Lyso-Sulfatide Binds Factor Xa and Inhibits Thrombin Generation by the Prothrombinase Complex

Blood coagulation reactions are strongly influenced by phospholipids, but little is known about the influence of sphingolipids on coagulation mechanisms. Lysosulfatide (lyso-SF) (sulfogalactosyl sphingosine) prolonged factor Xa (fXa) 1-stage plasma clotting assays, showing it had robust anticoagulant activity. In studies using purified clotting factors, lyso-SF inhibited >90% of prothrombin (II) activation for reaction mixtures containing fXa/factor Va (fVa)/II, and also inhibited II activation generation by fXa/ phospholipids and by Gla-domainless-fXa/fVa/phospholipids. When lyso-SF analogs were tested, results showed that N-acetyl-sulfatide was not anticoagulant, implying that the free amine group was essential for the anticoagulant effects of lyso-SF. Lyso-SF did not inhibit fXa enzymatic hydrolysis of small peptide substrates, showing it did not directly inhibit the fXa activity. In surface plasmon resonance studies, lyso-SF bound to immobilized inactivated fXa as well as inactivated Gla-domainless-fXa. Confirming this lyso-SF:fXa interaction, fluorescence studies showed that fluorescently-labeled-fXa in solution bound to lyso-SF. Thus, lyso-SF is an anticoagulant lipid that inhibits fXa when this enzyme is bound to either phospholipids or to fVa. Mechanisms for inhibition of procoagulant activity are likely to involve lyso-SF binding to fXa domain(s) that are distinct from the fXa Gla domain. This suggests that certain sphingolipids, including lyso-SF and some of its analogs, may down-regulate fXa activity without inhibiting the enzyme’s active site or binding to the fXa Gla domain.     

Cloning,expression of b-1,3-1,4 glucanase from Bacillus subtilis SU40 and the effect of calcium ion on the stability of recombinant enzyme: in vitro and in silico analysis

A new glucanolytic bacterial strain, SU40 was isolated, and identified as Bacillus subtilis on the basis of 16S rRNA sequence homology and phylogenetic tree analysis. The gene encoding β-1,3-1,4-glucanase was delineated, cloned into pET 28a+ vector and heterologously overexpressed in Escherichia coli BL21(DE3). The purified recombinant enzyme was about 24 kDa. The enzyme exhibited maximum activity (36.84 U/ml) at 60°C, pH 8.0 and maintained 54% activity at 80°C after incubation for 60 min. The enzyme showed activity against β-glucan, lichenan, and xylan. Amino acid sequence shared a conserved motif EIDIEF. The predicted three-dimensional homology model of the enzyme showed the presence of catalytic residues Glu105, Glu109 and Asp107, single disulphide bridge between Cys32 and Cys61 and three calcium binding site residues Pro9, Gly45 and Asp207. Presence of calcium ion improves the thermal stability of SU40 β-1,3-1,4-glucanase. Molecular dynamics simulation studies revealed that the absence of calcium ion fluctuate the active site residues which are responsible for thermostability. The high catalytic activity and its stability to temperature, pH and metal ions indicated that the enzyme β-1,3-1,4-glucanase by B. subtilis SU40 is a good candidate for biotechnological applications.     

Tissue-specific temporal exome capture revealed muscle-specific genes and SNPs in Indian buffalo (Bubalus bubalis)

Whole genome sequencing of buffalo is yet to be completed,and in the near future it may not be possible to identify an exome(coding region of genome) through bioinformatics for designing probes to capture it.In the present study,we employed in solution hybridization to sequence tissue specific temporal exomes(TST exome) in buffalo.We utilized cDNA prepared from buffalo muscle tissue as a probe to capture TST exomes from the buffalo genome.This resulted in a prominent reduction of repeat sequences(up to 40%) and an enrichment of coding sequences(up to 60%).Enriched targets were sequenced on a 454 pyro-sequencing platform,generating 101,244 reads containing 24,127,779 high quality bases.The data revealed 40,100 variations,of which 403 were indels and 39,218 SNPs containing 195 nonsynonymous candidate SNPs in protein-coding regions.The study has indicated that 80% of the total genes identified from capture data were expressed in muscle tissue.The present study is the first of its kind to sequence TST exomes captured by use of cDNA molecules for SNPs found in the coding region without any prior sequence information of targeted molecules.     

