A case history. NMR studies of the structure of a small protein, omega-conotoxin MVIIA.
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Amino Acid Sequence↗
Biomedical subjects
A J Edwards
Publications and source records attributed to A J Edwards.
The effects of vitamin C supplementation on biomarkers of oxygen radical generated damage in human volunteers with "low" or "high" cholesterol levels.
A human volunteer study was conducted to test the effect of vitamin C supplementation on biomarkers of oxygen radical-mediated damage in individuals with a range of serum cholesterol levels. A group of 48 non-smokers, 24 men and 24 women, was selected from a panel of over 100 volunteers to give as wide a range of serum cholesterol levels as possible. None of the volunteers was taking medication to control cholesterol levels and they maintained their normal dietary habits so as not to compromise their cholesterol status. Volunteers were allocated to three groups of 16, each consisting of four males with low cholesterol levels (< 6 mmol/L) matched for age and build with four males with high cholesterol levels (> 6 mmol/L) and eight females matched in the same way. A three-treatment, three-treatment period, cross-over design was adopted to take account of any temporal differences in response. The three treatments given were placebo, 60 mg vitamin C/day (the recommended daily allowance) and 6 g vitamin C/day. Each treatment was given for 14 days with 6 weeks between the treatment periods. All procedures were performed to the standards of Good Clinical Practice. Blood samples were taken at the end of each treatment period. Serum was assayed for cholesterol whilst vitamin C, total antioxidant capacity, lipid peroxidation breakdown products and ras p21 protein levels were measured in plasma. Lymphocytes were examined for DNA damage using the Comet assay and chromosome aberration test. The Comet assay was conducted with and without challenge with hydrogen peroxide and the chromosome aberration test with and without challenge with bleomycin. Vitamin C supplementation caused a statistically significant increase in plasma vitamin C concentrations and total antioxidant capacity but did not affect cholesterol levels or ras p21 protein levels. There was a non-significant dose-related decrease in lipid peroxidation breakdown products with vitamin C supplementation. No effect on DNA damage was observed in the Comet assay, either with or without hydrogen peroxide challenge, or in the chromosome aberration test without bleomycin. However, a statistically significant increase in bleomycin-induced aberrations was found after vitamin C supplementation. This may be due to effects of vitamin C on iron status. Comparison of male and female subjects showed statistically significant differences in plasma vitamin C levels, the antioxidant capacity of the plasma and the number of chromosome aberrations induced by bleomycin challenge of lymphocytes in vitro. The results were the same for both low and high cholesterol subjects. This study provides no evidence of a beneficial effect on any of the biomarkers studied of vitamin C supplementation over a short-term supplementation period of 2 weeks in a population of healthy, non-smoking individuals eating a nutritionally adequate diet.
Adult↗
Male-mediated F1 effects in mice exposed to 1,3-butadiene.
We examined the effects on dominant lethality, the incidence of fetal abnormalities and tumour incidence in surviving offspring of acute and subchronic exposure of male mice by inhalation to the industrial monomer, 1,3-butadiene. In the acute study, CD-1 mice were exposed to atmospheres containing 0 (n = 25), 1250 (n = 25) or 6250 ppm (n = 50) for 6 h, and each male was caged 5 days later for 1 week with two untreated virgin females. One of the females was killed humanely on day 17 of gestation. The other was allowed to deliver and rear her litter and the litters were monitored throughout adulthood. The killed female was examined for the number of live foetuses, the number of post implantation deaths (early and late) and the number and type of any gross malformations. In the subchronic study, males were exposed to 0 (n = 25), 12.5 (n = 25) or 1250 (n = 50) for 6 h per day on 5 days per week for 10 weeks and then mated the next morning. Mating and observation details were as for the acute study. Acute exposure to butadiene resulted in only a small decrease in implantations; after 10 weeks' subchronic exposure with either the high or low concentration, however, a wide variety of statistically significant effects was seen. At 1250' ppm, the number of implantations was reduced, dominant lethal mutations were induced, and the incidences of early and late deaths were increased; some of the live foetuses were malformed. The low dose also increased the frequency of malformations and late deaths but it did not affect the number of early deaths. Skeletal examination of malformed foetuses, randomly selected normal litter mates and controls confirmed the abnormalities seen at necropsy in malformed foetuses. However, karyotypic analysis of foetal liver from malformed foetuses, randomly selected normal litter mates and controls showed no karyotypic abnormalities. The number of gross suspected tumours in the F1 adults did not appear to reveal an increase over control values. Thus, butadiene is mutagenic in the germ cells of male mice, as shown by the induction of dominant lethality at 1250 ppm, and the frequencies of late deaths and congenital malformations appear to be increased at the subchronic level of 12.5 ppm and skeletal examination of malformed foetuses confirmed the macroscopic abnormalities.
