Oxidation of cholesterol moiety of low density lipoprotein in the presence of human endothelial cells or Cu+2 ions: identification of major products and their effects

Oxidation of lipoproteins is believed to play a key role in atherogenesis. In this study, low density lipoproteins (LDL) was subjected to oxidation in the presence of either human umbilical vein endothelial cells or with Cu+2 ions and the major oxides formed were identified. While cholesterol-alpha-epoxide (C-alpha EP) was the major product of cholesterol peroxidation in the presence of endothelial cells, cholest-3,5-dien-7-one (CD) predominated in the presence of Cu+2 ion. Both steroids were identified by gas chromatography/mass spectrometry. HDL cholesterol was resistant to oxidation. When tested on human skin fibroblasts in culture C-alpha EP (10 micrograms/ml) caused marked stimulation of 14C-oleate incorporation into cholesterol esters, while CD stimulated cholesterol esterification only mildly. These studies show that a) C-alpha EP is the major peroxidation product of LDL cholesterol moiety in the presence of endothelial cells and b) it causes marked stimulation of cholesterol esterification in cells. C-alpha EP may play a key role in increasing cholesterol esterification noted in atherogenesis.     

Incorporation of liposomal phytosterols into human cells in culture: a potential in vitro model for investigating pathological effects of phytosterolemia

A potential in vitro cell culture model was developed for studies concerning the pathological effect of phytosterolemia in which liposomal phytosterols were incorporated into human skin fibroblasts and hepatoblastoma (HepG2) cells. After incubation with phytosterols, fibroblasts and HepG2 cells contained a significant amount (20-27%) of phytosterols (campesterol and beta-sitosterol). Phytosterol accumulation caused a significant reduction in the cholesterol content of cells. Labeled sitosterol and cholesterol showed similar uptake with lower esterification of sitosterol when compared to cholesterol. Labeled sitosterol incorporated into LDL was esterified to a greater extent than sitosterol added as straight liposome. About 23% of the labeled sitosterol was converted into acidic products and 5.6% was present as 5 alpha-stanols in HepG2 cells.     

Biodegradative threonine dehydratase. Reduction of ferricyanide by an intermediate of the enzyme-catalyzed reaction.

The threonine-dependent reduction of ferricyanide catalyzed by the purified biodegradative threonine dehydratase of Escherichia coli has been studied. The rate of production of 2-oxobutyrate in the presence of ferricyanide was lower than that found in the absence of ferricyanide. The concentrations of threonine required for half-maximal effects for the reduction of ferricyanide and, in the presence of the dye, for 2-oxobutyrate production, were 3 mM and 9mM, respectively. Reduction of ferricyanide was accompanied by evolution of CO2, and even within a very short incubation time with the enzyme, the ratio of ferricyanide reduced over CO2 evolved was approximately 7. Stopping the enzyme activity after a brief exposure to threonine at pH 9.7 resulted in the accumulation of an intermediate (with a half-life of 4 min at 25 degrees C) which formed an adduct with N-ethylmaleimide; the accumulated intermediate, in the absence of N-ethylmaleimide, reduced ferricyanide with concomitant evolution of CO2. We conclude from these results that 2-aminocrotonate is the intermediate which serves as a source of reducing equivalent for ferricyanide, and nonstoichiometric amount of ferricyanide reduction may be attributed to some secondary reactions of ferricyanide with compounds derived from the oxidation product of 2-aminocrotonate.     

