Expression of recombinant human betaine: homocysteine S-methyltransferase for x-ray crystallographic studies and further characterization of interaction with S-adenosylmethionine

Elevated homocysteine as a result of dysfunctional metabolic enzymes is an independent risk factor for arteriosclerosis. Betaine:homocysteine S-methyltransferase (BHMT) (EC 2.1.1.5) is an important enzyme in the pathway of homocysteine metabolism in that it recycles methionine from homocysteine and nonfolate methyl donors. To initiate X-ray crystallographic structural studies, we created a BHMT expression construct for use in Escherichia coli that has a polyhistidine purification tag with no extraneous protein, usually found in commercial vectors, between the tag and protein sequence. The extra amino acids can hinder the crystallization process. A modified pET28b vector was designed to produce N-terminal polyhistidine-tagged proteins with a simple construction scheme having broad applicability because of the use of rare SapI cloning sites. BHMT expressed using this vector could be rapidly purified using metal chelate chromatography. Gel exclusion chromatography analysis showed that recombinant polyhistidine-tagged human BHMT is a tetramer. S-Adenosylmethionine (SAMe) has no effect on the recombinant BHMT's ability to methylate homocysteine nor does the enzyme appear to bind SAMe when examined by microcalorimetry.     

Inactivation of glyceraldehyde-3-phosphate dehydrogenase of human malignant cells by methylglyoxal

The effect of methylglyoxal on the activity of glyceraldehyde-3-phosphate dehydrogenase (GA3PD) of several normal human tissues and benign and malignant tumors has been tested. Methylglyoxal inactivated GA3PD of all the malignant cells (47 samples) and the degree of inactivation was in the range of 25-90%, but it had no inhibitory effect on this enzyme from several normal cells (24 samples) and benign tumors (13 samples). When the effect of methylglyoxal on other two dehydrogenases namely glucose 6-phosphate dehydrogenase (G6PD) and L-lactic dehydrogenase (LDH) of similar cells was tested as controls it has been observed that methylglyoxal has some inactivating effect on G6PD of all the normal, benign and malignant samples tested, whereas, LDH remained completely unaffected. These studies indicate that the inactivating effect of methylglyoxal on GA3PD specifically of the malignant cells may be a common feature of all the malignant cells, and this phenomenon can be used as a simple and rapid device for the detection of malignancy.     

Augmenting Chinese hamster genome assembly by identifying regions of high confidence

Chinese hamster Ovary (CHO) cell lines are the dominant industrial workhorses for therapeutic recombinant protein production. The availability of genome sequence of Chinese hamster and CHO cells will spur further genome and RNA sequencing of producing cell lines. However, the mammalian genomes assembled using shot‐gun sequencing data still contain regions of uncertain quality due to assembly errors. Identifying high confidence regions in the assembled genome will facilitate its use for cell engineering and genome engineering. We assembled two independent drafts of Chinese hamster genome by de novo assembly from shotgun sequencing reads and by re‐scaffolding and gap‐filling the draft genome from NCBI for improved scaffold lengths and gap fractions. We then used the two independent assemblies to identify high confidence regions using two different approaches. First, the two independent assemblies were compared at the sequence level to identify their consensus regions as ”high confidence regions“ which accounts for at least 78 % of the assembled genome. Further, a genome wide comparison of the Chinese hamster scaffolds with mouse chromosomes revealed scaffolds with large blocks of collinearity, which were also compiled as high‐quality scaffolds. Genome scale collinearity was complemented with EST based synteny which also revealed conserved gene order compared to mouse. As cell line sequencing becomes more commonly practiced, the approaches reported here are useful for assessing the quality of assembly and potentially facilitate the engineering of cell lines.     

PE-only/PE_PGRS proteins of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> contain a conserved tetra-peptide sequence DEVS/DXXS that is a potential caspase-3 cleavage motif

Mycobacterium tuberculosis H37Rv is an intracellular pathogen responsible for causing tuberculosis in humans. The M. tuberculosis genome has been shown to contain a very large and unique family of PE proteins made of two sub-families: PE-only and PE_PGRS proteins. These two subtypes of proteins play a crucial role in the pathogenesis of the microbe. However, despite numerous investigations, the role of these proteins in disease development remains obscure. In this study, sequence analysis with a search for short conserved motifs revealed a conserved tetra-peptide motif DEVS/DXXS at the PE domain of almost every PE-only and PE_PGRS protein. The motif was found at a distance of 43–46 amino acids from the N-terminal of PE_PGRS proteins, and at a distance of between 35 and 82 amino acids of the PE-only proteins. As phosphorylation of the serine residue of this tetra-peptide could yield a motif similar to the caspase-3 binding recognition sequence DEVD/E, the region from a representative PE_PGRS protein (PE_PGRS45) was docked to human caspase-3. Strong interactions of only the protein containing the phosphorylated motif (DEVpS/DXXpS) to caspase-3 were observed. This suggested that the conserved DEVS/DXXS motif could have evolved for phosphorylation and subsequent recognition by caspase-3. These findings have important implications in unravelling the role of these PE proteins in mycobacterial infection.     

