Effect of weight reduction on liver histology and biochemistry in patients with chronic hepatitis C

Background: Steatosis occurs in more than 50% of patients with chronic hepatitis C and is associated with increased hepatic fibrosis. In many of these patients the pathogenesis of steatosis appears to be the some as for patients with non-alcoholic fatty liver disease-that is, related to visceral adiposity and obesity. Methods: The effect of a three month weight reduction programme on liver biochemistry and metabolic parameters was examined in 19 subjects with steatosis and chronic hepatitis C. Paired liver biopsies were performed in 10 subjects, prior to and 3-6 months following the intervention, to determine the effect of weight loss on liver histology. Results: There was a mean weight loss of 5.9 (3.2) kg and a mean reduction in waist circumference of 9.0 (5.0) cm. In 16 of the 19 patients, serum alanine aminotransferase levels fell progressively with weight loss. Mean fasting insulin fell from 16 (7) to 11 (4) mmol/l (p

Hepatic xenobiotic metabolism of cylindrospermopsin in vivo in the mouse

Cylindrospermopsin (CYN) is a hepatotoxin isolated from the blue-green alga Cylindrospermopsis raciborskii. The role of both glutathione (GSH) and the cytochrome P450 enzyme system (P450) in the mechanism of toxicity of CYN has been previously investigated in in vitro systems. We have investigated the role of GSH and P450 in vivo in mice. Mice pre-treated with buthionine sulphoximine and diethyl maleate to deplete hepatic GSH prior to dosing with 0.2 mg/kg CYN showed a seven-day survival rate of 5/13 while the control group rate was 9/14. Dosing mice with 0.2 mg/kg CYN produced a small decrease in hepatic GSH with a characteristic rebound effect at 24 h, The magnitude of this effect is however small and combined with the non-significant difference in survival rates after GSH depletion suggest depletion of GSH by CYN could not be a primary mechanism for CYN toxicity, Conversely, pro-treatment with piperonyl butoxide, a P450 inhibitor, protected mice against CYN toxicity giving a survival rate of 10/10 compared with 4/10 in the control group (p < 0.05 Chi squared) and was protective at doses up to 0.8 mg/kg, suggesting activation of CYN by P450 is of primary importance in the mechanism of action. (C) 2002 Elsevier Science Ltd. All rights reserved.

Associations between human liver and kidney cadmium content and immunochemically detected CYP4A11 apoprotein

This present study was undertaken to assess potential effects of cadmium on CYP4A11 apoprotein in human liver and kidney as detected by Western blotting using a highly specific anti-peptide antibody. Liver and kidney cortex samples were autopsy specimens of 37 individuals (26 mates and I I females) whose ages ranged from 3 to 89 years. All were Caucasians who had not been exposed to cadmium in the workplace. Reduced CYP4A11 apoprotein levels were found in chronic hepatitis samples and in liver samples showing fatty changes. In contrast, increased CYP4A11 apoprotein levels were found in liver samples having higher cadmium content compared to the lower cadmium content samples. Increased CYP4A11 levels were also found in liver samples from female donors, compared to male donors; the difference being attributable to higher female liver cadmium burden. In distinction to liver, lowered CYP4A11 levels were seen in the kidney cortex samples which have high cadmium content, It is proposed here that the difference between the absolute cadmium burden of the liver and kidney samples may be responsible for the different patterns of expression of CYP4A11 in these two tissues. Further, since cadmium exposure may be associated with derangement in blood pressure control, it is interesting to note the possible relationship between altered CYP4A11-dependent production of arachidonic acid hydroxy and epoxy metabolites in kidney cortex and altered control of blood pressure. Our findings provide a possible link between these observations. (C) 2002 Elsevier Science Inc. All rights reserved.

Single-chain antibodies produced by phage display against the C-terminal 19 kDa region of merozoite surface protein-1of Plasinodium yoelii reduce parasite growth following challenge

Antibodies have the potential to be therapeutic reagents for malaria. Here we describe the production of a novel phage antibody display library against the C-terminal 19 kDa region of the Plasmodium yoelii YM merozoite surface protein-1 (MSP1(19)). In vivo studies against homologous lethal malaria challenge show an anti-parasite effect in a dose dependent manner, and analysis by plasmon resonance indicates binding to the antigen is comparable to the binding of a protective monoclonal antibody. The data support the lack of a need for any antibody Fc-related function and hold great significance for the development of a therapeutic reagent for malaria. (C) 2002 Elsevier Science Ltd. All rights reserved.

