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PubMedDate not supplied

Exposure to methyl tertiary-butyl ether from oxygenated gasoline in Stamford, Connecticut.

In 1993, state health officials in Connecticut invited the Centers for Disease Control and Prevention (CDC) to assist in an investigation of exposure to methyl tertiary-butyl ether in oxygenated gasoline in Stamford, Connecticut. Venous blood samples were collected from 14 commuters and from 30 other persons who worked in the vicinity of traffic or automobiles, and the samples were analyzed for methyl tertiary-butyl ether, tertiary-butyl alcohol, benzene, m-/p-xylene, o-xylene, and toluene. The highest levels of methyl tertiary-butyl ether in blood were measured among gasoline service station attendants (median = 15 micrograms/l, range = 7.6-28.9 micrograms/l). Blood levels of methyl tertiary-butyl ether were highly variable among persons who worked in car-repair shops (median = 1.73 micrograms/l, range = 0.17-36.7 micrograms/l) and were generally lowest among commuters (median = 0.11 micrograms/l, range = < 0.05-2.60 micrograms/l). Blood levels of methyl tertiary-butyl ether were correlated strongly with personal-breathing-zone samples of methyl tertiary-butyl ether and blood levels of other volatile organic compounds. This exposure information should prove useful to a future risk analysis of this high-volume chemical.

Air Pollutants↗

PubMed1994

The isolation and maintenance of the human pilosebaceous unit.

We have previously developed methods for the isolation and maintenance of human sebaceous glands and hair follicles. However, in long-term culture the maintenance of both is suboptimal. This may be due to a lack of stem cells, which are thought to be located in the bulge area of the hair follicle, and this region is not present in either model. Isolation of the entire pilosebaceous unit would retain this region, and may lead to improved maintenance of both structures. We describe a method for the isolation of viable, individual, pilosebaceous units by microdissection from human scalp face-lift skin. The viability of isolated pilosebaceous units has been determined by light microscopy, patterns of DNA synthesis by [methyl-3H] thymidine autoradiography, and lipogenesis by [U-14C] acetate uptake into lipids. When maintained for 7 days in supplemented Williams E medium, isolated pilosebaceous units showed a significant increase in length. This was due to the production of a keratinized hair fibre which grew at the in vivo rate of 0.3 mm/day. Light microscopy and [methyl-3H] thymidine autoradiography confirmed that after 7 days maintenance the hair follicle retained apparently normal morphology and patterns of DNA synthesis, However, the morphology of the sebaceous gland on maintenance was more variable, generally showing luminal keratinization. Moreover [methyl-3H] thymidine autoradiography of sebaceous glands showed a marked reduction on maintenance. The rates and patterns of lipogenesis by the whole pilosebaceous unit were, respectively, lower and different from those seen with isolated human sebaceous glands; this indicates that the bulk of pilosebaceous lipogenesis is derived from the hair follicle. Rates of recovery of [14C] from 2 mM-[U-14C] sodium acetate into thin-layer chromatography plates after 7 days maintenance decreased, although this was not statistically significant, indicating that rates of lipogenesis may fall on maintenance. Pilosebaceous units were maintained for 7 days on Gelfoam (an absorbable gelatin sponge) at the media-air interface. Initial results show a marked improvement in sebaceous gland morphology. It is possible, therefore, to obtain viable human pilosebaceous units by microdissection, and to maintain them in vitro for up to 7 days, with apparently full retention of hair follicle function, but only partial retention of sebaceous gland function.

Autoradiography↗

PubMed1995

Synergistic effect of intrastriatal co-administration of L-NAME and quinolinic acid.

Chronic dialytic intrastriatal co-administration of quinolinic acid (QUIN) and four concentrations of the nitric oxide synthase (NOS) inhibitor NG nitro-L-arginine methyl ester (L-NAME) produced variable results. Low concentrations of L-NAME (1 microM and 50 microM) co-administered with 15 mM QUIN produced lesions not significantly different from those produced by 15 mM QUIN alone. In contrast, higher concentrations of L-NAME (1 mM and 100 mM) co-administered with 15 mM QUIN produced striatal lesions significantly larger than those produced by 15 mM QUIN alone. Administered by itself, 100 mM L-NAME produced little striatal damage. These findings suggest that low levels of NOS inhibition have little or no effect on NMDA neurotoxicity in the striatum, whereas high levels of NOS inhibition increase NMDA-induced striatal lesion volume.

Analysis of Variance↗

PubMed1995

Interindividual variability in the urinary excretion of inorganic arsenic metabolites by C57 BL/6J mice: possible involvement of a thiol/disulfide exchange mechanism.

