Cyanase-mediated utilization of cyanate in Pseudomonas fluorescens NCIB 11764.

Pseudomonas fluorescens NCIB 11764 was capable of utilizing cyanate (OCN-) as a sole nitrogen source for growth. Crude cell extracts from cells grown on cyanate, but not on ammonium sulfate, were induced for an enzyme catalyzing cyanate conversion to ammonia. Enzymatic activity was shown to be bicarbonate dependent and specific for cyanate as a substrate, suggesting that cyanate utilization in this organism is facilitated by an enzyme resembling cyanase (cyanate amidohydrolase; EC 3.5.5.3), as described previously in Escherichia coli and Flavobacterium sp.     

Degradation of the metal-cyano complex tetracyanonickelate(II) by cyanide-utilizing bacterial isolates

Ten bacterial isolates capable of growth on tetracyanonickelate(II) [K2[Ni(CN)4]] (TCN) as the sole nitrogen source were isolated from soil, freshwater, and sewage sludge enrichments. Seven of the 10 were identified as pseudomonads, while the remaining 3 were classified as Klebsiella species. A detailed investigation of one isolate, Pseudomonas putida BCN3, revealed a rapid growth rate on TCN (generation time, 2 h), with substrate removal and growth occurring in parallel. In addition to TCN, all isolates were able to utilize KCN, although the latter was significantly more toxic; MICs ranged from 0.2 to 0.8 mM for KCN and greater than or equal to 50 mM for TCN. While growth occurred over a wide range of TCN concentrations (0.25 to 16 mM), degradation was most substantial under growth-limiting conditions and did not occur when ammonia was present. In addition, cells grown on TCN were found to accumulate nickel cyanide [Ni(CN)2] as a major biodegradation product. The results show that bacteria capable of growth on TCN can readily be isolated and that degradation (i) appears to parallel the capacity for growth on KCN, (ii) does not occur in the presence of ammonia, and (iii) proceeds via the formation of Ni(CN)2 as a biological metabolite.     

Characterization of single-stranded DNA-binding proteins from Mycobacteria. The carboxyl-terminal of domain of SSB is essential for stable association with its cognate RecA protein

Single-stranded DNA-binding proteins (SSB) play an important role in most aspects of DNA metabolism including DNA replication, repair, and recombination. We report here the identification and characterization of SSB proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis. Sequence comparison of M. smegmatis SSB revealed that it is homologous to M. tuberculosis SSB, except for a small spacer connecting the larger amino-terminal domain with the extreme carboxyl-terminal tail. The purified SSB proteins of mycobacteria bound single-stranded DNA with high affinity, and the association and dissociation constants were similar to that of the prototype SSB. The proteolytic signatures of free and bound forms of SSB proteins disclosed that DNA binding was associated with structural changes at the carboxyl-terminal domain. Significantly, SSB proteins from mycobacteria displayed high affinity for cognate RecA, whereas Escherichia coli SSB did not under comparable experimental conditions. Accordingly, SSB and RecA were coimmunoprecipitated from cell lysates, further supporting an interaction between these proteins in vivo. The carboxyl-terminal domain of M. smegmatis SSB, which is not essential for interaction with ssDNA, is the site of binding of its cognate RecA. These studies provide the first evidence for stable association of eubacterial SSB proteins with their cognate RecA, suggesting that these two proteins might function together during DNA repair and/or recombination.     

Mycobacterium tuberculosis RecA intein,a LAGLIDADG homing endonuclease,displays Mn(2+) and DNA-dependent ATPase activity

Mycobacterium tuberculosis RecA intein (PI-MtuI), a LAGLIDADG homing endonuclease, displays dual target specificity in response to alternative cofactors. While both ATP and Mn²⁺ were required for optimal cleavage of an inteinless recA allele (hereafter referred to as cognate DNA), Mg²⁺ alone was sufficient for cleavage of ectopic DNA sites. In this study, we have explored the ability of PI-MtuI to catalyze ATP hydrolysis in the presence of alternative metal ion cofactors and DNA substrates. Our results indicate that PI-MtuI displays maximum ATPase activity in the presence of cognate but not ectopic DNA. Kinetic analysis revealed that Mn²⁺ was able to stimulate PI-MtuI catalyzed ATP hydrolysis, whereas Mg²⁺ failed to do so. Using UV crosslinking, limited proteolysis and amino acid sequence analysis, we show that ³²P-labeled ATP was bound to a 14 kDa peptide containing the putative Walker A motif. Furthermore, the limited proteolysis approach disclosed that cognate DNA was able to induce structural changes in PI-MtuI. Mutation of the presumptive metal ion-binding ligands (Asp122 and Asp222) in the LAGLIDADG motifs of PI-MtuI impaired its affinity for ATP, thus resulting in a reduction in or loss of its endonuclease activity. Together, these results suggest that PI-MtuI is a (cognate) DNA- and Mn²⁺-dependent ATPase, unique from the LAGLIDADG family of homing endonucleases, and implies a possible role for ATP hydrolysis in the recognition and/or cleavage of homing site DNA sequence.     