Antagonists of protein kinase C inhibit rat retinal glutamate transport activity in situ

Neuronal and glial high-affinity transporters regulate extracellular glutamate concentration, thereby terminating synaptic transmission and preventing neuronal excitotoxicity. Glutamate transporter activity has been shown to be modulated by protein kinase C (PKC) in cell culture. This is the first study to demonstrate such modulation in situ, by following the fate of the non-metabolisable glutamate transporter substrate, D-aspartate. In the rat retina, pan-isoform PKC inhibition with chelerythrine suppressed glutamate uptake by GLAST (glutamate/aspartate transporter), the dominant excitatory amino acid transporter localized to the glial Muller cells. This effect was mimicked by rottlerin but not by Go6976, suggesting the involvement of the PKCdelta isoform, but not PKCalpha, beta or gamma. Western blotting and immunohistochemical labeling revealed that the suppression of glutamate transport was not due to a change in transporter expression. Inhibition of PKCdelta selectively suppressed GLAST but not neuronal glutamate transporter activity. These data suggest that the targeting of specific glutamate transporters with isoform-specific modulators of PKC activity may have significant implications for the understanding of neurodegenerative conditions arising from compromised glutamate homeostasis, e.g. glaucoma and amyotrophic lateral sclerosis.

Bioactivation of tamoxifen by recombinant human cytochrome P450 enzymes

Tamoxifen is a major drug used for adjuvant chemotherapy of breast cancer; however, its use has been associated with a small but significant increase in risk of endometrial cancer. In rats, tamoxifen is a hepatocarcinogen, and DNA adducts have been observed in both rat and human tissues. Tamoxifen has been shown previously to be metabolized to reactive products that have the potential to form protein and DNA adducts. Previous studies have suggested a role for P450 3A4 in protein adduct formation in human liver microsomes, via a catechol intermediate; however, no clear correlation was seen between P450 3A4 content of human liver microsomes and adduct formation. In the present study, we investigated the P450 forms responsible for covalent drug-protein adduct formation and the possibility that covalent adduct formation might occur via alternative pathways to catechol formation. Recombinant P450 3A4 catalyzed adduct formation, and this correlated with the level of uncoupling in the P450 incubation, consistent with a role of reactive oxygen species in potentiating adduct formation after enzymatic formation of the catechol metabolite. Whereas P450s 1AI, 2D6, and 3A5 generated catechol metabolite, no covalent adduct formation was observed with these forms. By contrast, P450 2136, 2C19, and rat liver microsomes catalyzed drug-protein adduct formation but not catechol formation. Drug protein adducts formed specifically with P450 3A4 in incubations using membranes isolated from bacteria expressing P450 3A4 and reductase, as well as in reconstitutions of purified 3A4, suggesting that the electrophilic species reacted preferentially with the P450 enzymes concerned.

Metabolism of tamoxifen by recombinant human cytochrome P450 enzymes: Formation of the 4-hydroxy, 4′-hydroxy andN-desmethyl metabolites and isomerization oftrans-4-hydroxytamoxifen

The cytochrome P450 (P450)-mediated biotransformation of tamoxifen is important in determining both the clearance of the drug and its conversion to the active metabolite, trans-4-hydroxytamoxifen. Biotransformation by P450 forms expressed extrahepatically, such as in the breast and endometrium, may be particularly important in determining tissue-specific effects of tamoxifen. Moreover, tamoxifen may serve as a useful probe drug to examine the regioselectivity of different forms. Tamoxifen metabolism was investigated in vitro using recombinant human P450s. Forms CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7 were coexpressed in Escherichia coli with recombinant human NADPH-cytochrome P450 reductase. Bacterial membranes were harvested and incubated with tamoxifen or trans-4-hydroxytamoxifen under conditions supporting P450-mediated catalysis. CYP2D6 was the major catalyst of 4-hydroxylation at low tamoxifen concentrations (170 +/- 20 pmol/40 min/0.2 nmol P450 using 18 muM tamoxifen), but CYP2B6 showed significant activity at high substrate concentrations (28.1 +/- 0.8 and 3.1 +/- 0.5 nmol/120 min/0.2 nmol P450 for CYP2D6 and CYP2B6, respectively, using 250 muM tamoxifen). These two forms also catalyzed 4'-hydroxylation (13.0 +/- 1.9 and 1.4 +/- 0.1 nmol/120 min/0.2 nmol P450, respectively, for CYP2B6 and CYP2D6 at 250 muM tamoxifen; 0.51 +/- 0.08 pmol/40 min/0.2 nmol P450 for CYP2B6 at 18 muM tamoxifen). Tamoxifen N-demethylation was mediated by CYP2D6, 1A1, 1A2, and 3A4, at low substrate concentrations, with contributions by CYP1B1, 2C9, 2C19 and 3A5 at high concentrations. CYP1B1 was the principal catalyst of 4-hydroxytamoxifen trans-cis isomerization but CYP2B6 and CYP2C19 also contributed.