Field performance of transgenic sugarcane produced using Agrobacterium and biolistics methods

Future genetic improvement of sugarcane depends, in part, on the ability to produce high‐yielding transgenic cultivars with improved traits such as herbicide and insect resistance. Here, transgenic sugarcane plants generated by different transformation methods were assessed for field performance over 3 years. Agrobacterium‐mediated (Agro) transgenic events (35) were produced using four different Agrobacterium tumefaciens strains, while biolistic (Biol) transgenic events (48) were produced using either minimal linearized DNA (LDNA) transgene cassettes with 5′, 3′ or blunt ends or whole circular plasmid (PDNA) vectors containing the same transgenes. A combined analysis showed a reduction in growth and cane yield in Biol, Agro as well as untransformed tissue culture (TC) events, compared with the parent clone (PC) Q117 (no transformation or tissue culture) in the plant, first ratoon and second ratoon crops. However, when individual events were analysed separately, yields of some transgenic events from both Agro and Biol were comparable to PC, suggesting that either transformation method can produce commercially suitable clones. Interestingly, a greater percentage of Biol transformants were similar to PC for growth and yield than Agro clones. Crop ratoonability and sugar yield components (Brix%, Pol%, and commercial cane sugar (CCS)) were unaffected by transformation or tissue culture. Transgene expression remained stable over different crop cycles and increased with plant maturity. Transgene copy number did not influence transgene expression, and both transformation methods produced low transgene copy number events. No consistent pattern of genetic changes was detected in the test population using three DNA fingerprinting techniques.     

Peripheral inhibin levels in relation to climatic variations and stage of estrous cycle in buffalo (Bubalus bubalis )

The present study investigated the peripheral plasma inhibin levels in relation to 1) the stage of estrous cycle and the effect of climatic variations. Blood samples were collected from cyclic buffalo (n=5) once daily for 32 consecutive days during the tropical hot humid (summer) and cold (winter) seasons. Estrus was recorded by parading a vasectomized bull as well as by plasma progesterone determination. In the winter season, peripheral inhibin concentrations which were lowest (0.35 +/- 0.02 ng/ml) during the mid-luteal phase of estrous cycle (Day 6 to Day 14, Day 0 = day of estrus) increased significantly (P < 0.02) to 0.47 +/- 0.04 ng/ml during the late luteal phase (Day -4 to Day -2) and then further to 0.52 +/- 0.03 ng/ml (P< 0.02) during the periestrus phase (Day -1 to Day 1). Inhibin concentrations then decreased significantly (P < 0.02) to 0.40 +/- 0.03 ng/ml during the early luteal phase (Day 2 to Day 5). In the summer season the differences in peripheral inhibin concentrations among different phases of estrous cycle were found to be nonsignificant. A comparison of the circulating inhibin concentrations between the two seasons indicated that inhibin concentrations were significantly higher in the late luteal phase (P < 0.01) and periestrus phase (P < 0.05) during the winter season compared with corresponding periods during the summer season. The present study suggests that peripheral inhibin concentrations change in the estrous cycle during cooler breeding season and that environmental heat stress can cause a reduction in peripheral inhibin concentrations.     

Shoot regeneration in stem expiants and its amenability to Agrobacterium tumefaciens mediated gene transfer in Brassica carinata

Immature stem segments of seven different genotypes of Brassica carinata produced shoots with variable frequencies when cultured in MS medium with BAP and picloram at 0.2 mg/l each. Line 171, which produced shoots with 100% efficiency from both cut ends of the expiant, was selected for testing the amenability of this regeneration protocol for genetic transformation. A non-oncogenic Agrobacterium tumefaciens containing plasmid PCV 730, a binary vector carrying resistance genes for kanamycin and hygromycin, was used. A cocultivation period of 4 d with a bacterial concentration of approximately 2.5×10 cells/ml, followed by a recovery period of 2 d, produced transformed shoots that could be selected and rooted in the presence of kanamycin at 15 mg/l. Transformation was confirmed by neomycin phospho-transferase assay and Southern blot analysis. Seed analysis of transformed plants indicated that kanamycin resistance was inherited in the progeny.Abbreviations BAP 6-Benzylaminopurine - Kn Kinetin - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid - ATP Adenosine triphosphate - MS Murashige and Skoog (1962) medium     

