Cloning of a Novel 6-Chloronicotinic Acid Chlorohydrolase from the Newly Isolated 6-Chloronicotinic Acid Mineralizing Bradyrhizobiaceae Strain SG-6C

A 6-chloronicotinic acid mineralizing bacterium was isolated from enrichment cultures originating from imidacloprid-contaminated soil samples. This Bradyrhizobiaceae, designated strain SG-6C, hydrolytically dechlorinated 6-chloronicotinic acid to 6-hydroxynicotinic acid, which was then further metabolised via the nicotinic acid pathway. This metabolic pathway was confirmed by growth and resting cell assays using HPLC and LC-MS studies. A candidate for the gene encoding the initial dechlorination step, named cch2 (for 6-chloronicotinic acid chlorohydrolase), was identified using genome sequencing and its function was confirmed using resting cell assays on E. coli heterologously expressing this gene. The 464 amino acid enzyme was found to be a member of the metal dependent hydrolase superfamily with similarities to the TRZ/ATZ family of chlorohydrolases. We also provide evidence that cch2 was mobilized into this bacterium by an Integrative and Conjugative Element (ICE) that feeds 6-hydroxynicotinic acid into the existing nicotinic acid mineralization pathway.     

Influence of PDZK1 on lipoprotein metabolism and atherosclerosis

PDZK1 is a scaffold protein containing four PDZ protein interaction domains, which bind to the carboxy termini of a number of membrane transporter proteins, including ion channels (e.g., CFTR) and cell surface receptors. One of these, the HDL receptor, scavenger receptor class B type I (SR-BI), exhibits a striking, tissue-specific dependence on PDZK1 for its expression and activity. In PDZK1 knockout (KO) mice there is a marked reduction of SR-BI protein expression (approximately 95%) in the liver, but not in steroidogenic tissues or, as we show in this report, in bone marrow- or spleen-derived macrophages, or lung-derived endothelial cells. Because of hepatic SR-BI deficiency, PDZK1 KO mice exhibit dyslipidemia characterized by elevated plasma cholesterol carried in abnormally large HDL particles. Here, we show that inactivation of the PDZK1 gene promotes the development of aortic root atherosclerosis in apolipoprotein E (apoE) KO mice fed with a high fat/high cholesterol diet. However, unlike complete SR-BI-deficiency in SR-BI/apoE double KO mice, PDZK1 deficiency in PDZK1/apoE double knockout mice did not result in development of occlusive coronary artery disease or myocardial infarction, presumably because of their residual expression of SR-BI. These findings demonstrate that deficiency of an adaptor protein essential for normal expression of a lipoprotein receptor promotes atherosclerosis in a murine model. They also define PDZK1 as a member of the family of proteins that is instrumental in preventing cardiovascular disease by maintaining normal lipoprotein metabolism.     

Role of procoagulant lipids in human prothrombin activation. 2. Soluble phosphatidylserine upregulates and directs factor X(a) to appropriate peptide bonds in prothrombin.

Activation of human prothrombin to thrombin (II(a)) by factor X(a) during blood coagulation requires proteolysis of two bonds and thus involves two possible activation pathways (parallel-sequential activation model). Hydrolysis of Arg(322)-Ile(323) produces meizothrombin (MzII(a)) as an intermediate, while hydrolysis of Arg(273)-Thr(274) produces prethrombin 2-fragment 1.2 (Pre2-F1.2). A soluble lipid, dicaproylphosphatidylserine (C6PS), enhances activation by 60-fold [Koppaka et al. (1996) Biochemistry 35, 7482]. We report here that C6PS binding to factor X(a) not only enhances the rate of activation but also alters the pathway. Activation was monitored using a chromogenic substrate (S-2238) to detect both II(a) and MzII(a) active site formation and SDS-PAGE to detect Pre2-F1.2 as well as II(a) and MzII(a). Of the four kinetic constants needed to describe activation, two (MzII(a) and Pre2-F1.2 consumption) were measured directly, and two (MzII(a) and Pre2-F1.2 formation) were obtained by fitting the three time courses simultaneously to the parallel-sequential reaction model. The time courses of II(a), MzII(a), and Pre2-F1.2 formations were all well described below the C6PS critical micelle concentration (CMC) by this activation model. The rate of Arg(322)-Ile cleavage leading to MzII(a) formation increased by 150-fold, while the rate of Arg(273)-Thr cleavage leading to Pre2-F1.2 formation was inhibited slightly. At concentrations of water-soluble C6PS above its CMC, all four proteolytic reactions increased in rate by 2-5-fold at the C6PS CMC. We conclude that soluble C6PS differentially affects the rate of individual bond cleavages during prothrombin activation in solution such that activation occurs almost exclusively via MzII(a) formation. Finally, C6PS enhanced the rates of all proteolytic reactions to within a factor of 3 of the enhancement seen with PS-containing membranes. We conclude that PS-containing membranes regulate prothrombin activation by factor X(a) mainly via interaction of individual PS molecules with factor X(a).     