Use of heterologously-expressed cytochomre P450 and glutathione transferase enzymes in toxicity assays

Our groups have had a long-term interest in utilizing bacterial systems in the characterization of bioactivation and detoxication reactions catalyzed by cytochrome P450 (P450) and glutathione transferase (GST) enzymes. Bacterial systems remain the first choice for initial screens with new chemicals and have advantages, including high-throughput capability. Most human P450s of interest in toxicology have been readily expressed in Escherichia coli with only minor sequence modification. These enzymes can be readily purified and used in assays of activation of chemicals. Bicistronic systems have been developed in order to provide the auxiliary NADPH-P450 reductase. Alternative systems involve these enzymes expressed together within bacteria. In one approach, a lac selection system is used with E. coli and has been applied to the characterization of inhibitors of P450s 1A2 and 1131, as well as in basic studies involving random mutagenesis. Another approach utilizes induction of the SOS (umu) response in Salmonella typhimurium, and systems have now been developed with human P450s 1A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4, which have been used to report responses from heterocyclic amines. S. typhimurium his reporter systems have also been used with GSTs, first to demonstrate the role of rat GST 5-5 in the activation of dihalomethanes. These systems have been used to compare these GSTs with regard to activation of dihaloalkanes and potential toxicity. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Locomotion at-1.0 degrees C: burst swimming performance of five species of Antarctic fish

We investigated the burst swimming performance of five species of Antarctic fish at -1.0degreesC. The species studied belonged to the suborder, Notothenioidei, and from the families, Nototheniidae and Bathydraconidae. Swimming performance of the fish was assessed over the initial 300 ms of a startle response using surgically attached miniature accelerometers. Escape responses in all fish consisted of a C-type fast start; consisting of an initial pronounced bending of the body into a C-shape, followed by one or more complete tail-beats and an un-powered glide. We found significant differences in the swimming performance of the five species of fish examined, with average maximum swimming velocities (U-max) ranging from 0.91 to 1.39 m s(-1) and maximum accelerations (A(max)) ranging from 10.6 to 15.6 m s(-2). The cryopelagic species, Pagothenia borchgrevinki, produced the fastest escape response, reaching a U-max and A(max) of 1.39 m s(-1) and 15.6 m s(-2), respectively. We also compared the body shapes of each fish species with their measures of maximum burst performance. The dragonfish, Gymnodraco acuticeps, from the family Bathdraconidae, did not conform to the pattern observed for the other four fish species belonging to the family Nototheniidae. However, we found a negative relationship between buoyancy of the fish species and burst swimming performance. (C) 2002 Elsevier Science Ltd. All rights reserved.

A new species of Phyllurus (Lacertilia : Gekkonidae) and a revised phylogeny and key for the Australian leaf-tailed geckos

Phyllurus gulbaru, sp. nov., is a highly distinct species of leaf-tailed gecko restricted to rocky rainforest of Pattersons Gorge, north-west of Townsville. The possession of a cylindrical, non-depressed, tapering original and regenerated tail separates P. gulbaru from all congeners except P. caudiannulatus. From this species P. gulbaru is separated by having a partially divided, as opposed to fully divided, rostral scale. Furthermore, the very small spinose body tubercles of P. gulbaru are in marked contrast to the large spinose body scales of P. caudiannulatus. An analysis of 729 bp of mitochondrial 12S rRNA and cytochrome b genes reveals P. gulbaru to be a deeply divergent lineage with closer affinities to mid-east Queensland congeners than the geographically neighbouring P. amnicola on Mt Elliot. In conservation terms, P. gulbaru is clearly at risk. Field surveys of Pattersons Gorge and the adjacent ranges indicate that this species is restricted to a very small area of highly fragmented habitat, of which only a small proportion receives a degree of protection in State forest. Further, there is ongoing, unchecked destruction of dry rainforest habitat by fire. Under current IUCN criteria, P. gulbaru warrants an Endangered ( B1, 2) listing.

C-terminal sequences in R-Ras are involved in integrin regulation and in plasma membrane microdomain distribution

The small GTPases R-Ras and H-Ras are highly homologous proteins with contrasting biological properties, for example, they differentially modulate integrin affinity: H-Ras suppresses integrin activation in fibroblasts whereas R-Ras can reverse this effect of H-Ras. To gain insight into the sequences directing this divergent phenotype, we investigated a panel of H-Ras/R-Ras chimeras and found that sequences in the R-Ras hypervariable C-terminal region including amino acids 175-203 are required for the R-Ras ability to increase integrin activation in CHO cells; however, the proline-rich site in this region, previously reported to bind the adaptor protein Nck, was not essential for this effect. In addition, we found that the GTPase TC21 behaved similarly to R-Ras. Because the C-termini of Ras proteins can control their subcellular localization, we compared the localization of H-Ras and R-Ras. In contrast to H-Ras, which migrates out of lipid rafts upon activation, we found that activated R-Ras remained localized to lipid rafts. However, functionally distinct H-Ras/R-Ras chimeras containing different C-terminal R-Ras segments localized to lipid rafts irrespective of their integrin phenotype. (C) 2003 Elsevier Inc. All rights reserved.

Factorizations of the complete graph into C-5-factors and 1-factors

In this paper, we show that K-10n can be factored into alpha C-5-factors and beta 1-factors for all non-negative integers alpha and beta satisfying 2alpha + beta = 10(n) - 1.