The 24-h urine of 75 C57 BL/6J mice injected s.c. with 0.5 mg/kg arsenic as sodium arsenite were examined for creatinine, S-adenosylmethionine (SAM), urea and inorganic arsenic metabolites including inorganic arsenic (ASi), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA). There was interindividual variability of about a 10-fold range in the 24-h urinary excretion of creatinine (80-642 micrograms/24 h, variability (cv) of 33%), SAM (7.5-67.2 micrograms/24 h, cv of 38%), urea (9.5-89.7 mg/24 h, cv of 36%), ASi (0.1-1.6 microgram/24 h, cv of 48%), MMA (0.17-2.1 micrograms/24 h, cv of 50%), DMA (0.73-8.13 micrograms/24 h, cv of 32%) and total arsenic metabolites (1.0-10.4 micrograms/24 h, cv of 31%). Interindividual differences, varying by as much as 5-7-fold, were also found in the urinary proportion of ASi (3-23%, cv of 41%) and MMA (5-22%, cv of 37%), but not in the urinary proportion of DMA (64-90%, cv of 7%). The 24-h urinary excretion of all arsenic metabolites correlated with the 24-h urinary excretion of urea (r = 0.81), creatinine (r = 0.88) and SAM (r = 0.83) as did the 24-h urinary excretion of urea with creatinine (r = 0.94) and SAM (r = 0.86), and the 24-h urinary excretion of creatinine with SAM (r = 0.94). Taken together, these results suggest that the overall intracellular glutathione (GSH)-dependent redox state, as reflected by the 24-h urinary excretion of SAM and creatinine, is involved in the interindividual variability in total arsenic metabolite excretion by C57 BL/6J mice. These preliminary results were also discussed with regard to the involvement of intracellular GSH-dependent redox state in the regulation of the reduction and of the methylation steps of arsenic, and to interindividual variability in the urinary excretion of total arsenic metabolites as a possible complicating factor in the biological monitoring of occupational exposure to arsenic.

Animals↗

PubMed1994

Hypomethylation of CCGG sites in the 3' region of H-ras protooncogene is frequent and is associated with H-ras allele loss in non-small cell lung cancer.

The methylation of MspI/HpaII sites flanking a variable tandem repeat in the 3' region of the c-Ha-ras protooncogene was analyzed in 74 DNA samples of non-small cell lung carcinomas and control lung tissues. Of 39 informative samples, 7 allelic deletions (18%) were found at the c-Ha-ras locus and of these, five (71%) showed hypomethylation of the nondeleted allele. Heterozygous DNA samples without allele loss revealed hypomethylation in 37% (12 of 32). Among 35 homozygotes, 9 showed hypomethylation (26%). We also analyzed c-Ha-ras mutations at codons 12, 13, and 61 by polymerase chain reaction and designed restriction fragment length polymorphism and found no mutation. Thus, c-Ha-ras mutations are not associated with the development of the detected abnormalities. We conclude that hypomethylation at specific sites in the 3' region is associated with loss of heterozygosity for the c-Ha-ras gene in non-small cell lung cancer. The finding that, in informative samples, hypomethylation occurs 2-3 times more frequently than allelic loss suggests that it might be a change contributing or predisposing to a genetic instability that can ultimately lead to c-Ha-ras allelic deletions found in tumor DNA.

Alleles↗

PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗

PubMed1999

N-cadherin-mediated adhesion and aberrant catenin expression in anaplastic thyroid-carcinoma cell lines.

The role of cadherins and catenins in the progression of thyroid carcinoma is unclear. We have investigated alpha-, beta- and gamma-catenins and p120(ctn) in relation to the expression of cadherins in human anaplastic thyroid-carcinoma cell lines (HTh7, HTh74, C643 and SW1736) with Western blotting and immunofluorescence. E-cadherin was lacking except in SW1736, which consisted of E-cadherin-positive (approx. 5%) and -negative cells. The alpha- and beta-catenin levels were similar to those of primary cultured non-neoplastic (E-cadherin-positive) human thyrocytes. In contrast, the expression of gamma-catenin was low and variable, correlating with the different levels of cytokeratin in the same cells (HTh74 > SW1736 > C643 > HTh7). p120(ctn) resolved as a doublet in Western blots; the approximately 100-kDa band also found in non-neoplastic epithelial cells was reduced whereas the approximately 115-kDa band, corresponding to the fibroblast-type isoform of p120(ctn), was neo-expressed. A DNA-demethylating agent, 5-aza-2'-deoxycytidine, up-regulated E-cadherin in SW1736 and gamma-catenin in SW1736 and C643, whereas the other cell lines were unresponsive; other catenins were not affected. The catenins were generally distributed along the cell borders. Immunostaining, cell-surface biotinylation and co-immunoprecipitation revealed that all cell lines expressed N-cadherin in connection with beta-catenin at the plasma membrane. Incubation with an N-cadherin antibody disrupted cell-cell adhesion. We conclude that E-cadherin-negative anaplastic thyroid-carcinoma cell lines display functional N-cadherin/beta-catenin complexes, partial or complete loss of gamma-catenin, and isoform shift of p120(ctn). The unequal expression of E-cadherin and gamma-catenin and the variable response to DNA de-methylation suggest that anaplastic thyroid carcinoma is not a uniform entity.