Abnormalities, Drug-Induced↗
Synthesis and analysis of the enantiomers of calmidazolium, and a 1H NMR demonstration of a chiral interaction with calmodulin.
Calmidazolium [R24571, 1-[bis(4-chlorophenyl)methyl]-3-[2-(2,4-dichlorophenyl)-2-[(2,4- dichlorophenyl)methoxy]ethyl]-1H-imidazolium chloride] is a potent calmodulin inhibitor. This paper describes the synthesis and properties of the enantiomers of calmidazolium from the enantiomers of miconazole [1(N)-(2-(2,4-dichlorobenzyloxy)-2-(2,4 dichlorophenyl))-ethyl imidazole], prepared from the racemate by chiral preparative scale high performance liquid chromatography. Overlap between ligand and protein resonances in the aromatic region of the 1H NMR spectrum of the calmidazolium-calmodulin complexes has been obviated by preparation of the protein with all of its nine phenylalanine rings deuterated (Phe-d5 calmodulin). This has been accomplished by the overexpression of calmodulin derived from Trypanosoma brucei rhodiesiense in E. coli in a medium supplemented with ring-deuterated phenylalanine. The kinetics of binding of each enantiomer are slow on the 1H NMR time scale as judged by the behaviour of the H2 resonance of Histidine-107, which is clearly visible under the sample conditions used. The aromatic spectral regions of the protein-bound (+) and (-) enantiomers contrast strikingly, reflecting differences in bound environment and/or conformation.
Calmodulin↗
Induction of polyploidy in human lymphocytes in vitro by excess adenine, but not by adenosine.
It is known that high levels of DNA precursors can be both clastogenic and mutagenic in cultured cell lines and in vivo. The purpose of the present study was to examine at an observational level the cytogenetic effects of adenine and adenosine in primary human cell cultures. Human peripheral blood lymphocytes from four donors were cultured and treated with a range of concentrations of adenine and adenosine. Although no increase in sister chromatid exchange (SCE) frequency was observed with either compound, there was a statistically significant, dose-related increase in the proportion of polyploid cells in cultures treated with adenine, but not in those treated with adenosine. Some of the polyploid metaphases found after adenine treatment contained diplochromosomes, suggesting that endoreduplication might have been involved in polyploid formation in these cells. It is concluded that a high level of adenine can cause genetic changes in human lymphocytes by interfering with mitosis, perhaps by disturbing the balance of DNA precursor pools.
Adenine↗
Human retinal microglia: expression of immune markers and relationship to the glia limitans.
The immunoreactivity, morphology and relationship to the glia limitans of microglia were investigated in flatmounts and sections of normal human retina, using immunogold histochemistry, electron microscopy (EM), and antibodies directed against CD45, major histocompatability complex class I (MHC-I), MHC-II, and human macrophage antigens. Immunoreactivity was evident for all antibodies tested, including MHC-I, which labeled both microglia and retinal vascular endothelium. Most consistent labeling was obtained using antibodies to CD45, MHC-II, and anti-human macrophage (S22) antigen. Immunoreactive cells were seen in the perivascular space (perivascular cells), where they were closely adherent to the vessel profile, and in the retinal parenchyma (microglia). Some parenchymal microglia were also vessel associated and by EM were seen to be closely related to the glia limitans (paravascular microglia). Paravascular microglia were shown by optical densitometry, to express higher levels of MHC antigens than neighboring, non-vessel associated, parenchymal microglia. In addition, paravascular microglia were macrophage (S22) antigen positive, while other parenchymal microglia did not express macrophage antigens. Quantitative data indicate that similar populations of microglia are immunoreactive to CD45, MHC-I, and MHC-II, while relatively few microglia (approximately 10%) are immunoreactive for human macrophage (S22) antigens, supporting previous suggestions that microglia are a heterogeneous population.
Adolescent↗
The effect of antioxidants on bleomycin treatment in in vitro and in vivo genotoxicity assays.
Antioxidants are thought to be important in protecting against damage from active oxygen species. The effects of the antioxidant nutrients vitamins C and E have been investigated after bleomycin treatment in the Salmonella typhimurium bacterial mutation assay, in the human peripheral lymphocyte chromosome aberration assay, and in the mouse micronucleus assay in peripheral blood and bone marrow cells. There were no protective effects from vitamins C and E in the bacterial mutation assay, but vitamin C and not vitamin E abolished chromosome damaging responses in human peripheral lymphocytes, and both vitamins reduced responses in micronuclei from peripheral blood cells in mice. This would suggest that in human cells in vitro and mouse cells in vivo these vitamins could have a protective role.