A propionitrile-induced nitrilase of <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. NDB 1165 and its application in nicotinic acid synthesis

Rhodococcus sp. NDB 1165, a nitrile-transforming organism was isolated from temperate forest soil of Himalayas. The nitrilase (EC 3.5.5.2) activity of this organism had higher substrate specificity toward aromatic nitriles (benzonitrile, 3-cyanopyridine and 4-cyanopyridine) and unsaturated aliphatic nitrile (acrylonitrile) in comparison to saturated aliphatic nitriles (acetonitrile, propionitrile, butyronitrile and isobutyronitrile) nitrile and arylacetonitrile (phenylacetonitrile and indole-3-acetonitrile). The nitrilase of Rhodococcus sp. NDB 1165 was inducible in nature and propionitrile proved to be an efficient inducer. However, the salts of ferrous and cobalt ions had an inhibitory effect. Under optimized reaction conditions (pH 8.0 and temperature 45°C) the nitrilase activity of this organism was 2.39 ± 0.07 U/mg dry cell mass (dcm). The half-life of this enzyme was 150 min and 40 min at 45°C and 50°C respectively. However, it was quite stable at 40°C and around 58 % activity was retained even after 6 h at this temperature. The V _max and K _m value of this nitrilase were 1.67 μmol/ml min and 0.1 M respectively using 3-cyanopyridine as substrate. However, the decrease in V _max and K _m values (0.56 μmol/ml min and 0.02 M, respectively) were ␣observed at >0.05 M 3-cyanopyridine which revealed that this enzyme experienced uncompetitive inhibition at higher substrate concentrations. Under optimized reaction conditions, 1.6 M 3-cyanopyridine was successfully converted in to nicotinic acid using 2.0 mg resting cells (dcm)/ml reaction mixture in 11 h. This is the highest production of nicotinic acid i.e. 8.95 mg/mg resting cells (dcm)/h as compared to nitrilase systems reported hitherto.     

Detection of a true breeding homeotic gene mutant Pps-1 with partially petaloid sepals in opium poppy (Papaver somniferum L.) and its genetic behavior

A spontaneous true breeding homeotic gene mutant Pps-1 with distinct partial petaloid sepals was detected in the population of downy mildew (DM)-resistant elite accession I-14 during our studies for the identification of disease resistance sources in opium poppy. The trait was found to be stable and inherited truly in the subsequent generations. Genetic studies were carried out through systematic reciprocal crosses with the parental wild-type genotype I-14, and segregation pattern of phenotypic characteristics in F(1) and F(2) populations clearly indicated single recessive nuclear gene control of the mutant character. The studies have demonstrated that the mutant phenotype is due to mutations at the Pps-1 locus that possibly corresponds to B-class function (according to ABC model) with negative control function. The mutant Pps-1 being single-whorl homeotic mutant might greatly help in providing insight into mechanisms of flower development in opium poppy.     

An improved delta-centralization method for population stratification

Dadd et al. [Hum Hered 2010;69:285-294] recently criticized our delta-centralization (DC) method of controlling for population stratification (PS) and concluded that DC does not work. To explore our method, the authors simulated data under the Balding-Nichols (BN) model, which is more general than the model we had used in our simulations. They determined that the DC method underestimated the PS parameter (δ) and inflated the type I error rates when applied to BN-simulated data, and from this they concluded that the DC method is invalid. However, we argue that this conclusion is premature. In this paper, we (1) show why δ is underestimated and type I error rates are inflated when BN-simulated data are used, and (2) present a simple adjustment to DC that works reasonably well for data from both kinds of simulations. We also show that the adjusted DC method has appropriate power under a range of scenarios.