87. During A. suum infection, the number of isolated basophils and histamine levels peaked at 14 to 21 days postinfection and then showed a significant decrease. Percent-specific histamine released from PBBs by infected swine was significantly greater than that released by control pigs. The L3L4ES antigen and secretagogues effectively induced specific/nonspecific histamine release from PBBs and should facilitate future investigations of porcine basophils.

Constitutive expression of the steroid sulfatase gene supports the growth of MCF-7 human breast cancer cells in vitro and in vivo.

Many human breast tumors are driven by high intratumor concentrations of 17beta-estradiol that appear to be locally synthesized. The role of aromatase is well established, but the possible contribution of the steroid sulfatase (STS), which liberates estrogens from their biologically inactive sulfates, has been inadequately assessed and remains unclear. To evaluate the role of STS further, we transduced estrogen-dependent MCF-7 human breast cancer cells with a retroviral vector directing the constitutive expression of the human STS gene. Gene integration was confirmed by Southern hybridization, production of the appropriately sized messenger RNA by Northern hybridization, and expression of functional protein by metabolism of [(3)H]estrone sulfate to [(3)H]estrone. Maximum velocity estimates of estrone formation are 64.2 pmol estrone/mg protein.h in STS-transduced cells (STS Clone 20), levels comparable to those seen in some human breast tumors. Lower levels of endogenous activity are seen in MCF-7 cells (13.0 pmol estrone/mg protein.h) and in cells transduced with vector lacking the STS gene (Vector 3 cells; 12.0 pmol estrone/mg protein.h). 17beta-Estradiol sulfate induces expression of the progesterone receptor messenger RNA only in STS Clone 20 cells, whereas estrone sulfate produces the greatest stimulation of anchorage-independent growth in these cells. STS Clone 20 cells retain responsiveness to antiestrogens, which block the ability of estrogen sulfate to increase the proportion of cells in both the S and G(2)/M phases of the cell cycle. Consistent with these in vitro observations, only STS Clone 20 cells exhibit a significant increase in the proportion of proliferating tumors in nude ovariectomized mice supplemented with 17beta-estradiol sulfate. The primary activity in vivo appears to be from intratumor STS, rather than hepatic STS. Surprisingly, 17beta-estradiol sulfate appears more effective than 17beta-estradiol when both are administered at comparable concentrations. This effect, which is seen only in STS Clone 20 cells, may reflect differences in the cellular pharmacology of exogenous estrogens compared with those released by the activity of intracellular STS. These studies directly demonstrate that intratumor STS activity can support estrogen-dependent tumorigenicity in an experimental model and may contribute to the promotion of human breast tumors.

Coping strategies in families of children with sickle cell disease.

OBJECTIVE: To examine the impact of family environment, morbidity, and socioeconomic status (SES) on coping strategies in families of children with sickle cell disease. DESIGN: A cross-sectional study. METHODS: The study sample consisted of 40 mothers, 24 fathers, 40 patients, and 28 healthy siblings from 40 African-American families, each of which had only one child with sickle cell disease. Data were collected through the use of structured interviews utilizing a demographic questionnaire, the Family Environment Scale (FES), and the COPE. RESULTS: The results indicate that support-seeking coping strategies were employed more often by mothers in more cohesive families and siblings in less cohesive families, while increased growth (resilience) was experienced by siblings in less conflicted families. Patients in more religious families reported greater use of religion as a means of coping. Mothers of mildly affected patients used acceptance more frequently than those of severely affected patients, and fathers of severely affected patients more often sought emotional support. Mothers, fathers, and siblings in low SES families reported greater use of denial than did those in high SES families. CONCLUSION: Additional psychosocial research involving families (including fathers and siblings) dealing with sickle cell disease is needed to facilitate the utilization of adaptive coping strategies, thereby enhancing individual and family adjustment.

Acetylcholinesterase activity in chronic renal failure.