Cadherins↗

PubMed1983

True histiocytic lymphoma. A report of four cases.

Clinical, morphologic, cytochemical, immunologic, and ultrastructural features of four cases of true histiocytic lymphoma are described. The neoplastic cells were large, ranging from 20 to 45 mu in diameter with round, folded, or convoluted nuclei, and abundant eosinophilic cytoplasm. They exhibited diffuse nonspecific esterase activity. Diffuse acid phosphatase activity was present in two cases so tested. Muramidase activity was present in half of the cases. Finely granular PAS-positive material was seen in the cytoplasm. Methyl green-pyronin positivity was variable. An occasional neoplastic cell showed erythropagocytosis in one case. Malignant cells either contained no cytoplasmic immunoglobulins (three cases) or had immunoglobulins of multiple classes (one case). Surface markers were studied in two cases; they were absent in one case, and were of multiple classes in another case. Ultrastructurally the neoplastic cells had lysosomal granules in three cases so examined, and phagolysosomes, phagocytized material and residual bodies in one of three cases so studied. Patients ranged in age from 28 to 60 years. Two patients had extralymphatic tumors. Survival of more than 5 years was seen in one patient.

Adult↗

PubMed1999

Influences on skatole formation from tryptophan in the pig colon.

Variable amounts of skatole (3-methyl-indole) are formed by microbes out of tryptophan in the colon of pigs. It is resorbed and accumulated in fat, leading to a fecal odor of the meat. We investigated the mechanisms by which differences in diet composition lead to variations in skatole concentrations in blood plasma and fat. The experiments were based on the hypothesis that tryptophan is derived from mucosa cell debris from the small intestine. It was found, that gut cell mitosis is stimulated by the growth factor IGF-I. This factor increases when energy in the diet is high. In addition, the mitotic rate is elevated when high amounts of purines are available in the diet, allowing a more rapid DNA- and RNA-synthesis. Thus, high energy combined with high purines in the diet lead to a remarkable increase of gut cell mitosis which is accompanied by an increase of apoptosis. These apoptotic cells ultimately provide the substrate for skatole formation. In consequence, a dramatically rise of skatole in blood plasma and fat was measurable.

Animals↗

PubMedDate not supplied

Neurophysiological markers as early signs of organophosphate neurotoxicity.

The central and peripheral nervous system effects of acute and subchronic exposure to three organic phosphoro-acid esters (dimethoate, dichlorvos, parathion-methyl) were studied. CNS-dependent variables included mean EEG amplitude, mean frequency of the EEG, and the activity (power density) of six component frequency bands. Peripheral nervous system evaluations included determinations of conduction velocity, and both relative and absolute refractory periods. Cholinesterase activity was measured in blood, brain and other organs. The results indicate that acute large doses of these agents produce substantial changes in these measures of CNS and PNS function. In subchronic experiments it was found that a six weeks administration of 1/50 LD50 of the chemicals induced early functional changes in both the central and the peripheral nervous systems. It is recommended that when cholinesterase inhibition is detected in humans, functional evaluations of CNS and PNS should follow.

Animals↗

PubMed2006

Cardiac regulation in the socially monogamous prairie vole.

Social experiences, both positive and negative, may influence cardiovascular regulation. Prairie voles (Microtus ochrogaster) are socially monogamous rodents that form social bonds similar to those seen in primates, and this species may provide a useful model for investigating neural and social regulation of cardiac function. Cardiac regulation has not been studied previously in the prairie vole. Radiotelemetry transmitters were implanted into adult female prairie voles under anesthesia, and electrocardiographic parameters were recorded. Autonomic blockade was performed using atenolol (8 mg/kg ip) and atropine methyl nitrate (4 mg/kg ip). Several variables were evaluated, including heart rate (HR), HR variability and the amplitude of respiratory sinus arrhythmia. Sympathetic blockade significantly reduced HR. Parasympathetic blockade significantly increased HR, and reduced HR variability and the amplitude of respiratory sinus arrhythmia. Combined autonomic blockade significantly increased HR, and reduced HR variability and respiratory sinus arrhythmia amplitude. The data indicate that autonomic function in prairie voles shares similarities with primates, with a predominant vagal influence on cardiac regulation. The current results provide a foundation for studying neural and social regulation of cardiac function during different behavioral states in this socially monogamous rodent model.