Selective and tandem amplification of a member of the metallothionein gene family in Candida glabrata

Metallothioneins constitute a multigene family in the yeast Candida glabrata. Two genes, designated metallothionein-I (MT-I) and one member of the metallothionein-II family (MT-II), were cloned and sequenced previously (Mehra, R. K., Garey, J. R., Butt, T. R., Gray, W. R., and Winge, D. R. (1989) J. Biol. Chem. 264, 19747-19753). Southern analysis of the genomic DNA samples from different wild-type isolates indicated that the MT-I gene was always present as a single copy but multiple (3-9) and tandemly arranged copies of one MT-II gene were present in different strains. Strains of C. glabrata highly resistant to copper salts were obtained by repeated culturing of wild-type isolates in medium containing increasing concentrations of copper sulfate. These strains showed further stable chromosomal amplification (greater than 30 copies) of the MT-II gene. The MT-I gene remained as a single copy. Amplified copies of the MT-II gene were always arranged tandemly. One of the copper-resistant strains acquired more copies of the MT-II gene by apparent duplication of the chromosome carrying this gene. The size of the amplification unit was 1.25 kilobases. The principal MT-I and -II genes of C. glabrata were shown to map to different chromosomes by electrophoretic karyotypic analysis. The length of chromosome carrying MT-II gene increased appreciably in strains exhibiting the highest amplification of this gene. Northern analysis showed increased basal levels of MT-II mRNA in strains having highly amplified MT-II locus.     

Emergence of artemisinin-resistant Plasmodium falciparum with kelch13 C580Y mutations on the island of New Guinea

The rapid and aggressive spread of artemisinin-resistant Plasmodium falciparum carrying the C580Y mutation in the kelch13 gene is a growing threat to malaria elimination in Southeast Asia, but there is no evidence of their spread to other regions. We conducted cross-sectional surveys in 2016 and 2017 at two clinics in Wewak, Papua New Guinea (PNG) where we identified three infections caused by C580Y mutants among 239 genotyped clinical samples. One of these mutants exhibited the highest survival rate (6.8%) among all parasites surveyed in ring-stage survival assays (RSA) for artemisinin. Analyses of kelch13 flanking regions, and comparisons of deep sequencing data from 389 clinical samples from PNG, Indonesian Papua and Western Cambodia, suggested an independent origin of the Wewak C580Y mutation, showing that the mutants possess several distinctive genetic features. Identity by descent (IBD) showed that multiple portions of the mutants’ genomes share a common origin with parasites found in Indonesian Papua, comprising several mutations within genes previously associated with drug resistance, such as mdr1, ferredoxin, atg18 and pnp. These findings suggest that a P. falciparum lineage circulating on the island of New Guinea has gradually acquired a complex ensemble of variants, including kelch13 C580Y, which have affected the parasites’ drug sensitivity. This worrying development reinforces the need for increased surveillance of the evolving parasite populations on the island, to contain the spread of resistance.     

Purification and properties of purine hydroxylase II from Aspergillus nidulans

Purine hydroxylase II from Aspergillus nidulans has been purified to near homogeneity. The enzyme has a pI of 5.7, a molecular weight of 300,000, and two subunits with molecular weight of 153,000 each. The enzyme contains 2 FAD, 2 molybdenum atoms, and 4 (2 Fe-2S) iron-sulfur centers per molecule and exhibits broad specificity for reducing and oxidizing substrates. Among the more notable characteristics are the ability to oxidize hypoxanthine and nicotinic acid but not xanthine and virtually complete inactivity with oxygen. Moreover, while the enzyme is inactivated by borate and methanol, it is very resistant to cyanide and arsenite and it not inactivated by allopurinol. At infinite concentrations of reducing and oxidizing substrates, the Km for hypoxanthine was 119 microM, for nicotinic acid was 136 microM, and for NAD+ was 525 microM.     

Adverse health effects in workers exposed to trace/toxic metals at workplace

Widespread use of metals in industrial activities has enhanced the occupational exposure to toxic metals as well as the health risks of metal hazards to humans. Elemental analysis in human tissues is the most common application of biological monitoring for screening, diagnosis and assessment of such exposures and risk. Among various biopsy materials, blood, hair, nail, teeth and body fluids may be used as bioindicators for this purpose. The present paper deals with the determination of Pb, Cr, Ni, Mn, Fe, Cu and Zn elemental concentration in workers exposed to these metals at workplace by atomic absorption spectrophotometry, with adequate quality control measures using hair as biopsy material. The study group includes the male workers such as welders, foundry man, fitter, hammer man, machine man, cupola man etc., besides office workers of locomotive workshop in Ajmer and surrounding areas exposed to different metals. Age and sex matched controls of persons working in the same area of work in offices etc. and not exposed to metal pollution were selected for valid comparison. It is proposed to validate the use of hair as a biological marker for assessing metal body burden of workers. In our study significant correlations have been found between skin disease and Cr, Mn, Fe, Cu; chest pain and Pb; hypertension and Cu, Mn; mental stress and Mn, Ni, Cu, Zn; liver problem and Ni; indigestion and Cr; Ni, diabetes and Cr, Mn, Ni; tuberculosis and Zn; breathing trouble and Cr, Mn, Fe, Ni, Zn. The advantages of choosing hair as a biopsy material are also given.