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.     

Mapping of the MYC gene to band 8q24.12----q24.13 by R-banding and distal to fra(8)(q24.11), FRA8E, by fluorescence in situ hybridization

The MYC gene was mapped to R-banded human prometaphase chromosomes and to chromosomes expressing fra(8)(q24.11) by fluorescence in situ hybridization. By high-resolution banding analysis, the fluorescent signals were localized to R-positive band q24.12----q24.13 of the long arm of chromosome 8. Furthermore, the signals were localized near the middle part, q24.12----q24.13, of the distal portion of fra(8)(q24.11) expression. Thus, the precise localization of MYC was to the subband 8q24.12----q24.13.     

The platelet open canalicular system: a final common pathway.

Channels of the surface-connected, open canalicular system (OCS) of human platelets serve as the pathway for transport of substances into the cells and as conduits for the discharge of alpha granule products secreted during the platelet release reaction. The purpose of the present study was to determine if both functions of the OCS can take place simultaneously. Suspensions of washed platelets were exposed to thrombin at 1 U/ml for 5, 60, or 180 seconds in the presence of fibrinogen molecules coupled to particles of colloidal gold (Fgn/Au). The samples were fixed in a low concentration of glutaraldehyde and embedded in L.R. White resin to preserve antigenicity. Thin sections were exposed to a rabbit polyclonal antibody to human fibrinogen followed by an anti-rabbit IgG coupled to 5-nm gold beads. Thrombin caused Fgn/Au particles to bind to platelets and enter channels of the surface-connected OCS. Endogenous fibrinogen detected by immunogold 5-nm beads were localized to alpha granules in resting platelets and 5 seconds after thrombin stimulation. At 60 seconds and 3 minutes Fgn/Au particles were present in swollen alpha granules, as well as OCS channels. Fibrinogen gold beads were evident in alpha granules and OCS channels connected to the platelet surface. The 18- to 20-nm Fgn/Au particles were in the same channels of the OCS as fibrinogen gold beads. The OCS is a final common pathway for uptake of particulates and discharge of secretory products in thrombin-activated human platelets.     

Sequence of the gene encoding phosphoglycerate mutase from Saccharomyces cerevisiae

The gene encoding yeast phosphoglycerate mutase was isolated, and its sequence was determined. The gene specifies a protein of 246 amino acids, and contains no introns. The sequence shows a strong codon bias. The upstream untranslated portion of the gene contains a CT-rich block such as is found in many highly expressed yeast genes, but does not have the associated CAAG sequence.     

Evidence for a multigene family involved in bile acid 7-dehydroxylation in Eubacterium sp. strain VPI 12708.

Eubacterium sp. strain VPI 12708 is a human intestinal isolate which has an inducible bile acid 7-dehydroxylation activity. At least two cholic acid-induced polypeptides, with molecular masses of 27,000 and 45,000 daltons, respectively, coelute with bile acid 7-dehydroxylation activity. The 45,000-dalton polypeptide appears to be encoded by a cholic acid-induced mRNA species of greater than 6 kilobases, which suggests that the gene coding for this polypeptide is part of a larger operon. A gene has been cloned which flanks the gene encoding the 45,000-dalton polypeptide, in the upstream (5') direction. This gene appears to encode a second 27,000-dalton polypeptide. The gene bears striking homology at both the nucleotide (80%) and deduced amino acid sequence (89%) levels with the gene which encodes the 27,000-dalton polypeptide that has been shown previously to be involved in the bile acid 7-dehydroxylation reaction sequence. The implications of this homology and the possible function(s) of the two homologous genes in bile acid 7-dehydroxylation are discussed. Evidence is presented which suggests that the two homologous genes involved in bile acid 7-dehydroxylation may be part of a larger multigene family in Eubacterium sp. strain VPI 12708.     

Mineralization of Trichloroethylene by Heterotrophic Enrichment Cultures

Microbial consortia capable of aerobically degrading more than 99% of exogenous trichloroethylene (TCE) (50 mg/liter) were collected from TCE-contaminated subsurface sediments and grown in enrichment cultures. TCE at concentrations greater than 300 mg/liter was not degraded, nor was TCE used by the consortia as a sole energy source. Energy sources which permitted growth included tryptone-yeast extract, methanol, methane, and propane. The optimum temperature range for growth and subsequent TCE consumption was 22 to 37°C, and the pH optimum was 7.0 to 8.1. Utilization of TCE occurred only after apparent microbial growth had ceased. The major end products recovered were hydrochloric acid and carbon dioxide. Minor products included dichloroethylene, vinylidine chloride, and, possibly, chloroform.     