Analysis of Variance↗

PubMed2005

Floral benzenoid carboxyl methyltransferases: from in vitro to in planta function.

Benzenoid carboxyl methyltransferases synthesize methyl esters (e.g., methyl benzoate and methyl salicylate), which are constituents of aromas and scents of many plant species and play important roles in plant communication with the surrounding environment. Within the past five years, eleven such carboxyl methyltransferases were isolated and most of them were comprehensively investigated at the biochemical, molecular and structural level. Two types of enzymes can be distinguished according to their substrate preferences: the SAMT-type enzymes isolated from Clarkia breweri, Stephanotis floribunda, Antirrhinum majus, Hoya carnosa, and Petunia hybrida, which have a higher catalytic efficiency and preference for salicylic acid, while BAMT-type enzymes from A. majus, Arabidopsis thaliana, Arabidopsis lyrata, and Nicotiana suaveolens prefer benzoic acid. The elucidation of C. breweri SAMT's three-dimensional structure allowed a detailed modelling of the active sites of the carboxyl methyltransferases and revealed that the SAM binding pocket is highly conserved among these enzymes while the methyl acceptor binding site exhibits some variability, allowing a classification into SAMT-type and BAMT-type enzymes. The analysis of expression patterns coupled with biochemical characterization showed that these carboxyl methyltransferases are involved either in floral scent biosynthesis or in plant defense responses. While the latter can be induced by biotic or abiotic stress, the genes responsible for floral scent synthesis exhibit developmental and rhythmic expression pattern. The nature of the product and efficiency of its formation in planta depend on the availability of substrates, the catalytic efficiency of the enzyme toward benzoic acid and/or salicylic acid, and the transcriptional, translational, and post-translational regulation at the enzyme level. The biochemical properties of benzenoid carboxyl methyltransferases suggest that the genes involved in plant defenses might represent the ancestor for the presently existing floral genes which during evolution gained different expression profiles and encoded enzymes with the ability to accept structurally similar substrates.

Amino Acid Sequence↗

PubMed2004

Differential contribution of active site residues in substrate recognition sites 1 and 5 to cytochrome P450 2C8 substrate selectivity and regioselectivity.

Selected active site residues in substrate recognition sites (SRS) 1 and 5 of cytochrome P450 2C8 (CYP2C8) were mutated to the corresponding amino acids present in CYP2C9 to investigate the contribution of these positions to the unique substrate selectivity and regioselectivity of CYP2C8. The effects of mutations, singly and in combination, were assessed from changes in the kinetics of paclitaxel 6alpha-hydroxylation, a CYP2C8-specific pathway, and the tolylmethyl and ring hydroxylations of torsemide, a mixed CYP2C9/CYP2C8 substrate. Within SRS1, the single mutation S114F abolished paclitaxel 6alpha-hydroxylation, while the I113V substitution resulted in modest parallel reductions in K(m) and V(max). Mutations in SRS5 (viz., V362L, G365S, and V366L) reduced paclitaxel intrinsic clearance (V(max)/K(m)) by 88-100%. Torsemide is preferentially metabolized by CYP2C9, and it was anticipated that the mutations in CYP2C8 might increase activity. However, methyl and ring hydroxylation intrinsic clearances were either unchanged or decreased by the mutations, although hydroxylation regioselectivity was often altered relative to wild-type CYP2C8. The mutations significantly increased (28-968%) K(m) values for both torsemide methyl and ring hydroxylation but had variable effects on V(max). The effects of the combined mutations in SRS1, SRS5, and SRS1 plus SRS5 were generally consistent with the changes produced by the separate mutations. Mutation of CYP2C8 at position 359 (S359I), a site of genetic polymorphism in CYP2C9, resulted in relatively minor changes in paclitaxel- and torsemide-hydroxylase activities. The results are consistent with multiple substrate binding orientations within the CYP2C8 active site and a differential contribution of active site residues to paclitaxel and torsemide binding and turnover.

Aryl Hydrocarbon Hydroxylases↗

PubMed2003

Isobutene polymerization using a chelating diborane co-initiator.

