Bioremediation of Hexachlorocyclohexane Isomers,Chlorinated Benzenes,and Chlorinated Ethenes in Soil and Fractured Dolomite

Groundwater at an industrial site is contaminated with α hexachlorocyclohexane (HCH) and γ -HCH (i.e., lindane) (0.3 to 0.5 ppm). Other contaminants in the 1 to 15 ppm range include 1,2,4-trichlorobenezene (TCB), 1,2-dichlorobenzene (DCB), 1,3-DCB, 1,4-DCB, chlorobenzene (CB), benzene, trichloroethene (TCE), and cis-1,2-dichloroethene (cDCE). The aquifer consists of a shallow layer of soil over fractured dolomite, where most of the contaminant mass resides. The objective of this study was to compare (1) anaerobic reductive dechlorination of the polychlorinated contaminants, followed by aerobic biodegradation of the daughter products (mainly DCBs, CB, and benzene); and (2) aerobic biodegradation of α - and γ -HCH, TCB, DCBs, CB, and benzene, followed by anaerobic reduction of TCE and cDCE to ethene. Conventional wisdom suggests that sequential anaerobic and aerobic conditions are desirable for bioremediating sites contaminated by mixtures of polychlorinated organics. The results of this microcosm study suggest that a sequential aerobic and anaerobic approach may be more successful, although implementing this in the field presents some major challenges. In the dolomite microcosms incubated under aerobic conditions first (59 days), α - and γ -HCH were biodegraded close to the maximum contaminant level for lindane; all of the aromatic compounds were consumed; and there was partial removal of TCE and cDCE (presumptively via cometabolism). The subsequent switch to anaerobic conditions (day 101) yielded reductive dechlorination of the remaining TCE; a significant level of ethene was produced, although some cDCE and VC persisted. In contrast, sequential anaerobic (393 days) and aerobic treatment (498 days) for the dolomite microcosms was ineffective in completely removing the aromatic compounds, α -HCH, cDCE, and VC. For the soil microcosms, both treatment sequences were effective, most likely reflecting a greater abundance of the necessary microbes and electron donor in this part of the site.     

Sequence analysis of the respiratory syncytial virus phosphoprotein gene.

A recombinant cDNA plasmid (pRSA3) containing an almost full-length copy of the mRNA encoding respiratory syncytial virus phosphoprotein was identified in a cDNA library prepared with mRNA from respiratory syncytial virus-infected cells. The cDNA insert was sequenced, and a protein of 27,150 daltons was deduced from the DNA sequence. The protein is relatively acidic, containing two clusters of acidic amino acids, one in the middle of the molecule and the other at the C-terminus. It is devoid of both cysteine and tryptophan. There was no other potential reading frame within the phosphoprotein gene of respiratory syncytial virus. This situation is unlike that with Sendai virus, a paramyxovirus, which has a nonstructural C protein encoded by a second overlapping reading frame near the 5' end of the mRNA for phosphoprotein.     

Amino acid sequence of human respiratory syncytial virus nucleocapsid protein.

Amino acid sequence of the human respiratory syncytial (RS) virus nucleocapsid (NC) protein, deduced from the DNA sequence of a recombinant plasmid, is presented. The cDNA plasmid (pRSB11) has 1412 bp of RS viral NC sequence and lacks six nucleotides of the 5' end of mRNA. There is a single long open reading frame encoding 467 amino acids. This 51540 dal protein is rich in basic amino acids and has no homologies with other known viral capsid proteins.     

Purification and some properties of inducible N-acetylglucosamine kinase from Candida albicans

N-Acetylglucosamine kinase (ATP:2-acetamido-2-deoxy-D-glucose 6-phosphotransferase, EC 2.7.1.59) catalyzes the first reaction in the inducible N-acetylglucosamine catabolic pathway of Candida albicans, an obligatory aerobic yeast. As a part of continuing biochemical studies concerning the regulation of gene expression in a simple eukaryote, N-acetylglucosamine kinase has been purified and characterized biochemically. The enzyme has been purified about 300-fold from the crude extract and its molecular weight of 75 000 has been determined by Sephadex G-100 gel filtration. Isolation and analysis procedures are described. The kinase reaction is optimal within a pH range of 7--8. The enzyme is strictly specific for GlcNAc as phosphate acceptor; ATP is the phosphoryl group donor for the kinase reaction and to a lesser extent dATP and CTP. Km values for GlcNAc and ATP are 1.33 mM and 1.82 mM, respectively. The enzyme required Mg2+, which may be replaced by other bivalent metal ions such as Mn2+, Ca2+, Ba2+ and Co2+ for a lesser degree of effectiveness. The purified enzyme is extremely sensitive to thermal denaturation and becomes completely inactive by heating at 65% C for 2 min. The enzyme is also inactivated by sulphydryl reagents such as p-chloromercuribenzene sulfonic acid and N-ethylmaleimide.     

