Nasal tumors in rats following long-term inhalation exposure to 1,4-dichlorobutene-2 (DCB).

This study was conducted to elucidate the time- and dose-response relationships of long-term, low-level 1,4-dichlorobutene-2 (DCB) inhalation exposure to nasal tumor induction in rats. Male Crl:CD BR rats were exposed 6 hours per day, 5 days week to 0, 0.1, 0.3, or 1.0 ppm DCB for up to 19 months; some rats were sacrificed at various time intervals during the study. After 19 months of exposure, surviving rats were held without treatment for an additional 5 months. Tissues from the respiratory tract, lymph nodes, and brain were evaluated microscopically. Compound-related non-neoplastic lesions were observed in the nasal cavities of rats in the 1.0 ppm group after three months of exposure and in the other two groups after twelve months of exposure. The lesions were progressive in severity and frequency. A statistically significant increase in benign nasal tumors (adenomas) occurred in rats from all three DCB-exposed groups. The adenomas occurred in the respiratory region of the nasal cavity and were first observed in the 1.0 ppm group at study month 10. Malignant nasal tumors occurred in the olfactory region of the nasal cavity and were statistically increased at 1.0 ppm.

Characterization of a split respiratory pathway in the wheat "take-all" fungus, Gaeumannomyces graminis var. tritici.

This article describes the first detailed analysis of mitochondrial electron transfer and oxidative phosphorylation in the pathogenic filamentous fungus, Gaeumannomyces graminis var. tritici. While oxygen consumption was cyanide insensitive, inhibition occurred following treatment with complex III inhibitors and the alternative oxidase inhibitor, salicylhydroxamic acid (SHAM). Similarly, maintenance of a Deltapsi across the mitochondrial inner membrane was unaffected by cyanide but sensitive to antimycin A and SHAM when succinate was added as the respiratory substrate. As a result, ATP synthesis through complex V was demonstrated to be sensitive to these two inhibitors but not to cyanide. Analysis of the cytochrome content of mitochondria indicated the presence of those cytochromes normally associated with electron transport in eukaryotic mitochondria together with a third, b-type heme, exhibiting a dithionite-reduced absorbance maxima at 560 nm and not associated with complex III. Antibodies raised to plant alternative oxidase detected the presence of both the monomeric and dimeric forms of this oxidase. Overall this study demonstrates that a novel respiratory chain utilizing the terminal oxidases, cytochrome c oxidase and alternative oxidase, are present and constitutively active in electron transfer in G. graminis tritici. These results are discussed in relation to current understanding of fungal electron transfer and to the possible contribution of alternative redox centers in ATP synthesis.

Developmental regulation of the plant mitochondrial matrix located HSP70 chaperone and its role in protein import.

Changes in the level of the mitochondrial chaperone mtHSP70 have been investigated in pea (Pisum sativum) leaf mitochondria by Western blot analysis and quantified by scanning densitometry. As pea leaves develop (from 6 days to 30 days of age) the levels of mtHSP70 decrease. Analysis of the levels of the alpha subunit of the F1ATPase show that the levels of this protein remain constant throughout the same developmental period, whereas the levels of the alternative oxidase increase. In vitro import of the alternative oxidase precursor protein into pea leaf mitochondria from day 6 to day 30 leaves and quantification by scanning densitometry indicates that protein import efficiency decreases with increasing maturity of the plant cell. Results are discussed in terms of how changing levels of the mtHSP70 chaperone, as a result of plant cell development, influence the efficiency of protein import.

Characterisation of the paxillin-binding site and the C-terminal focal adhesion targeting sequence in vinculin.