Lewis acidic diborane 1 (J. Am. Chem. Soc. 1999, 121, 3244-3245) is highly effective for both proton- and cationogen-initiated isobutene polymerization in hydrocarbon media at low temperature. Reactions of diborane 1 with cumyl chloride and cumyl methyl ether were studied by variable-temperature 1H and 19F NMR spectroscopy. At low temperatures stable ion pairs 2a and 2b are formed; at higher temperatures these ion-pairs form phenyl-1,3,3-trimethylindan (3) with concomitant release of HCl to form 1 in the case of 2a or degradation of the anion (2b). Reaction between Ph3C-Cl and diborane 1 resulted in the generation of an ion-pair 4 consisting of the Ph3C cation very weakly associated with the chelated, mu-Cl counteranion as revealed by X-ray crystallography.

Journal Article↗

PubMed2003

Frequent aberrant promoter hypermethylation of O6-methylguanine-DNA methyltransferase and death-associated protein kinase genes in immunodeficiency-related lymphomas.

Aberrant promoter hypermethylation is a mechanism of tumour suppressor gene inactivation. We explored aberrant promoter hypermethylation of multiple genes in 88 human immunodeficiency virus (HIV)-non Hodgkin lymphomas (NHL), 25 post-transplant lymphoproliferative disorders (PTLD) and five common variable immunodeficiency (CVI)-related NHL. Twenty-six of 79 (32.9%) HIV-NHL, eight of 14 (57.1%) PTLD and two of five (40.0%) CVI-NHL showed aberrant hypermethylation of O6-methylguanine-DNA methyltransferase (MGMT). Aberrant hypermethylation of death-associated protein-kinase (DAP-K) occurred in 70 of 84 (83.3%) HIV-NHL, 19 of 25 (72.0%) PTLD and three of five (60.0%) CVI-NHL. These data implicate MGMT and DAP-K hypermethylation in lymphomagenesis of immunodeficient hosts. In particular, promoter hypermethylation of DAP-K represents the most frequent molecular alteration yet identified in immunodeficiency-related lymphomas.

Apoptosis Regulatory Proteins↗

PubMed1985

Simplex optimization of extractive alkylation procedures for organic acids in aqueous samples.

Simplex optimization procedures may be used to optimize the yield from an extractive alkylation procedure. Three variables from the derivatization reaction (pH, methylating agent concentration, and phase transfer agent concentration) were chosen for optimization because they interacted with each other and could not be optimized by conventional means. The yield for methylation of adipic acid was doubled with relatively little effort. This procedure may be easily applied to other systems.

Acids↗

PubMed1981

Uptake and metabolism of noradrenaline by blood vessels of perinephritic hypertensive dogs.

Tissue noradrenaline content as well as uptake and metabolism of tritiated exogenous noradrenaline were studied comparatively, in vitro in mesenteric arteries, and in saphenous veins of normotensive and perinephritic hypertensive dogs. The influence of cocaine, iproniazid and 3'-4'-dihydroxy-2-methyl-propiophenone (U-0521) on these variables was also investigated. The concentration of (-)-7-3H-noradrenaline used was 1.08 microM. No changes were observed in noradrenaline content, uptake and metabolism in saphenous vein strips obtained from normotensive or hypertensive animals. However, in mesenteric artery strips obtained from hypertensive dogs, a marked reduction in endogenous noradrenaline content was observed as well as a reduction in noradrenaline accumulation (20 weeks after surgery). The deamination pattern was also modified in these strips: the formation of DOPEG was markedly diminished and the formation of DOMA was increased. These results agree well with the degeneration of the sympathetic innervation of the mesenteric arteries of hypertensive dogs described by Azevedo et al. (1981).

Animals↗

PubMed2006

Optimization of bilayer floating tablet containing metoprolol tartrate as a model drug for gastric retention.

The purpose of the present study was to develop an optimized gastric floating drug delivery system (GFDDS) containing metoprolol tartrate (MT) as a model drug by the optimization technique. A 2(3) factorial design was employed in formulating the GFDDS with total polymer content-to-drug ratio (X1), polymer-to-polymer ratio (X2), and different viscosity grades of hydroxypropyl methyl cellulose (HPMC) (X3) as independent variables. Four dependent variables were considered: percentage of MT release at 8 hours, T50%, diffusion coefficient, and floating time. The main effect and interaction terms were quantitatively evaluated using a mathematical model. The results indicate that X1 and X2 significantly affected the floating time and release properties, but the effect of different viscosity grades of HPMC (K4M and K10M) was nonsignificant. Regression analysis and numerical optimization were performed to identify the best formulation. Fickian release transport was confirmed as the release mechanism from the optimized formulation. The predicted values agreed well with the experimental values, and the results demonstrate the feasibility of the model in the development of GFDDS.

Absorption↗