Twenty healthy subjects and 39 Chronic Renal Failure patients (CRF-patients) maintained on chronic hemodialysis were used in this investigation to study the changes in acetylcholinesterase (AChE) activity of red blood cells (RBCs). The CRF-patients were all undergoing hemodialysis treatment. AChE activity from the CRF-patients was determined before and after dialysis. An additional objective was to study the effect of chronic renal failure on human red blood cell aging. Blood samples were drawn from controls and CRF-patients in tubes containing EDTA or sodium heparin as an anticoagulant. Red blood cells were purified to avoid interference with monocytes, reticulocytes and leukocytes. The purified RBCs were subfractionated into young (y) (1.08-1.09), mid (m) (1.09-1.11) and old (o) (1.11-1.12) percoll density (g/mL) fractions using a discontinous percoll gradient. The mean +/- SD AChE per gram hemoglobin (U/g Hgb) activities in whole blood (WB), purified human red blood cells (PRBCs), young human red blood cells (y-RBCs), mid age human red blood cells (m-RBCs) and old human red blood cells (o-RBCs) in CRF-patients were 31.2+/-3.43, 29.3+/-3.26, 30.4+/-3.91, 25.1+/-5.25, 17.1+/-6.02 in females and 29.8+/-5.39, 28.8+/-5.29, 28.7+/-5.29, 23.7+/-5.39 and 16.0+/-5.60 in males. AChE activity from CRF-patients were higher than that found in the control subjects. The aging of human RBCs in both the controls and CRF-patients showed a progressive reduction in AChE activity. AChE activity of RBCs from female CRF-patients were significantly higher (p < 0.05) than that of the female control subjects. The RBCs isolated from male CRF-patients showed a higher AChE activity than control males, but a significant difference was only observed with the mid-age-cells. These studies further indicate that AChE activity remained insignificantly different in the various density based age subfractions of RBCs of both CRF-patients and controls.

Comparison of calcium import as a function of contraction in the aortic smooth muscle of Sprague-Dawley, Wistar Kyoto and spontaneously hypertensive rats.

Genetic variations of far-reaching consequences have been established between spontaneously hypertensive rats (SHR) and their controls, Wistar Kyoto rats (WKY). The SHR strain is the most widely used model for the study of genetic hypertension. Calcium homeostasis in the vascular smooth muscle (VSM) is controlled by calcium channels and calcium pumps located in both VSM and the overlying endothelial cells that line the large blood vessels and the heart. Hypertension adversely affects calcium homeostasis. Investigations on the import of calcium from extracellular spaces with alpha1-adrenergic stimulation as a function of contractility of VSM cells in SHR and WKY were made and compared with the contractility observed in VSM cells of Sprague-Dawley (CD) rats. Experiments were performed on rings from thoracic aortas of three strains with endothelial lining intact or removed to discern the paracrine control of endothelium on contractility in response to calcium import. The internal stores of Ca2+ were depleted by repeated alpha 1-adrenergic stimulation with phenylephrine (PE) and refilling of these stores was prevented by cyclopiazonic acid (CPA) and/or thapsigargin (TG), two known inhibitors of Ca2+ATPase, the enzyme that drives sarcoplasmic calcium pumps. The two components of tonic muscular contraction, T I and T II, which are known to be due to the flow of Ca2+ from the extracellular gradient controlled via the poly-phosphoinositide cascade and nifedipine sensitive Ca2+ channels were found to be variable among these strains. Implications of these variations are discussed in this report

Acetylcholinesterase: an enzymatic marker of human red blood cell aging.

The purpose of this investigation was to determine whether acetylcholinesterase (AChE) can be used as a marker of cell aging in human red blood cells (RBCs). This study used consented subjects; both males and females in an age range of 21-42 years. The blood samples (8-9 mL) were drawn in tubes containing sodium heparin or EDTA as anticoagulants. To avoid contamination with other cells, (lymphocytes, monocytes and reticulocytes), RBCs were purified (PRBC) by Hypaque-Ficoll gradient technique. The PRBCs were subfractionated into young (y) (1.08-1.09), mid (m) (1.09-1.11) and old (o) (1.11-1.12) percoll density (g/mL) fractions using a discontinuous percoll gradient. The mean +/- 1 SD AChE per gram hemoglobin (U/g Hgb) activities in whole blood (WB) purified human red blood cells (PRBCs), young human red blood cells (y-RBCs), mid age human red blood cells (m-RBCs) and old human red blood cells (o-RBCs) were 27.4 +/- 2.98, 26.0 +/- 2.33, 25.5 +/- 1.64, 20.3 +/- 3.84, 14.6 +/- 3.42 in males and 26.3 +/- 4.44, 24.8 /- 4.83, 26.4 +/- 4.59, 24.0 +/- 5.50 and 12.4 +/- 7.09 in females respectively. Although there was variation in the data, the results indicated that old human red blood cells showed significantly (p<.05) lower AChE activity compared to young human red blood cells of both sexes. These preliminary but novel observations suggest that AChE can be an excellent enzymatic marker for RBC aging in man.

Resilience in siblings of children with sickle cell disease.