Regeneration of Indian ginseng plantlets from stem callus

The development of stem callus mediated plant regeneration system for Withania somnifera is described. Maximum callus proliferation was obtained on Murashige and Skoog medium supplemented with 2.26 μM 2,4-D. Three-week-old, white, friable callus was used for shoot regeneration. The maximum shoot regeneration (6.2 ± 0.34 shoots/explant) was achieved in four weeks when callus was cultured on MS medium fortified with 4.44 μM BA and 0.57 μM IAA. Regenerated shoots were excised and multiplied (8.4 ± 0.43 shoots/explant) on MS medium supplemented with 4.44 μM of BA. Multiple shoots were divided into single shoots and were rooted (5.1 ± 0.49 rootlets/shoot) on half strength MS medium supplemented with 9.84 μM of IBA. After a hardening phase of 3 weeks the plantlets were transferred to the field. This revised version was published online in June 2006 with corrections to the Cover Date.     

DNA methylation and structural and functional bimodality of vertebrate promoters

Human promoters divide into 2 classes, the low CpG (LCG) and the high CpG (HCG), based on their CpG dinucleotide content. The LCG class of promoters is hypermethylated and is associated with tissue-specific genes, whereas the HCG class is hypomethylated and associated with broadly expressed genes. By analyzing several chordate genomes separated for hundreds of millions of years, here we show that the divide between low CpG and high CpG promoters is conserved in several distantly related vertebrate taxa (including human, chicken, frog, lizard, and fish) but not in close invertebrate outgroups (sea squirts). Furthermore, LCG and HCG promoters are distinctively associated with tissue-specific and broadly expressed genes in these distantly related vertebrate taxa. Our results indicate that the function of DNA methylation on gene expression is conserved across these vertebrate taxa and suggest that the 2 classes of promoters have evolved early in vertebrate evolution, as a consequence of the advent of global DNA methylation.     

Bioaugmentation potential of a vinyl chloride-assimilating Mycobacterium sp., isolated from a chloroethene-contaminated aquifer

An aerobic bacterium, Mycobacterium sp. strain TRW-2 that assimilated vinyl chloride (VC) or ethene (ETH) as the sole carbon source was isolated from a chloroethene-degrading enrichment culture. The strain TRW-2 also degraded cis-dichloroethene (cis-DCE) in mineral salts medium, but only when VC was present as the primary carbon source. However, no degradation of trans-dichloroethene or trichloroethene occurred in either the presence or absence of added VC. The measured growth yield values were 6.53 and 14.1g protein/mol of VC and ETH utilized, respectively. Inoculation by strain TRW-2 in microcosms prepared with aquifer samples resulted in rapid degradation of VC, whereas native bacteria degraded negligible amounts of VC within the same time period, thus suggesting bioaugmentation potential of the isolate. Phylogenetic analysis of the 16S rDNA sequence of the isolate revealed 98% sequence similarity to the members of the genus Mycobacterium. In summary, the isolate's ability to degrade VC, cis-DCE, and ETH and also its ability to survive and degrade VC in the presence of other microorganisms is relevant to the remediation of VC-impacted aquifers.     

Targeted Molecular Sequencing Revealed Allelic Heterogeneity of <Emphasis Type="Italic">BRAF</Emphasis> and <Emphasis Type="Italic">PTPN11</Emphasis> Genes among Arab Noonan Syndrome Patients