Atropisomeric quinazolin-4-one derivatives are potent noncompetitive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists

Piriqualone (1) was found to be an antagonist of AMPA receptors. Structure activity optimization was conducted on each of the three rings in 1 to afford a series of potent and selective antagonists. The sterically crowded environment surrounding the N-3 aryl group provided sufficient thermal stability for atropisomers to be isolated. Separation of these atropisomers resulted in the identification of (+)-38 (CP-465,022), a compound that binds to the AMPA receptor with high affinity (IC50 = 36 nM) and displays potent anticonvulsant activity.     

Null alleles at the Huntington disease locus: implications for diagnostics and CAG repeat instability

PCR amplification of the CAG repeat in exon 1 of the IT15 gene is routinely undertaken to confirm a clinical diagnosis of Huntington disease (HD) and to provide predictive testing for at-risk relatives of affected individuals. Our studies have detected null alleles on the chromosome carrying the expanded repeat in three of 91 apparently unrelated HD families. Sequence analysis of these alleles has revealed the same mutation event, leading to the juxtaposition of uninterrupted CAG and CCG repeats. These data suggest that a mutation-prone region exists in the IT15 gene bounded by the CAG and CCG repeats and that caution should be exercised in designing primers that anneal to the region bounded by these repeats. Two of the HD families segregated null alleles with expanded uninterrupted CAG repeats at the lower end of the zone of reduced penetrance. The expanded repeats are meiotically unstable in these families, although this instability is within a small range of repeat lengths. The haplotypes of the disease-causing chromosomes in these two families differ, only one of which is similar to that reported previously as being specific for new HD mutations. Finally, no apparent mitotic instability of the uninterrupted CAG repeat was observed in the brain of one of the HD individuals.     

Osteopontin reduces polyspermy during in vitro fertilization of porcine oocytes

This study was designed to determine the role of osteopontin (SPP1) in in vitro fertilization (IVF) in swine. The initial objective was to evaluate the effect of various concentrations of SPP1 (0, 0.001, 0.01, 0.1 and 1 microg/ml) on spermatozoa and oocytes during IVF. The results demonstrate that SPP1 reduced the rate of polyspermy in a dose-dependent manner (P < 0.05). SPP1 also reduced both the number of sperm in oocytes as compared to the control and the number of spermatozoa bound to the zona pellucida (ZP) (P < 0.05). High doses of SPP1 (1 microg/ml) reduced penetration and male pronucleus formation as compared to the control (P < 0.05). Interestingly, compared to the control group, medium doses of SPP1 increased fertilization efficiency (42.6% and 44.6% vs. 31.6%; P < 0.05), representing a 41% improvement for 0.1 microg/ml SPP1). The ZP of 0.1 microg/ml SPP1-treated oocytes was more difficult to digest than control oocytes (P < 0.05). The percentage of acrosome-reacted spermatozoa bound to the ZP during IVF increased after 4 h of 1.0 microg/ml SPP1 treatment compared to 0 or 0.1 microg/ml SPP1. SPP1 did not have an effect on sperm motility, progressive motility, and sperm viability. To confirm that the reduction of polyspermy was specific to SPP1, a mixture of pregnancy-associated glycoproteins was included in the IVF protocol and shown to have no effect on polyspermy. Furthermore, Western blotting demonstrated that a 50-kDa SPP1 form was present in the oviducts on Days 0, 3, and 5 in pregnant and nonpregnant gilts, and the concentration of SPP1 on Day 0 was higher than on Days 3 and 5. The current study represents the first report to demonstrate that SPP1 plays an important role in the regulation of pig polyspermic fertilization; it decreases polyspermy and increases fertilization efficiency during IVF.     

Growth model for raceway pond cultivation of Desmodesmus sp. MCC34 isolated from a local water body

Biofuel production by microalgae has the advantage of higher biomass productivity over land crops. The selection of potential microalgae depends on the growth in outdoor mass cultivation during different seasons, which can be predicted by a mathematical model. Here, freshwater green algae were isolated from a local water body in Pilani, Rajasthan, India (geographical coordinates: 28°22′N 75°36′E) and characterized by microscopy and ribosomal RNA analysis. The strain was submitted to the Indian Agricultural Research Institute's microbial culture collection (IARI, India) and identified as Desmodesmus sp. MCC34. This strain, along with a fresh water green algae (Chlorella minutissima), two marine green algae species (Dunaliella salina and Dunaliella tertiolecta) and two nitrogen fixing cyanobacteria (Nostoc muscorum and Anabaena doliolum), were screened for lipid productivity and growth kinetics under culture room and raceway pond conditions. Desmodesmus sp. MCC34 showed the highest specific growth rate (0.26 day^?1), biomass production (1.9 g L^?1) and lipid productivity (103 mg L^?1 day^?1). The optimal temperature and saturating light intensity for maximal growth of Desmodesmus sp. MCC34 were 35 °C and 75 μmol m^?2 s^?1 with molar extinction coefficient of 0.22 m² g^?1, respectively. Desmodesmus sp. MCC34 was then subjected to outdoor cultivation in a 20‐m long raceway pond for 18 days during March and November 2013. The areal biomass productivity and volumetric biomass productivity were 13946.23 kg ha^?1 year^?1 and 56.94 mg L^? 1day^?1 during the month of March, decreasing to 6262.28 kg ha^?1 year^?1 and 25.57 mg L^? 1day^?1 during the month of November. A mathematical model was constructed to explain the relationship between biomass production and growth parameters such as temperature, light intensity and nutrient concentration. The productivity values predicted with the proposed model correspond well with the experimental data, suggesting the validity of the model.