Acetone-regulated synthesis and degradation of cytochrome P450E1 and cytochrome P4502B1 in rat liver [corrected

The regulation of CYP2E1 and 2B1 was studied by following mRNA levels, catalytic activities and the subcellular distribution of the apoproteins in rat liver 0, 6, 12, 24, 48 and 96 h after a single intragastric dose of acetone. No changes were observed in hepatic CYP2E1 mRNA levels at any time after acetone treatment, whereas rapid rises were observed in the microsomal amount of CYP2E1 protein and CYP2E1-catalyzed 4-nitrophenol hydroxylase and carbon-tetrachloride-initiated lipid-peroxidation activities. However, CYP2E1-dependent catalytic activities declined much faster than the immunodetectable CYP2E1 protein, suggesting that this cytochrome P-450 is inactivated prior to degradation. Similar results were seen in primary hepatocyte cultures. By contrast, concomitant changes in levels of CYP2B1 and CYP2B1-dependent O-depentylation of pentoxyresorufin were observed in the same microsomal preparations. Investigation of the degradative mechanism of both CYP2E1 and CYP2B1 by immunoquantitation of the proteins in lysosomes and by immunohistochemistry indicated their degradation via an autophagic-lysosomal pathway. The data suggest that CYP2E1 is acutely inactivated in the endoplasmic reticulum and that degradation of this isozyme occurs, at least in part, by the lysosomal route. By contrast, CYP2B1 is principally controlled at the level of synthesis.     

In situ generation of iminodiacetic acid groups on nanoporous alumina for the reversible immobilization of enzymes and other biomolecules

Nanoporous alumina membranes were silanized with aminopropylsilane and iminodiacetic acid (IDA) groups were generated in situ by reaction with iodoacetate. The membranes were mounted in standard filter holders, connected to a HPLC system and saturated with selected metal ions. Cu(II) allowed the capture of chicken muscle lactate dehydrogenase with such stability, repeatability and reproducibility that Michaelis–Menten kinetics could be studied. The IDA surface was stable for months and could be depleted and regenerated with metal ions multiple times without appreciable loss of capacity. The binding of lactate dehydrogenase influenced the backpressure to the extent that could be expected for a monolayer according to Poiseuilles law.     

The opioid systems - Panacea and nemesis

This mini-review outlines the opioid systems and their roles primarily as related to reward and compulsive drug/alcohol intake. The central role is taken by the mu-opioid receptor, target for opiate analgesics and also a central target in compulsive alcohol abuse, alcoholism. The mu-opioid receptor and the cognate opioid neuropeptides from proenkephalin and proopiomelancortin are members of a superfamily of opioid systems, each with unique and still to be defined roles in the central nervous system.     

A linkage group on pig chromosome 4 comprising the loci for blood group L, GBA, ATP1B1 and three microsatellites

Restriction fragment length polymorphisms (RFLPs) were described for the porcine loci for β-glucosidase (GBA) and the β-polypeptide 1 of the Na⁺, K⁺-transporting ATPase (ATP1B1). Linkage analyses using a three-generation pedigree provided evidence for the assignment of ATP1B1, GBA and two microsatellite loci (S0001 and S0067) to a previously described linkage group comprising the loci for blood group L (EAL) and an anonymous microsatellite (S0097). The linear order of the six markers was determined with confidence by multipoint analyses and the length of the linkage group was estimated at 88 CM. This linkage group was assigned to pig chromosome 4 on the basis of a previous physical localization of the ATP1B1 gene. In situ hybridization data for S0001 presented in this study were consistent with a localization on chromosome 4 and suggested a regional localization to 4pl2-pl3. The present study reveals conflicting data concerning the genetic localization of the K88 loci controlling the expression of the receptors for the E. coli pilus antigens. One group has reported data suggesting a loose linkage between K88 and EAL, now mapped to chromosome 4, whereas two other groups have found linkage between K88 and the transferrin locus (TF), mapped to chromosome 13 by in situ hybridization.     

Mercury in blood cells—Altered elemental profiles

The study was composed of 27 persons that displayed vague symptoms similar to those of the victims of Minamata and Iraq. Skew distributions of mercury were observed in individual erythrocytes and neutrophil granulocytes when measured by PIXE. Mercury could not be detected in the platelets. The erythrocytes also displayed lowered concentrations of magnesium and zinc, together with increased concentrations of calcium and strontium. The neutrophils displayed decreased concentrations of magnesium and zinc and increased concentrations of calcium, strontium, and iron. The presence of mercury and the altered elemental profiles in the erythrocytes and the neutrophil granulocytes are suggested as early signs of exposure.     

Lipid bilayer polypeptide interactions studied by molecular dynamics simulation

A model membrane with a polypeptide alpha-helix inserted has been simulated by molecular dynamics at a temperature well above the gel/liquid crystalline phase transition temperature. Order parameters of the lipids and other equilibrium and dynamic quantities have been calculated. Three systems, polyglycine constrained into an alphahelical configuration, glycophorin with similarly conformationally constrained backbone and finally glycophorin free to change its backbone conformation, have been studied. In all cases there was an ordering of the chains close to the helix. This effect was, however, much smaller for glycophorin with its rather bulky side chains than for polyglycine. The dynamics of the lipids were affected by the neighbouring helix, not drastically however. Lateral diffusion and reorientational time correlations of lipids close to the helix were slower than for the bulk ones, but not more than two or three times. Thus, we did not find any evidence of bound or frozen boundary lipids.     

Rapid lateral diffusion of lectin-labelled glycoconjugates in the human colonic adenocarcinoma cell line HT29

The lateral diffusion of lectin-labelled glycoconjugates was studied in the human colon carcinoma cell line HT29 using fluorescence photobleaching techniques. HT29 cells were grown in either Dulbecco's modified Eagle's medium with glucose (25 mM; DMEM-Glu) or with galactose (25 mM; DMEM-Gal). Cell cultivation in the DMEM-Gal medium was assumed to promote a transformation of the cells to become small-intestinal-like with characteristic microvilli and associated enzymes. The diffusion of glycoconjugates labelled with fluoresceinated Triticum vulgaris agglutinin (Wheat germ agglutinin; WGA), Ricinus communis agglutinin-I (RCA-I), Concanavalia ensiformis agglutinin (ConA), Ulex europaeus agglutinin-I (UEA-I) and Arachis hypogaea agglutinin (PNA) was in all cases rapid, with a diffusion constant (D) ranging between 0.4 and 0.8×10^-8 cm² s^-1. As a comparison the diffusion of the fluorescent synthetic lipid analog diI-C₁₄ was characterized by D=0.8 – 1.0 × 10^–8 cm² s^-1. The diffusion of lectin-labelled surface components could not be related to the presence of microvilli on HT29 cells grown in DMEM-Gal, which ought to yield an apparently lower diffusion rate. The results indicate either that surface glycoconjugates in HT29 cells are dominated by glycolipid, or that the labelled glycoproteins are more or less free to diffuse in the plane of the membrane.