A thermolyzed diet increases oxidative stress, plasma alpha-aldehydes and colonic inflammation in the rat

A thermolyzed diet has the potential of providing exogenous oxidative stress in the form of advanced glycation end-products (AGE) and decreased thiamin. There is then a possibility that it could result in intracellular exposure to alpha-oxoaldehydes (glyoxal and methylglyoxal (MG)) with metabolic and genetic consequences. Two groups of Fischer 344 rats were fed the following diets: group A was given an AIN93G diet (control diet), while group B was given a thermolyzed AIN93G diet for 77 days. At the end of 77 days TK activity in red blood cells; glyoxal/MG levels in the plasma; glyoxal/MG HI protein adducts and dicarbonyls in the plasma, liver and colon tissues; glutathione levels of whole blood; and oxidative stress/inflammatory markers in the colon were measured. The thermolyzed diet resulted in: decreased thiamin status, increased plasma levels of glyoxal/MG and their adducts, increased protein dicarbonyls in the liver and plasma, lowered blood glutathione levels, increased infiltration of macrophages and increased colon nitrotyrosine levels. The thermolyzed diet increased the body burden of AGEs and decreased the thiamin status of the rats. This increased endogenous alpha-oxoaldehydes and oxidative stress has the potential to injure tissues that have low levels of antioxidant defenses such as the colon.     

Growth responses and Hyoscyamine content ofDatura innoxia under the influence of coal-smoke pollution

This study evaluated the impact of pollution on growth responses inDatura innoxia. Coal-smoke emissions were produced by the Badarpur Thermal Power Plant in Delhi, India. At the polluted site, the size of roots and leaves as well as the number of branches and leaves per plant increased, but shoot lengths and leaf areas were lower, compared with control plants. The net photosynthesis rate, stomatal resistance, and the amount of pigments (chlorophyll a, b, and carotenoids) were less in pollution-affected plants, while stomatal conductance and intercellular CO₂ concentration were higher in these plants. Explants from both sites (polluted and non-polluted), grown in vitro on various combinations of auxin (2,4-D, NAA) and cytokinin (BAP, KN), showed the maximum response on a medium containing NAA (0.1 mg L^-1) with BAP (5.0 mg L^-1). Hyoscyamine content was higher in all parts (root, stem, leaf, and regener-ants) of the polluted plants.     

Recent Selective Sweeps in North American Drosophila melanogaster Show Signatures of Soft Sweeps

Adaptation from standing genetic variation or recurrent de novo mutation in large populations should commonly generate soft rather than hard selective sweeps. In contrast to a hard selective sweep, in which a single adaptive haplotype rises to high population frequency, in a soft selective sweep multiple adaptive haplotypes sweep through the population simultaneously, producing distinct patterns of genetic variation in the vicinity of the adaptive site. Current statistical methods were expressly designed to detect hard sweeps and most lack power to detect soft sweeps. This is particularly unfortunate for the study of adaptation in species such as Drosophila melanogaster, where all three confirmed cases of recent adaptation resulted in soft selective sweeps and where there is evidence that the effective population size relevant for recent and strong adaptation is large enough to generate soft sweeps even when adaptation requires mutation at a specific single site at a locus. Here, we develop a statistical test based on a measure of haplotype homozygosity (H12) that is capable of detecting both hard and soft sweeps with similar power. We use H12 to identify multiple genomic regions that have undergone recent and strong adaptation in a large population sample of fully sequenced Drosophila melanogaster strains from the Drosophila Genetic Reference Panel (DGRP). Visual inspection of the top 50 candidates reveals that in all cases multiple haplotypes are present at high frequencies, consistent with signatures of soft sweeps. We further develop a second haplotype homozygosity statistic (H2/H1) that, in combination with H12, is capable of differentiating hard from soft sweeps. Surprisingly, we find that the H12 and H2/H1 values for all top 50 peaks are much more easily generated by soft rather than hard sweeps. We discuss the implications of these results for the study of adaptation in Drosophila and in species with large census population sizes.     

Effect of statin therapy on serum trace element status in dyslipidaemic subjects.

Patients previously not treated with a lipid-lowering agent (n = 20; mean age 49.15 +/- 3.28 years) were treated with either 10 mg/day of Simvastatin (n = 11), or Atorvastatin (n = 9) for 4 months. Fourteen additional patients were recruited from the same clinic at the same hospital as a control group. The medication of these latter patients was unaltered for 4 months and the same parameters were measured as for the statin groups. Serum concentrations of zinc, copper, caeruloplasmin, selenium, glutathione peroxidase (GPx) and C-reactive protein (CRP) were measured together with their lipid profiles pre- and post-treatment. In addition to reducing serum total and low-density lipoprotein (LDL) cholesterol (p < 0.0001), statin treatment was associated with a significant reduction in mean serum zinc (9%, p = 0.03), copper (9%, p < 0.01), caeruloplasmin (24%, p < 0.05), and median CRP (45%, p < 0.03). Similar changes were not observed in the control patients. No significant effects were observed for serum selenium, copper/caeruloplasmin ratio, or GPx (p > 0.05) in either statin or control groups. These changes may be related to the known anti-inflammatory properties of the statin class of drugs.