Effect of ultraviolet B (302 nm) irradiation on viability, metabolic and detoxification functions of goat hepatocytes – in vitro study

The object of the present study was to investigate the effect(s) of UV-B irradiation on the functional integrity, metabolic and detoxifying capacity of the isolated goat hepatocytes. Isolated goat hepatocytes were subjected to UV-B irradiation invitro for 0, 250, 500, 1250, 2500 and 7500 Joules/m² which correspond to the irradiation time of 0, 1, 2, 5, 10 and 30 min. Cells were then analysed for Viability (Trypan blue exclusion test [TBE], 3-[4,5-dimethylthiozol-2yl]-2,5-diphenyltetrazolium bromide [MTT] assay, Membrane integrity (Lactate dehydrogenase [LDH] leakage, Lipid peroxidation) Detoxification (Ureagenesis, Cytochrome P450 activity [CYP450, Diazepam metabolism] and Glutathione-S-Transferase [GST] activity. The results show that there was no difference in functional, metabolic as well as detoxifying parameters of the hepatocytes when irradiated from 0–1250 Joules/m², whereas a significant alteration was appreciable in the parameters such as LDH leakage, lipid peroxidation, and CYP450 activity when irradiated beyond 1250 Joules/m². Our present findings suggest that the biologically compatible and feasible dose of UV-B irradiation for xenotransplantation appears to be 1250 Joules/m².     

Effect of Sargassum polycystum (Phaeophyceae)-sulphated polysaccharide extract against acetaminophen-induced hyperlipidemia during toxic hepatitis in experimental rats

The effect of Sargassum polycystum crude extract on lipid metabolism was examined against acetaminophen-induced (800 mg/kg body wt., intraperitoneally) hyperlipidemia during toxic hepatitis in experimental rats. The animals intoxicated with acetaminophen showed significant elevation in the levels of cholesterol, triglycerides and free fatty acid in both serum and liver tissue. The levels of tissue total lipids and serum LDL-cholesterol were also elevated with depleted levels of serum HDL-cholesterol and tissue phospholipid. The acetaminophen-induced animals showed significant alterations in the activities of lipid metabolizing enzymes serum lecithin cholesterol acyl transferase (LCAT) and hepatic triglyceride lipase (HTGL). The levels of liver tissue fatty acids (saturated, mono and polyunsaturated) such as palmitic acid, stearic acid, oleic acid, linoleic acid, arachidonic acid and linolenic acid monitored by gas chromatography were considerably altered in acetaminophen intoxicated animals when compared with control animals. The prior oral administration of Sargassum polycystum (200 mg/kg body wt./day for a period of 15 days) crude extract showed considerable prevention in the severe disturbances of lipid profile and metabolizing enzymes triggered by acetaminophen during hepatic injury. Liver histology also showed convincing supportive evidence regarding their protective nature against fatty changes induced during acetaminophen intoxication. Thus the present study indicates that the protective nature of Sargassum polycystum extract may be due to the presence of active compounds possessing antilipemic property against acetaminophen challenge. (Mol Cell Biochem 276: 89–96, 2005)     

Selection of Optimal Host Strain for Molecular Pathogenesis Studies on <Emphasis Type="Italic">Cryptococcus gattii</Emphasis>

Encapsulated yeast, Cryptococcus gattii (Cg) is a primary and emerging fungal pathogen in North America. It has a predilection for invading the central nervous system of both healthy and immunocompromised humans and animals. Recently, we initiated molecular pathogenesis studies in Cg strain NIH444 (ATCC 32609). In this report, we compared the biology and pathogenic potential of NIH444 to those of WM276, an Australian environmental isolate that is being used for the whole genome-sequencing project. Our data indicated that NIH444 is comparatively more virulent in a mouse model of cryptococcosis than is WM 276. We found robust mating of NIH444, and no mating of WM276, when tested against Cg MATa strain, NIH198. WM276 but not NIH444 was defective in filamentation and sporulation (haploid fruiting). Interestingly, NIH444 has a VGII/AFLP6 genotype similar to that of the genotype of the recent outbreak strains from Vancouver Island, British Columbia, Canada. Additionally, comparisons of nucleotide sequences of various genes also showed differences between NIH444 and WM276. Based on these observations, we conclude that NIH444 should remain the strain of choice for understanding Cg pathogenesis, especially on the North American continent.     