This pilot study was conducted to identify factors responsible for promoting resilience in siblings of children with sickle cell disease. Twenty siblings (10-17 years of age) of children (5-13 years) with sickle cell disease were selected from the Pediatric Clinic of Howard University Center for Sickle Cell Disease. The siblings responded to questionnaires, and the data obtained was analyzed by chi-square for association. The results indicated that age, birth order, and gender had no effect on resilience in the siblings. However, family size, number of parents in the home, sibling's knowledge of the illness, degree of morbidity of the illness, socioeconomic status of the family, and parents' attitudes and childrearing practices were all found to affect resilience. These findings provide additional insight into the psychosocial aspects of, and genetic counseling for sickle cell disease, as well as for other chronic genetic disorders.

In vitro mutagenicity of water contaminants in complex mixtures.

Trihalomethanes, Carbon tetrachloride and trichloroethylene were tested in single, binary and multi-complex mixtures using standard tester strains TA1535, TA1537, TA98 and TA100 of Salmonella typhimurium with and without addition of an in vitro metabolizing fraction S-9. Chloroform (CHCl3) was found to be mutagenic in all strains without S-9 activation. However, when tested with Bromoform (15%), which was nonmutagenic singly, the combined effect of the mixture was nonmutagenic. CCl4 was a direct mutagen (without S-9) in all strains except TA 1535. When combined with 85% CHCl3, only strains TA1535 and TA1537 were mutagenic. When tested with mammalian activation (S-9), CCl4 was mutagenic in all strains. However, when tested with CHCl3 (CHCl3 and CCl4-85:15), the mutagenic capability was lost. With or without S-9 Activation multi-complex mixture of CHCl3, CCl4 and TCE (85:8:7) was mutagenic for a narrow range of doses in all strains.

Tissue specific regulation of renal N-nitrosodimethylamine-demethylase activity by testosterone in BALB/c mice.

Nitrosodimethylamine (NDMA), like several other nitrosamines, is activated by the enzymes--mixed-function oxidases--present in the tissue microsomal fractions, producing mutagenic and carcinogenic effects. Previous studies in BALB/c mice have shown an age, sex and androgenic regulation of NDMA-induced mutagenicity. The present study was designed to test the correlation between renal NDMA-demethylase activity and previously published reports on NDMA-induced mutagenicity. Renal and hepatic NDMA-demethylases were determined from the microsomal fractions by quantitating formaldehyde. Renal NDMA-demethylase showed the presence of two isozymes, I and II, with Km values of 0.6 +/- 0.2 and 20.2 +/- 6.8 mM respectively. Isozyme I was detected in adult males and first appeared at the onset of puberty; it was absent in adult females and in immature mice. Renal isozyme II was detected in both males and females and was independent of age. Testosterone treatment of adult females resulted in the appearance of renal isozyme I. Castration of adult males caused a dramatic decrease in activity, whereas testosterone administration to such castrates increased activity, of renal isozyme I. Hepatic NDMA-demethylase activities were independent of age, sex or testosterone treatment. In conclusion, these results show an age, sex and tissue specific regulation of renal NDMA activity. Renal and hepatic NDMA-demethylase activities correlated positively with earlier studies on NDMA-induced mutagenesis and carcinogenesis.

Regulatory effects of testosterone and 17 beta-oestradiol on the metabolism of dimethylnitrosamine by renal and hepatic microsomal enzymes from BALB/c mice.

Previous investigations with BALB/c mice have demonstrated that no sex-related differences exist in the ability of liver microsomal fractions (S-9) to biotransform dimethylnitrosamine (DMN) to its active mutagenic metabolites as evidenced by bacterial screening assays. In contrast, kidney microsomal enzymes from adult male BALB/c mice and not from females, castrates, and immature animals, were capable of activating DMN. The present study was designed to test the effects of testosterone and oestradiol on DMN bioactivation by hepatic or renal microsomal enzymes. Mutagenic assays were performed using liver and kidney microsomal enzymes with the histidine deficient mutant Salmonella typhimurium TA100. Results indicate that testosterone treatment of female BALB/c mice resulted in an increase in the ability of their renal microsomal enzymes to metabolize DMN to its active mutagenic intermediates. Renal microsomal enzymes from female mice treated with 17 beta-oestradiol had no effect on DMN metabolism. However, the ability of the renal microsomal enzymes treated with 17 beta-oestradiol to bioactivate DMN was significantly decreased in males.

Mutagenicity of benzo(a)pyrene in uninduced tissues from BALB/c mice and Sprague-Dawley rats as an index of possible health risks using the Salmonella mutagenicity assay.