A new domain family in the superfamily of alkaline phosphatases

During the course of our large-scale genome analysis a conserved domain, currently detectable only in the genomes of Drosophila melanogaster, Caenorhabditis elegans and Anopheles gambiae, has been identified. The function of this domain is currently unknown and no function annotation is provided for this domain in the publicly available genomic, protein family and sequence databases. The search for the homologues of this domain in the non-redundant sequence database using PSI-BLAST, resulted in identification of distant relationship between this family and the alkaline phosphatase-like superfamily, which includes families of aryl sulfatase, N-acetylgalactosomine-4-sulfatase, alkaline phosphatase and 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGM). The fold recognition procedures showed that this new domain could adopt a similar 3-D fold as for this superfamily. Most of the phosphatases and sulfatases of this superfamily are characterized by functional residues Ser and Cys respectively in the topologically equivalent positions. This functionally important site aligns with Ser/Thr in the members of the new family. Additionally, set of residues responsible for a metal binding site in phosphatases and sulphtases are conserved in the new family. The in-depth analysis suggests that the new family could possess phosphatase activity.     

Regional adiposity, body composition and central body fat distribution of 10-16 years old Bengalee boys of Nimta, North 24 Parganas, West Bengal, India

A cross-sectional study of 502 Bengalee boys aged 10-16 years of Nimta, North 24 Parganas, West Bengal, India, was undertaken to study regional adiposity, body composition and central body fat distribution. The subjects were classified into seven age groups: 10-10.9 years (n = 74), 11-11.9 (n = 53), 12-12.9 (n = 87), 13-13.9 (n = 116), 14-14.9 (n = 58), 15-15.9 (n = 57), 16-16.9 (n = 57). In general, there was a significant linear increasing trend from 10 to 16 years for all the anthropometric variables. There was a net increase of 30.5 cm and 22.8 kg in mean height and weight, respectively, between 10 and 16 years of age. Mean BMI increased by 3.7 kg/m2 during the same period. Among circumferences, the largest increase was in hip followed by chest while the smallest increase was in mid upper arm, between 10 and 16 years of age. Subscapular skinfold showed the largest increase followed by abdomen and suprailiac skinfolds, while the increase was least in forearm skinfold. Significant linear increasing trend was observed for all the body composition measures. The largest increase in percent of body fat (PBF) was observed between ages 10 and 11 years while mean fat mass (FM), fat free mass (FFM) and fat mass index (FMI) increased the most between 14 to 15 years. However, an overall decreasing trend was observed, in mean waist-to-hip ratio (WHR) from 10 to 16 years. Boys aged 10 years had the highest mean WHR while those aged 15 years had the lowest mean WHR. There was an increase in mean WHR among 16 years old boys.     

Berberine, a strong polyriboadenylic acid binding plant alkaloid: spectroscopic, viscometric, and thermodynamic study

The interaction of berberine with single stranded poly(rA) structure was investigated using a combination of spectrophotometric, spectrofluorimetric, circular dichroic, viscometric, and thermodynamic studies. The interaction process was characterized by typical hypochromic and bathochromic effects in the absorption spectrum of berberine, enhancement of fluorescence intensity of berberine, increase of viscosity, and perturbation of circular dichroic spectrum of single stranded poly(rA). Scatchard plot obtained from spectrophotometric analysis showed that berberine bound strongly to single stranded poly(rA) in a non-cooperative manner. In contrast, berberine does not show any significant effect (i) in its absorbance and fluorescence spectra on binding to double stranded poly(rA), (ii) alter the circular dichroic spectrum of double stranded poly(rA), or (iii) increase of viscosity of double stranded poly(rA) indicating that it does not bind at all to double stranded poly(rA) structure. Thermodynamic parameters indicated that the binding of the alkaloid to single stranded poly(rA) is an endothermic process and entropy driven. All these findings, taken together clearly support that berberine binds strongly to single stranded poly(rA) structure by a mechanism of partial intercalation leading to its use in gene regulation in eukaryotic cells.     

Isolation of Potentially Pathogenic Escherichia coli O157:H7 from the Ganges River

Escherichia coli serotype O157:H7 was detected among bacteria collected from the Ganges River. O157:H7 isolates tested positive for stx₁, stx₂, and eae gene sequences. Identification of potentially pathogenic isolates from extensively used source water indicates that O157:H7 may be a significant but as yet underacknowledged public health concern in India.