The interaction site of Flap Endonuclease-1 with WRN helicase suggests a coordination of WRN and PCNA

Werner and Bloom syndromes are genetic RecQ helicase disorders characterized by genomic instability. Biochemical and genetic data indicate that an important protein interaction of WRN and Bloom syndrome (BLM) helicases is with the structure-specific nuclease Flap Endonuclease 1 (FEN-1), an enzyme that is implicated in the processing of DNA intermediates that arise during cellular DNA replication, repair and recombination. To acquire a better understanding of the interaction of WRN and BLM with FEN-1, we have mapped the FEN-1 binding site on the two RecQ helicases. Both WRN and BLM bind to the extreme C-terminal 18 amino acid tail of FEN-1 that is adjacent to the PCNA binding site of FEN-1. The importance of the WRN/BLM physical interaction with the FEN-1 C-terminal tail was confirmed by functional interaction studies with catalytically active purified recombinant FEN-1 deletion mutant proteins that lack either the WRN/BLM binding site or the PCNA interaction site. The distinct binding sites of WRN and PCNA and their combined effect on FEN-1 nuclease activity suggest that they may coordinately act with FEN-1. WRN was shown to facilitate FEN-1 binding to its preferred double-flap substrate through its protein interaction with the FEN-1 C-terminal binding site. WRN retained its ability to physically bind and stimulate acetylated FEN-1 cleavage activity to the same extent as unacetylated FEN-1. These studies provide new insights to the interaction of WRN and BLM helicases with FEN-1, and how these interactions might be regulated with the PCNA–FEN-1 interaction during DNA replication and repair.     

Solution conformation of Substance P antagonists-[D-Arg1, D-Trp7,9, Leu11]-SP, [D-Arg1, D-Pro2, D-Trp7,9, Leu11]-SP and [D-Pro2, D-Trp7,9]-SP by CD, NMR and MD simulations

Substance P (SP) is an important neuropeptide involved in pain transmission and induction of inflammation. Its antagonists are being extensively investigated for their non-narcotic analgesic and anti-inflammatory activity. With a view towards better understanding the structural requirements of these analogs for efficient interaction with the SP receptor, the conformation of three SP antagonists [D-Arg1, D-Trp7,9, Leu11]-SP, [D-Arg1, D-Pro2, D-Trp7,9, Leu11]-SP and [D-Pro2, D-Trp7,9]-SP has been studied by CD, NMR and molecular dynamics (MD) simulations. All three peptides exhibit a high dependence of structure on the solvent. The molecules tend to adopt beta-turns in solvents like DMSO and H2O and form helices in a hydrophobic environment. A direct relation between the helix forming potential of these antagonists with their receptor binding potency has been observed.     

Crystal structure of human E1 enzyme and its complex with a substrate analog reveals the mechanism of its phosphatase/enolase activity

Enolase-phosphatase E1 (MASA) is a bifunctional enzyme in the ubiquitous methionine salvage pathway that catalyzes the continuous reactions of 2,3-diketo-5-methylthio-1-phosphopentane to yield the aci-reductone metabolite using Mg2+ as cofactor. In this study, we have determined the crystal structure of MASA and its complex with a substrate analog to 1.7A resolution by multi-wavelength anomalous diffraction and molecular replacement techniques, respectively. The structures support the proposed mechanism of phosphatase activity and further suggest the probable mechanism of enolization. We establish a model for substrate binding to describe in detail the enzymatic reaction and the formation of the transition state, which will provide insight into the reaction mechanisms of other enzymes in the same family.