Noonan syndrome (NS) is a very rare heterogenous genetic disorder often characterized by short stature, facial dysmorphisms, congenital heart defects and learning disabilities in affected children. In the current study, we sought to discover the disease causal mutations, inherited or de novo, for Noonan Syndrome among Arab patients. We screened the coding regions and splice sites of 10 known RAS/MAP Kinase pathway genes in 17 NS-trios and 100 random healthy volunteers by oilgonucleotide chip testing and Sanger sequencing methods. We found pathogenic heteroallelic de novo mutations in BRAF or PTPN11 gene in 7/17 (41.17%) of NS patients. None of the 200 chromosomes of healthy volunteers had those pathogenic mutations. Genotype-phenotype analysis showed positive correlation between BRAF and PTPN11 gene mutations and classical NS clinical manifestations. Characteristic facies is the major observed clinical manifestation among PTPN11-mutation positive cases (c.236A>G, c.854T>C, c.923A>G), whereas both characteristic facies and ectodermal manifestations are seen as dominant clinical features among BRAF-mutation positive cases (c.730A>C, c.770A>G, c.1406G>A). In addition to genotyping and clinical phenotyping, we performed computational structural analysis to examine the impact of amino acid substitution mutations on the conformation and folding of BRAF and PTPN11 proteins. Our results suggested that BRAF (c.730A>C, c.770A>G, c.1406G>A) and PTPN11 (c.236A>G, c.854T>C, c.923A>G) gene mutations elicits structural and functional alterations at protein level, which would eventually lead to dysregulation of RAS-MAPK signaling cascade, which plays critical a role in cell cycle and senescence. In conclusion, our study suggest that molecular screening of BRAF and PTPN11 genetic mutations in Arab NS patients may assist in deriving competitive outcomes related to clinical phenotyping and disease diagnosis.     

In vitro cytotoxicity of Gymnema montanum in human leukaemia HL‐60 cells; induction of apoptosis by mitochondrial membrane potential collapse

Objectives

Gymnema montanum Hook, an Indian Ayurvedic medicinal plant, is used traditionally to treat a variety of ailments. Here, we report anti‐cancer effects and molecular mechanisms of ethanolic extract of G. montanum (GLEt) on human leukaemia HL‐60 cells, compared to peripheral blood mononuclear cells.

Materials and methods

HL‐60 cells were treated with different concentrations of GLEt (10–50 μg/ml) and cytotoxicity was assessed by MTT assay. Levels of lipid peroxidation, antioxidants, mitochondrial membrane potential and caspase‐3 were measured. Further, apoptosis was studied using annexin‐V staining and the cell cycle was analyzed by flow cytometry.

Results

GLEt had a potent cytotoxic effect on HL‐60 cells (IC₅₀‐20 μg/ml), yet was not toxic to normal peripheral blood mononuclear cells. Exposure of HL‐60 cells to GLEt led to elevated levels of malonaldehyde formation, but to reduced glutathione, superoxide dismutase, catalase and glutathione peroxidase activities (P < 0.05). Induction of apoptosis was confirmed by observing annexin‐V positive cells, associated with loss of mitochondrial membrane potential. Cell cycle arrest at G0/G1 was observed in GLEt‐treated HL‐60 cells, indicating its potential at inducing their apoptosis.

Conclusions

Findings of the present study suggest that G. montanum induced apoptosis in the human leukaemic cancer cells, mediated by collapse of mitochondrial membrane potential, generation of reactive oxygen species and depletion of intracellular antioxidant potential.

     

Tolerable amounts of amino acids for human supplementation: summary and lessons from published peer-reviewed studies

Amino acid supplementation may be indicated to correct for insufficient amino acid intake in healthy individuals, and in specific physiological or pathophysiological situations. However, there is a concern to not supplement beyond the tolerable upper intake level (UL) by determining parameters of no-observed-adverse-effect level (NOAEL) or lowest-observed-adverse-effect level (LOAEL) for each amino acid. Since the NOAEL and LOAEL values are at least one order of magnitude different when comparing the values obtained in rats and humans, the aim of this review is to evaluate to what extent the amino acid UL measured in the rat model, when referenced to the dietary usual consumption (UC) and dietary requirement (RQ) for indispensable amino acids, may be used as an approximation of the UL in humans. This review then compares the ratios of the NOAEL or LOAEL over UC and RQ in the rat model with the same ratios calculated in humans for the nine amino acids (arginine, serine, glycine, histidine, leucine, lysine, methionine, phenylalanine, and tryptophan) for which this comparison can be done. From the calculations made, it appears that for these 9 amino acids, the calculated ratios for rats and humans, although rather different for several amino acids, remains for all of them in the same order of magnitude. For tryptophan, tyrosine, and valine, the ratios calculated in rats are markedly different according to the sex of animals, raising the view that it may be also the case in humans.