Paxillin and vinculin are cytoskeletal proteins that colocalise to focal adhesions, specialised regions of the cell involved in attachment to the extracellular matrix. These two molecules form part of a complex of proteins that link the actin network to the plasma membrane. Paxillin has been shown to bind directly in vitro to the C-terminal region of vinculin (Turner et al. (1990). J. Cell Biol. 111, 1059-1068), which also contains a focal adhesion targeting sequence (Bendori et al. (1989). J. Cell Biol. 108, 2383-2393). In the present study, we have used a series of vinculin deletion mutants to map more precisely the sites in vinculin responsible for paxillin binding and focal adhesion localisation. A glutathione-S-transferase fusion protein spanning vinculin residues 881-1000 was sufficient to support 125I-paxillin binding in a gel-blot assay while no detectable binding was observed to a fusion protein spanning residues 881-978. Transfection experiments using cDNAs encoding chick vinculin residues 398-1066 and 398-1028 demonstrated that amino acids C-terminal to residue 1028 were not necessary for targeting to focal adhesions. In contrast, a vinculin polypeptide expressed from a cDNA encoding residues 398-1000 failed to localise to focal adhesions in stably transfected NIH3T3 cells. We have therefore identified a region of 50 amino acids (residues 979-1028) within the C-terminal region of vinculin that contains both the paxillin-binding site and the focal adhesion targeting sequence. This region is highly conserved in human and chicken vinculin and is likely to be important in regulation of the assembly of focal adhesions.

Ethylene: receptors and action.

The characterisation and purification of the ethylene binding protein from developing cotyledons of Phaseolus vulgaris is described. Polyclonal antibodies to this protein recognise homologous proteins in peas, tomatoes and Arabidopsis. Direct binding assays and results from immunological studies indicate that more binding protein is present in abscission zones of Phaseolus than in petioles; ethylene treatment increases binding site abundance in abscission zones. Binding sites for ethylene in peas. Arabidopsis and rice are described indicating that there exist two classes differing only in their rate constants of association and dissociation. Arabidopsis mutants wholly insensitive to ethylene may be receptor deficient and their possible use in receptor studies is assessed. It is proposed that those binding sites with high rate constants of association are functional receptors. The sites with low rate constants of association may be receptors but may also represent receptor precursors or internalised receptors.

Alterations in lipid metabolism and cellular morphology in the liver of female rats treated with ethmozine.

Alterations in lipid metabolism and cellular morphology in the liver were examined in female rats treated with 100 mg Ethmozine/kg/day for 7 days. The incorporation of either [3H]acetate with [methyl-14C]choline, or [methyl-14C]methionine was used to monitor the effect of the drug on neutral and phospholipid syntheses. Ethmozine (ETH) reduced the incorporation of choline into phosphatidylcholine (PC) by 50%, but the transmethylation of phosphatidylethanolamine to form PC was unaffected. The formation of lyso-PC was reduced by one-half irrespective of the donor radiolabel. An accumulation of both micro- and macrovesicles (fat) was found in the centri- and midlobular zones of the liver, which is likely the result of increased synthesis and decreased secretion of triacylglycerol (TAG). Incorporation of acetate into TAG was increased fivefold by ETH treatment, and to a lesser degree into cholesterol and cholesterylester/squalene.

Chronic inhalation toxicity/carcinogenicity study in rats exposed to fluorocarbon 113 (FC-113).

Groups of 100 male and 100 female Crl:CDBR rats were exposed by whole-body inhalation to FC-113 (1,1,2-trichloro-1,2,2-trifluoroethane) for 6 hr a day, 5 days a week for 24 months. Average exposure concentrations (+/- 1 SD) were 0.0 (control), 2000 +/- 100, 10,000 +/- 500, and 20,000 +/- 1000 ppm (v/v), respectively. Body weights were consistently lower in both male and female rats in the 20,000 ppm exposure group after approximately 1 and 4 months' exposure, respectively, and in female rats after 12 months' exposure at 10,000 ppm. Observations of appearance and behavior, mortality, and clinical laboratory measurements were unremarkable during the 24-month exposure period. Despite exposure levels as high as 20,000 ppm, only occasional slight increases in urinary fluoride were seen. Microscopic examination of tissues from rats examined during and at the end of the 24-month study revealed no evidence of compound-related toxicity or carcinogenicity. Based mainly on a 5 to 10% decrease in body weight gain at the 10,000 and 20,000 ppm exposure levels, the no-observed-effect level for FC-113 in this study was 2000 ppm.