Phosphorylation of insulin-like growth factor I receptor by insulin receptor tyrosine kinase in intact cultured skeletal muscle cells

The interaction between insulin and insulin-like growth factor I (IGF I) receptors was examined by determining the ability of each receptor type to phosphorylate tyrosine residues on the other receptor in intact L6 skeletal muscle cells. This was made possible through a sequential immunoprecipitation method with two different antibodies that effectively separated the phosphorylated insulin and IGF I receptors. After incubation of intact L6 cells with various concentrations of insulin or IGF I in the presence of [32P]orthophosphate, insulin receptors were precipitated with one of two human polyclonal anti-insulin receptor antibodies (B2 or B9). Phosphorylated IGF I receptors remained in solution and were subsequently precipitated by anti-phosphotyrosine antibodies. The identities of the insulin and IGF I receptor beta-subunits in the two immunoprecipitates were confirmed by binding affinity, by phosphopeptide mapping after trypsin digestion, and by the distinct patterns of expression of the two receptors during differentiation. Stimulated phosphorylation of the beta-subunit of the insulin receptor correlated with occupancy of the beta-subunit of the insulin receptor by either insulin or IGF I as determined by affinity cross-linking. Similarly, stimulation of phosphorylation of the beta-subunit of the IGF I receptor by IGF I correlated with IGF I receptor occupancy. In contrast, insulin stimulated phosphorylation of the beta-subunit of the IGF I receptor at hormone concentrations that were associated with significant occupancy of the insulin receptor but negligible IGF I receptor occupancy. These findings indicate that the IGF I receptor can be a substrate for the hormone-activated insulin receptor tyrosine kinase activity in intact L6 skeletal muscle cells.     

Photolabeling of membrane-bound Torpedo nicotinic acetylcholine receptor with the hydrophobic probe 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine

The hydrophobic, photoactivatable probe 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ([125I]TID) was used to label acetylcholine receptor rich membranes purified from Torpedo californica electric organ. All four subunits of the acetylcholine receptor (AChR) were found to incorporate label, with the gamma-subunit incorporating approximately 4 times as much as each of the other subunits. Carbamylcholine, an agonist, and histrionicotoxin, a noncompetitive antagonist, both strongly inhibited labeling of all AChR subunits in a specific and dose-dependent manner. In contrast, the competitive antagonist alpha-bungarotoxin and the noncompetitive antagonist phencyclidine had only modest effects on [125I]TID labeling of the AChR. The regions of the AChR alpha-subunit that incorporate [125I]TID were mapped by Staphylococcus aureus V8 protease digestion. The carbamylcholine-sensitive site of labeling was localized to a 20-kDa V8 cleavage fragment that begins at Ser-173 and is of sufficient length to contain the three hydrophobic regions M1, M2, and M3. A 10-kDa fragment beginning at Asn-339 and containing the hydrophobic region M4 also incorporated [125I]TID but in a carbamylcholine-insensitive manner. Two further cleavage fragments, which together span about one-third of the alpha-subunit amino terminus, incorporated no detectable [125I]TID. The mapping results place constraints on suggested models of AChR subunit topology.     

Platelet-activating factor induces glycogen degradation in fetal rabbit lung in utero

The role of platelet-activating factor (PAF, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in initiating glycogen breakdown in the fetal rabbit lung was assessed by intraperitoneal administration of this potent ether-linked glycerophospholipid. Forty-five min after in utero injection of PAF (2.5 X 10(-7) mol), fetal pulmonary and hepatic glycogen concentrations were reduced from 326 to 256 and from 9.8 to 6.6 micrograms of glycogen/mg protein, respectively. Glycolytic activity was similarly increased as judged by an elevation of lactate (2-fold) in lung, liver, and plasma upon PAF injection. These actions of PAF were dose- and time-dependent. The glycogenolytic response did not occur when an equimolar dose of the inactive enantiomer, D-PAF was injected. Pretreatment of the fetus with a specific PAF receptor antagonist, SRI-63-441, prevented the PAF response. We have previously demonstrated (Hoffman, D. R., Truong, C. T., and Johnston, J. M. (1986) Biochim. Biophys. Acta 879, 88-96) that PAF biosynthesis and PAF concentrations increase significantly on day 24 of fetal rabbit lung development. A concurrent decrease in pulmonary glycogen concentration at this point of gestation is potentially reflective of the PAF-induced action. Thus, these observations would suggest a role for PAF in the normal physiology of fetal lung maturation.     

Mixtures of a series of homologous hydrophobic peptides with lipid bilayers: a simple model system for examining the protein-lipid interface

The interactions of several members of a homologous series of peptides with the phospholipid bilayer have been examined by using fluorescence and deuterium NMR spectroscopy, differential scanning calorimetry, and measurements of water-to-bilayer partition coefficients. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers and tripeptides of the form Ala-X-Ala-O-tert-butyl are used as a model system to probe the influence of amino acid side-chain substitution on the insertion of peptides into membranes and the behavior of peptide/bilayer mixtures. Tripeptides with X = Gly, Ala, Phe, and Trp have been examined. All of the tripeptides are water soluble, and all partition into DMPC bilayer vesicles to some extent. The Gly-containing peptide is the least soluble and the Trp-containing peptide the most soluble in the bilayer. The extent of perturbation of the bilayer structure induced by the peptides parallels their bilayer solubility: the Gly and Ala peptides act as simple impurities while peptides containing bulky aromatic rings cause a phase separation. Changes in the fluorescence properties of the Trp analogue upon incorporation into the bilayer indicate that the Trp side chain is probably immersed in the hydrocarbon region of the bilayer. Peptides of this form should serve as easily modifiable model systems with which to examine details of how the bilayer environment affects peptide conformation, as well as how hydrophobic peptides affect the bilayer structure.