Analysis of Variance↗
Production responses to various doses and ratios of estradiol benzoate and trenbolone acetate implants in steers and heifers.
At each of three locations, 400 steers and an equal number of heifers were randomized to 10 treatment groups. The purpose of the studies was to evaluate the response of feedlot steers and heifers to single implants containing a combination of estradiol benzoate (EB) and trenbolone acetate (TBA) at two different ratios each at three doses. The selected ratios corresponded to 1E2(estradiol-17 beta):5TBA and 1E2:10TBA. The two ratios were each tested at three different EB/TBA doses (1:5 at 20/70, 40/140, and 60 mg/210 mg, 1:10 and 14/100, 28/200, and 42 mg/300 mg). The test groups were compared to those given each of the compounds alone (60 mg of EB or 300 mg of TBA), as well as to groups reimplanted with Synovex S or Synovex H implants and untreated controls. Steers (P < .01) and heifers (P < .05) implanted with the 1:10 E2:TBA implants gained faster and had better feed conversion (FC) than their counterparts given 1:5 E2:TBA over the 140-d trial. The results indicated that both estradiol benzoate and trenbolone acetate contributed to the efficacy of the combination implant. Contour plots of ADG and FC indicate that increasing the amount of EB above approximately 36 and 37 mg does not significantly increase the response of steers. The results of these studies indicate that the 28 EB/200 TBA dose is close to optimal for growth promotion and feed conversion in both heifers and steers. In steers, carcass value was increased (P < .01) in all test groups except the group give TBA only. Despite a slight reduction in marbling score and percentage of Choice carcasses, carcasses of steers treated with either 28 mg of EB/200 mg of TBA or 42 mg of EB/300 mg of TBA were more valuable (P < .05) than carcasses from steers in any of the 1:10 ratio EB/TBA groups. Carcass values for groups reimplanted with Synovex S or Synovex H or implanted with EB alone were not significantly different from those for groups implanted with any dose of the 1:10 EB/TBA ratio.
Anabolic Agents↗
Inflammatory response to clostridial vaccines in feedlot cattle.
In 24 individually housed feedlot steers that weighed 275 kg, clostridial vaccines were compared for size of injection site reactions, systemic inflammatory response caused by vaccination, and impact on feed consumption. Vaccines were a bacterin/toxoid containing antigens from multiple clostridial species (7-way), and a Clostridium perfringens type C and D toxoid; sterile physiologic saline solution was used as the control. Injection site reactions were observed in all vaccinated cattle. Differences in size of lesions were found, with cattle given the 7-way vaccine (7-way group) having the largest lesions. The inflammatory-response to vaccination, measured by serum concentration of the acute-phase protein haptoglobin, was significantly (P < 0.05) increased in both vaccinated groups over that of controls. However, the 7-way group had higher postinjection values than did the group given type C and D toxoid. The 7-way group had a significant (P < 0.05) 20% decrease in feed consumption in response to the second injection of vaccine. These findings are indicative of potential negative effects of multiple clostridial vaccinations.
Acute-Phase Reaction↗
The effect of simultaneous exposure to bromodeoxyuridine and methyl methanesulphonate on sister-chromatid exchange frequency in cultured human lymphocytes.
Assessment of genotoxicity in cultured cells or in experimental animals through the measurement of sister-chromatid exchanges (SCEs) commonly requires their simultaneous exposure to both the test agent and bromodeoxyuridine (BrdUrd). This dual exposure could lead to modified responses because of either synergistic or antagonistic interactions. Differences in protocol may also have their effect. There is, for example, time for DNA repair to take place in protocols in which there is separate exposure to the test agent and BrdUrd, such as human genetic monitoring studies. In this study, human lymphocyte cultures have been used to investigate the effect of the duration of simultaneous exposure to the mutagen methyl methanesulphonate (MMS) and to BrdUrd on SCE incidence. There was a direct relationship between SCE frequency and the time of simultaneous exposure to MMS and BrdUrd that was not dependent on either the total culture time or the total time of exposure to BrdUrd. This suggestion of an interaction between MMS and BrdUrd in inducing SCEs has important implications for the interpretation of SCE data in both experimental and human monitoring studies.
Analysis of Variance↗
Male-mediated F1 effects in mice exposed to 1,3-butadiene.