Guinea pig respiratory response to isocyanates.

Exposure to some isocyanates (e.g., toluene diisocyanate) has been associated with development of respiratory sensitization. In this study, guinea pig respiratory response to protein conjugates of isocyanatoethyl methacrylate (IEM) and isocyanatoethyl propionate (IEP) was evaluated. Guinea pigs were exposed to daily induction exposures with an aerosol of bovine serum albumin (BSA) or BSA conjugated with IEM or IEP. After approximately 2 weeks significant increases in respiratory rate occurred in the guinea pigs exposed to the isocyanate conjugates. The number of animals responding was related to the degree of conjugation of isocyanate to protein. No response to unconjugated BSA was observed. The isocyanates conjugated to another carrier, guinea pig serum albumin (GSA), elicited responses. In guinea pigs responding to BSA-IEM, 0.01 ppm IEM monomer did not elicit responses; 0.1 to 0.4 ppm IEM vapor elicited responses similar to conjugates but which were delayed; 0.5 and 0.6 ppm induced irritation responses. An IEM polymer aerosol that contained less than 0.004% monomer did not elicit a response. These data suggest a response threshold. Guinea pig developing responses to either of the isocyanate conjugates displayed cross-reactions to challenge with the other. A conjugate of BSA with hexyl isocyanate (HI) did not induce cross-responses in guinea pigs reactive to BSA-IEM. Application of BSA-IEP or IEP monomer to the scratched skin of guinea pigs that responded by inhalation to BSA-IEP resulted in immediate wheal and flare responses not seen in unexposed animals. All of these findings suggest induction of Type I hypersensitivity (asthmatic) directed toward the isocyanate portion of the conjugate and not the protein.

The effect of acute and chronic ethanol intake on hepatic glycerolipid biosynthesis in the hamster.

The effect of acute and chronic ethanol intake on hepatic glycerolipid biosynthesis in the hamster was studied by in vivo and in vitro techniques. The results were compared with those from control hamsters receiving isocaloric amounts of glucose. Both chronic and acute ethanol intake elevated serum and hepatic triglyceride concentrations and induced a rapid rise in the capacity of neutral glycerolipid formation from sn[1,3-14C]glycerol-3-phosphate by hamster liver homogenate and microsomal fractions. Ethanol intake also produced a corresponding increase in the incorporation of [1,3-14C]glycerol into hepatic neutral glycerolipids by the intact animal. The ethanol-induced rise in the capacity of neutral glycerolipid production by liver as measured in vivo and in vitro correlated well with an increase in hepatic phosphatidate phosphohydrolase activity. Therefore, the rise in hepatic and serum triglyceride levels associated with ethanol intake may be explained in part by an increase in the activity of the enzyme.

The effect of ethanol on glycerolipid biosynthesis by primary monolayer cultures of adult rat hepatocytes.

This study evaluates the effects of ethanol exposure on glycerolipid production and release by hepatocyte monolayers. Glycerolipid formation from [1,3-14C]glycerol was increased in monolayers exposed to ethanol (1--50 mM) for 6 h. Monolayers exposed to 1 mM ethanol for 24 h also exhibited a rise in glycerolipid formation from either [1,3-14C]glycerol or [1-14C]palmitate; however, higher ethanol concentrations produced a dose dependent decrease in glycerolipid formation. Glycerolipids released into the medium by monolayers declined after all periods of ethanol exposure. The effects of ethanol on the enzymatic reactions involved in glycerolipid biosynthesis were determined in homogenates prepared from monolayers exposed to ethanol. Two enzymes, phosphatidate phosphohydrolase and glycerol kinase, exhibited ethanol-induced alterations in enzyme activity; however, only glycerol kinase activity correlated well with monolayer glycerolipid formation. These ethanol-induced alterations in enzyme activities and glycerolipid biosynthesis were reduced by simultaneously exposing monolayers to pyrazole or cycloheximide.