Expression of two cytochromes P450 involved in carcinogen activation in a human colon cell line

Cytochrome P450 is known to cause carcinogen activation and correspondingly increased cancer risk in animal models. In order to determine whether P450 in the colon may be involved in cancer development in the human, the human colon cell line LS174T was examined for the presence of various cytochromes P450. Two isozymes of P450 were identified in the human cell line. Expression of P450IAl or IA2 was increased by treatment of the cell line with benzanthracene; the induction was demonstrated by an increase in RNA hybridizing to a probe for P4501Al and by ethoxyresorufin deethylation activity. Western analysis of microsomes isolated from human colon tissue also demonstrated the presence of P4501A1, as well as a form which cross-reacted to an antibody to human P450IIC9. Another isozyme, P450IIE1, was identified by polymerase chain reaction amplification of RNA from LS174T cells. These results underscore the presence of cytochromes P450 in colonic tissue and provide a basis for the involvement of isozyme-specific P450 mediated reactions in carcinogenesis of the colon.Some of the data presented here were taken from a thesis submitted by D.K.H. in partial fulfillment of the requirements for the Ph.D. degree in the University of Texas Graduate School of Biomedical Sciences.     

Evidence for a role of calmodulin in the regulation of prolactin gene expression

Epidermal growth factor (EGF) stimulates prolactin (PRL) gene expression in GH3 cells in a Ca2+-dependent manner (White, B. A., and Bancroft, F. C. (1983) J. Biol. Chem. 258, 4618-4622). The present report shows that the phenothiazine, calmidazolium (compound R 24571), blocks the ability of EGF plus Ca2+ to increase levels of PRL mRNA. Calmidazolium inhibition of this response is dose dependent in the range of 0.05-1.00 microM. Total inhibition of the response was consistently obtained at a level of calmidazolium (0.5 microM) that had no effect on total cytoplasmic RNA synthesis, total cytoplasmic protein synthesis, cell viability, or extent of EGF plus Ca2+-induced cell aggregation. The drug inhibited the increase in PRL mRNA when given immediately before or 48 h after treatment with EGF plus Ca2+. Another calmodulin inhibitor, W13, similarly blocked the ability of EGF plus Ca2+ to stimulate PRL mRNA, whereas the less active analog, W12, had little effect. These results implicate Ca2+-binding proteins such as calmodulin in the mechanism of action of EGF in GH3 cells, and, therefore, provide further evidence for a role of intracellular Ca2+ in the regulation of the expression of a specific eukaryotic gene, the PRL gene.     

Functional properties of palmaris longus muscles of rhesus monkeys transplanted as index finger flexors

This experiment with skeletal muscle autografts in monkeys was designed to retest previous findings that transplanted skeletal muscle can regenerate to a functional degree in primates without predenervation and to test a new hypothesis that increased functional demands on regenerated muscle grafts in monkeys may result in improved functional capacity of the grafts. Rhesus monkey index flexors were replaced with free palmaris longus muscle autografts with microneural anastomoses between the graft motor nerve and the severed profundus motor nerve. One monkey was taught selective index flexion before grafting and continued with this program after grafting to test the effect of training on the graft. Mature grafts were evaluated for in vivo contractile properties and by histology and histochemistry and were compared with a group of normal Rhesus palmaris longus muscles. The results reconfirm the capacity of nonpredenervated monkey skeletal muscle grafts to regenerate and to achieve some contractile ability and suggest that training of free muscle grafts may enhance recovery of their functional and structural properties.     

The mechanism of action of 4-hydroxynonenal in cell injury

The effect of the C9 ketoaldehyde, 4-hydroxynonenal (HNE), a cytotoxic product of lipid peroxidation, on DNA, RNA and protein synthesis has been investigated in cells in culture. Macromolecular synthesis is powerfully inhibited by this agent which readily enters the lipid-rich membranes and is considerably more toxic than the polar ketoaldehyde, methyl glyoxal (MG). The entry of HNE into membranes lowers their glutathione GSH content. This is associated with an increased lipid peroxidation measured in vitro which is blocked by added GSH or alpha-tocopherol. It is proposed that this latter sequence of events is the underlying cause of the cytopathic effect of HNE in cells in culture.     

Reconstitution of cytokeratin filaments in vitro: further evidence for the role of nonhelical peptides in filament assembly

The in vitro renaturation and assembly of cytokeratin molecules to form intermediate filaments (IF) illustrates that these molecules contain all of the structural information necessary for IF information. These molecules contain nine structural domains: the amino- and carboxyterminal extra helical regions, and three conserved extra helical segments that separate four helical rod-like domains. Chymotrypsin treatment of these molecules removes the end-peptide domains and inhibits the self-assembly process. We have examined the renaturation and assembly of cytokeratin molecules using solution conditions that favor the presence of intermediate forms of IF organization. Dialysis against low salt buffers revealed the presence of bead-like chains of filaments in which the 6-8-nm beads are separated by a distance of 21 nm. These data suggest that a lateral stagger of protofilaments was among the primary events in IF assembly. Chymotrypsin-modified cytokeratin enriched for alpha-helix barely initiated a turbidity increase at conditions favoring self-assembly. Addition of small amounts of intact cytokeratin accelerated the rate and extent of this reaction. These results indicate that the nonhelical peptides on intact cytokeratin potentiate the assembly of IF by orientating the stagger of laterally associated protofilaments.     

Changes with time in the distribution of virus and PR protein around single local lesions of TMV infected tobacco

Summary ELISA was used to determine PR la protein and TMV accumulation in local necrotic lesions produced on salicylic acid and water sprayed Nicotiana tabacum cv Xanthi-nc leaves. The amount of PR la protein produced is the result of an interaction between the salicylic acid treatment and lesion growth. The implication of these observations for experiments investigating the relationship between PR proteins and resistance are discussed.The distribution of TMV and PR la protein in and around single local necrotic lesions up to 14 days after inoculation was measured by ELISA. The highest concentration of TMV was in the centre of the lesion and decreased rapidly with distance from the centre. In contrast there was very little PR la protein in the centre of the lesion, the largest amounts were just outside the centre, and the concentration then decreased with distance from the centre. This is the distribution that might be expected for a substance closely associated with the restriction of virus spread.     

Comparison of fatty acid content and DNA homology of the filamentous gliding bacteriaVitreoscilla,Flexibacter, Filibacter

DNA hybridization experiments showed that there was a high degree of homology amongVitreoscilla strains but not with DNA fromFilibacter limicola. Flexibacter spp were much more heterogeneous indicating a low genetic similarity. These results were also reflected in the membrane fatty acids of the bacteria. TheVitreoscilla strains were very similar with the 16:17c fatty acid being dominant. The membrane fatty acids ofF. limicola were dominated by a15:0 and a17:0 components which provided additional support for its relatedness to the genusBacillus. There was much greater diversity in the fatty acid patterns of theFlexibacter spp.F. aurantiacus, F. ruber andF. elegans shared the common dominant fatty acids 16:17c with theVitreoscilla strains, but this was replaced by the 16:16c acid inF. flexilis. F. ruber was distinguished by the absence of branched odd-chain monounsaturated fatty acids andF. elegans by the dominance of the -OH i15:0 acid. Precise determination of fatty acid double bond positions and geometry are essential for correct interpretation of increasingly complex ecological and taxonomic data sets.