We examined the effects on dominant lethality and the incidence of fetal abnormalities of acute and subchronic exposure of male mice by inhalation to the industrial monomer 1,3-butadiene. Investigation of the effect on tumour incidence in surviving offspring is still in progress. In the acute study, CD-1 mice were exposed to atmospheres containing 0 (n = 25), 1250 (n = 25) or 6250 ppm (n = 50) for 6 h, and each male was caged five days later for one week with two untreated virgin females. One of the females was killed humanely on day 17 of gestation. The other was allowed to deliver and rear her litter; the litters are being monitored for life. The killed female was examined for the number of live fetuses, the number of post-implantation deaths (early and late) and the number and type of any gross malformations. In the subchronic study, males were exposed to 0 (n = 25), 12.5 (n = 25) or 1250 ppm (n = 50) for 6 h per day on 5 days per week for 10 weeks and then mated immediately. Mating and observation were conducted as in the acute study. Acute exposure to butadiene resulted in only a small decrease in implantations; after 10 weeks' subchronic exposure to either the high or the low concentration, however, a wide variety of statistically significant effects was seen. At 1250 ppm, the number of implantations was reduced, dominant lethal mutations were induced, and the incidences of early and late deaths were increased; some of the live fetuses had abnormalities. The low dose also increased the frequency of abnormalities and late deaths, but it did not affect the number of early deaths. Thus, butadiene is mutagenic in the germ cells of male mice, as shown by the induction of dominant lethality at 1250 ppm, and the frequencies of late deaths and congenital abnormalities appear to be increased at the subchronic level of 12.5 ppm.
Abnormalities, Drug-Induced↗
Microbial-dependent gamma delta receptor-bearing T-cell localization?
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Animals↗
LFA-1-dependent OKT3-driven T cell clusters in common variable immunodeficiency.
The triggering of the TCR/CD3 complex by anti-CD3 (OKT3) antibody leads to the formation of T cell clusters. In cultures of T lymphocytes from most normal individuals, the peak of cluster formation occurs at 24 h, but with cells from patients with common variable immunodeficiency (CVI) it was seen earlier at 4-9 h; in addition, the clusters were larger than normal, particularly at 9 h. Cluster formation by CVI and normal cells was dependent on temperature and divalent cations, but did not require Fc receptors. Since OKT3 clustering is known to be dependent on the LFA-1/ICAM-1 adhesion system, the effect of monoclonal antibodies directed against these molecules was tested. A potent inhibitor was the antibody against the common beta chain of the integrin family (CD18), but of four MoAbs against the alpha chains (CD11), three inhibited and one stimulated T cell aggregate formation. Increased expression of LFA-1 or ICAM-1 on CVI patients' T cells could not be demonstrated. The accelerated clustering was therefore probably due to an increase in the proportion of cells carrying the activated form of LFA-1. The formation of large numbers of homotypic lymphocyte clusters might reduce the effective interaction between B and T cells, thus contributing to the depression of immunoglobulin synthesis observed in this disease.
Adolescent↗
Adhesion receptors are differentially expressed on developing thymocytes and epithelium in human thymus.
The thymic microenvironment consists of a network of interrelated cells of epithelial, fibroblastic, endothelial, and hemopoietic origin. Within this environment, the development of specific T-lymphocyte subpopulations partially depends on the selective interaction of T-cell precursors with such cells. Human thymic epithelial cell strains, generated with a defective retroviral vector containing simian virus 40 (SV40) large T antigen and the neomycin resistance gene or by transfection with an SV40 plasmid defective in the origin of replication, provide useful tools for understanding the mechanisms contributing to the control of T-cell maturation. Because interepithelial, epithelial-macrophage, and lymphocyte-epithelial cell interactions are important for thymocyte differentiation, the distribution of integrin and nonintegrin adhesion receptors on these cells and on developing thymocytes in vivo and in vitro has been examined in detail. Our results indicate that the transformed human thymic epithelial cell strains express the common very late antigen (VLA)-beta 1 receptor and unique alpha chains VLA-2, VLA-3, and VLA-6. The cells are also positive for LFA-3 and ICAM-1 and weakly express beta 3, beta 4, and VNR alpha. They do not express the Leu-cellular adhesion molecules (CAM). This phenotypic profile on cultured thymic epithelium generally corresponds to the distribution of integrin and other receptor molecules on thymic epithelial cells in tissue sections. The majority of thymocytes also express the integrin VLA-beta 1 and -beta 2 chains as well as VLA-4, VLA-6, and LFA-1 alpha(L). Three-color flow cytometric analyses show differential levels of expression of these adhesion receptors on human thymocyte subsets. Taken together with the immunohistochemical localization of extracellular matrix molecules, these studies suggest that both the distribution of receptor-ligand pairs and the level of expression of adhesion molecules may influence T-cell development within the thymus.