The mutagenicity of benzo(a)pyrene [B(a)P] in uninduced tissues from Sprague-Dawley rats and BALB/c mice resulted in no age, sex or tissue-related differences when S9 preparations from lung, kidney and spleen were used in the Salmonella mutagenicity assay. Liver S9 fractions from both species resulted in a significantly greater number of His+ revertants (TA98) per plate than observed on the control plates (spontaneous reversion rate). Liver homogenates from adult Sprague-Dawley rats showed a significantly lower potential to activate B(a)P than homogenates from adult BALB/c mice. In both species, male liver microsomal enzymes had a greater potential to activate B(a)P than female microsomal enzymes. These data indicated that in uninduced tissues B(a)P may not be a very powerful mutagenic agent. More detailed in vitro and in vivo studies are needed to assess the precise health risks associated with this environmental pollutant.

Dimethylnitrosamine metabolism: I. In vitro activation of dimethylnitrosamine to mutagenic substance(s) by hepatic and renal tissues from three inbred strains of mice.

The potential of hepatic and renal homogenates from three inbred strains of mice (BALB/c, C57BL and DBA) to activate dimethylnitrosamine (DMN) was investigated. Microsomal enzyme (S-9) preparations of liver and kidney from mature and immature mice were used in the Ames Salmonella mutagenicity assay. No age or sex-related differences in the formation of active mutagenic DMN Metabolites by liver microsomal enzymes were observed within any of the three inbred strains. In contrast, mature male kidney S-9 fractions from all three strains had a significantly greater potential to activate DMN than mature female and immature animals. Testosterone treatment resulted in no apparent changes in the ability of hepatic tissue to biotransform DMN to its mutagenic metabolites among age and sex classes. However, after testosterone treatment, renal microsomal fractions from mature female mice of all three strains did not differ significantly from their male counterparts in their ability to transform DMN to mutagenic metabolites.

Dimethylnitrosamine metabolism: II. In vitro activation of dimethylnitrosamine by hepatic and renal tissues from crosses among BALB/c, DBA and C57BL mice.

Crosses among BALB/c, C57BL and DBA mice were performed to investigate the genetic mechanisms involved in metabolism of DMN by renal and hepatic tissues. Liver S-9 fractions from parental strain DBA had the greatest potential to activate DMN and liver fractions from parental strain BALB/c had the lowest. No age or sex-related differences were observed within strain. Crossing of either C57BL or DBA to BALB/c mice resulted in F1 hybrids with liver microsomal enzymes that gave results similar to the BALB/c parental strain. There were no sex or age differences within crossbred strains in the potential of liver to activate DMN. In contrast male DBA and C57BL parental mice renal S-9 fractions did not differ significantly from each other but did differ significantly from male BALB/c renal fractions and from female and immature animals of all strains. Crossing of either DBA or C57BL mice with BALB/c mice resulted in male F1 hybrids whose renal S-9 fractions did not differ significantly from males of the parental BALB/c strain. In all instances, male renal S-9 fractions had a significantly greater potential to activate DMN than female or immature animals. F1 DBA X C57BL hybrids had renal S-9 fractions that did not differ significantly from the parental strains. These data suggest that the gene(s) for low DMN metabolism of BALB/c mice are apparently dominant over the genes from both DBA and C57BL. The exact genetic or physiological mechanism needs further elucidation.

Drosophila alcohol dehydrogenase: developmental studies on cryptic variant lines.

Thirty-five cryptic variant lines were used to examine the mechanisms involved in genetic modulation of alcohol metabolism in Drosophila. Late third-instar larval, preemergence pupal, and adult stages cultured at 18 and 28 C were examined. Spectrophotometric analyses for native alcohol dehydrogenase (ADH) activity and residual ADH activity after treatment with guanidine hydrochloride and heat were performed. Differential response of cryptic variants to treatment with the denaturants during development suggested that this variation may have an adaptive significance.

Hormonal regulation of the metabolism of carcinogens in renal tissue of BALB/c mice.

The role of androgens in the regulation of carcinogen metabolism in the renal tissue of BALB/c mice was investigated. Kidney microsomal enzyme preparations from mature and immature animals were used in mutagenic studies using the Ames test. Androgen receptors (cytosolic and nuclear) were also evaluated. The results show that the microsomal enzymes from mature males had greater potential to biotransform dimethylnitrosamine than did the microsomal enzymes from mature females or immature animals. Testosterone treatment of mature females or immature animals resulted in a significant increase in the mutagenic ability of their renal microsomal enzymes. Androgen receptors were detected in kidney cytosols of mature and immature animals (both males and females); however, nuclear androgen receptors were detected only in the mature males. Testosterone treatment resulted in a significant accumulation of nuclear androgen receptors in the kidneys of mature females and immature animals. The relationships among mutagenic activity, androgen receptors, the levels of N-demethylase (an enzyme responsible for conversion of dimethylnitrosamine to its active metabolite), dietary fat, and the carcinogen metabolism are discussed.