Antigens, Polyomavirus Transforming↗
A comparison of smokers and nonsmokers with respect to oncogene products and cytogenetic parameters.
Human monitoring studies can be valuable tools for assessing the adverse effects of chemicals. Cytogenetic parameters have been frequently employed but are rarely related directly to possible adverse health effects. Recently, the measurement of oncoprotein levels in plasma has been proposed as a possible and more appropriate indicator of exposure and carcinogenic risk but, unlike chromosome damage, little is known about the effects of possible confounding factors. This study compared the effect of smoking on chromosome aberrations, sister chromatid exchange, and plasma ras oncoprotein levels, in forty humans not otherwise known to be exposed to any specific chemical hazard. No effect was found on any of these end points, with the exception of a moderate, statistically nonsignificant elevation of sister chromatid exchange levels. It is concluded that smoking is unlikely to be a confounding factor in human monitoring studies using oncoprotein levels as an end point.
Chromosome Aberrations↗
Enzymic control of the expression of the X determinant (CD15) in human myeloid cells during maturation: the regulatory role of 6-sialytransferase.
To establish the basis for the reduced expression of the X determinant on leukemic blasts and the changes in antigenic expression that occur during myeloid maturation, the presence on myeloid cells of X and related structures was examined in conjunction with studies on the activities of the glycosyltransferases involved in their biosynthesis. Expression of X and sialyl-X was weak on blasts in comparison with neutrophils despite the presence of the requisite precursor structures. Much higher levels of 3-fucosyltransferase activity were found in blasts than in neutrophils when nonsialylated substrates were used, but, whereas the enzyme in neutrophils reacted equally well with 3'-sialylated and nonsialylated acceptors, the enzyme in blasts showed a marked preference for nonsialylated substrates. 6'-Sialyltransferase activity was strong in blasts but was not detectable in neutrophils, whereas a much lower level of 3'-sialyltransferase activity was present in both blasts and neutrophils. Dimethyl sulfoxide-induced maturation of HL60 cells was associated with (1) a decrease in both 6'-sialyltransferase and 3-fucosyltransferase activities, (2) a change in the substrate specificity of 3-fucosyltransferase towards that found in mature cells, and (3) increased cell surface expression of sialyl-X. These results suggest that the reduced expression of X in myeloblasts is related to the presence of the strong 6'-sialyltransferase, which uses the precursor substrate at the expense of the 3-fucosyltransferase and prevents the synthesis of X and sialyl-X. The developmental regulation of the levels of 3'- and 6'-sialyltransferases, and the level and specificity of the 3-fucosyltransferases, therefore controls the expression of X and its degree of sialylation.
Acute Disease↗
A human glioma cell line retaining expression of GFAP and gangliosides, recognized by A2B5 and LB1 antibodies, after prolonged passage.
A malignant astrocytoma was cultured from a tissue biopsy taken at surgical resection, and serially passaged 65 times over a period of 4 years. Early culture cells showed a variety of morphological types, but were all positively labelled with a polyclonal antibody directed against glial fibrillary acidic protein (GFAP). As the number of passages increased small, GFAP-positive cells became the predominant cell type. Flow cytometry demonstrated changes in GFAP expression between early and late passages; early passages showing greater fluorescence intensity than those at later passages. All cells, however, remained positive for GFAP. In addition, this line (IPSB-18) was both vimentin and glutamine synthetase positive and no change in the expression of these two proteins was detectable on continued sequential passaging. Fibronectin was not detectable at any stage. As the number of passages increased, the population doubling time was shortened from 72 h at passage 5, to 28 h at passage 30. This line is unusual in its retention of GFAP expression through 65 sequential passages; the majority of astrocytoma lines lose this capacity after a few passages in vitro. The monoclonal antibody, A2B5, which recognizes surface gangliosides, has been previously shown to identify sub-set type 2 astrocytes in normal neural tissue in vitro. The expression of A2B5 gangliosides by 5-20% of GFAP-positive IPSB-18 cells, and their co-expression with the ganglioside GD3 identified by the LB1 monoclonal antibody, shows that cells with a similar phenotype to the type 1 and type 2 astrocytes of optic nerve are present in cultured gliomas, even after long term sequential passaging.
Antibodies, Monoclonal↗