Identification of microRNAs and their targets in Finger millet by high throughput sequencing.

MicroRNAs are short non-coding RNAs which play an important role in regulating gene expression by mRNA cleavage or by translational repression. The majority of identified miRNAs were evolutionarily conserved; however, others expressed in a species-specific manner. Finger millet is an important cereal crop; nonetheless, no practical information is available on microRNAs to date. In this study, we have identified 95 conserved microRNAs belonging to 39 families and 3 novel microRNAs by high throughput sequencing. For the identified conserved and novel miRNAs a total of 507 targets were predicted. 11 miRNAs were validated and tissue specificity was determined by stem loop RT-qPCR, Northern blot. GO analyses revealed targets of miRNA were involved in wide range of regulatory functions. This study implies large number of known and novel miRNAs found in Finger millet which may play important role in growth and development.

Anti-coagulant activity of a metalloprotease: further characterization from the Indian cobra (Naja naja) venom.

A high molecular mass, non toxic metalloprotease the NN-PF3 with the bound Ca(2+) and Zn(2+) from the Naja naja venom has been studied further for its anticoagulant property. The molecular mass by MALDI-TOF mass spectrometry was 67.81 kDa. The NN-PF3 exhibited fibrin(ogen)olytic activity. In addition to fibrinogen, NN-PF3 hydrolyzed blood and plasma clot with the later hydrolyzed about one fold higher. The alpha polymer of fibrin was preferentially hydrolyzed over the alpha chain but the beta chain and gamma-gamma dimer remained untouched. It was devoid of plasminogen activation property. It prolonged the activated partial thromboplastin time, prothrombin time and the thrombin clotting time of citrated human plasma. It did not affect the thrombin activity. In mice, defibrinogentaion, prolonged bleeding time (P < 0.01) and reduced fibrinogen level were observed following intravenous injection. Human plasma or alpha2-macroglobulin did not, but the polyvalent anti-venom inhibited the NN-PF3 activity. In contrast to most snake venom metalloproteases, it did not degrade extra cellular matrix proteins.

Factor Xa-like and fibrin(ogen)olytic activities of a serine protease from Hippasa agelenoides spider venom gland extract.

In the recent past, a low molecular mass serine protease, the Hag-protease that caused pro-coagulant activity and as well as local toxicity was isolated and characterized from the Hippasa agelenoides spider venom gland extract (Devaraja et al., Toxicon 52:130-138, 2008). In the current study, the pro-coagulant property has been investigated further and the results are presented. The Hag-protease reduced the re-calcification time of citrated human plasma. It reduced the activated partial thromboplastin time (APTT), and prothrombin time (PT) suggesting its participation in common pathway of blood coagulation. Interestingly, it coagulated the citrated human plasma in the absence of CaCl(2) but, it was lacking thrombin-like activity as it did not clot the purified fibrinogen. Strikingly, the enzyme coagulated the factor X deficient congenital human plasma, suggesting the factor Xa-like activity. However, the cumulative augmented activity was observed in presence of CaCl(2) and phospholipids. Further, the Hag-protease preferentially hydrolyzed the Aalpha chain and then the Bbeta-chain, but not the gamma-chain. As a result, truncated fibrinogen generated was lacking in the polymerization property. It hydrolyzed all the subunits of partially cross-liked fibrin clot (alpha-polymer, alpha-chain, beta-chain, and gamma-gamma dimers). Further, at low concentrations, the Hag-protease stimulated the aggregation of human platelets in platelet rich plasma, but at high concentrations caused spontaneous clumping. In contrast, it inhibited the collagen induced aggregation of washed human platelets. In summary, the present study for the first time reporting the factor Xa-like activity of a serine protease especially from the spider venom that exhibited opposing effects on hemostasis, the pro-coagulant activity and the anti-coagulant activity including fibrin(ogen)olytic and platelet aggregation inhibition activities.

Purification and characterization of a proteinase inhibitor from field bean, Dolichos lablab perpureus L.

A proteinase inhibitor resembling Bowman-Birk family inhibitors has been purified from the seeds of cultivar HA-3 of Dolichos lablab perpureus L. The protein was apparently homogeneous as judged by SDS-PAGE, PAGE, IEF, and immunodiffusion. The inhibitor had 12 mole% 1/2-cystine and a few aromatic amino acids, and lacks tryptophan. Field bean proteinase inhibitor (FBPI) exhibited a pI of 4.3 and an Mr of 18,500 Da. CD spectral studies showed random coiled secondary structure. Conformational changes were detected in the FBPI-trypsin/chymotrypsin complexes by difference spectral studies. Apparent Ka values of complexes of inhibitor with trypsin and chymotrypsin were 2.1x10(7) M(-1) and 3.1x10(7) M(-1), respectively. The binary and ternary complexes of FBPI with trypsin and chymotrypsin have been isolated indicating 1:1 stoichiometry with independent sites for cognate enzymes. Amino acid modification studies showed lysine and tyrosine at the reactive sites of FBPI for trypsin and chymotrypsin, respectively.

Inhibition of human class 3 aldehyde dehydrogenase, and sensitization of tumor cells that express significant amounts of this enzyme to oxazaphosphorines, by chlorpropamide analogues.

In some cases, acquired as well as constitutive tumor cell resistance to a group of otherwise clinically useful antineoplastic agents collectively referred to as oxazaphosphorines, e.g. cyclophosphamide and mafosfamide, can be accounted for by relatively elevated cellular levels of an enzyme, viz. cytosolic class 3 aldehyde dehydrogenase (ALDH-3), that catalyzes their detoxification. Ergo, inhibitors of ALDH-3 could be of clinical value since their inclusion in the therapeutic protocol would be expected to sensitize such cells to these agents. Identified in the present investigation were two chlorpropamide analogues showing promise in that regard, viz. (acetyloxy)[(4-chlorophenyl)sulfonyl]carbamic acid 1,1-dimethylethyl ester (NPI-2) and 4-chloro-N-methoxy-N-[(propylamino)carbonyl]benzenesulfonamide (API-2). Each inhibited NAD-linked benzaldehyde oxidation catalyzed by ALDH-3s purified from human breast adenocarcinoma MCF-7/0/CAT cells (IC50 values were 16 and 0.75 microM, respectively) and human normal stomach mucosa (IC50 values were 202 and 5 microM, respectively). The differential sensitivities of stomach mucosa ALDH-3 and breast tumor ALDH-3 to each of the two inhibitors can be viewed as further evidence that the latter is a subtle variant of the former. Human class 1 (ALDH-1) and class 2 (ALDH-2) aldehyde dehydrogenases were much less sensitive to NPI-2; IC50 values were >300 microM in each case. API-2, however, did not exhibit a similar degree of specificity; IC50 values for ALDH-1 and ALDH-2 were 7.5 and 0.08 microM, respectively. Each sensitized MCF-7/0/CAT cells to mafosfamide; the LC90 value decreased from >2 mM to 175 and 200 microM, respectively. Thus, the therapeutic potential of combining NPI-2 or API-2 with oxazaphosphorines is established.

Effect of cooking on proteinase inhibitors of Dolichos lablab bean (Dolichos lablab perpureus L.).

Proteinase inhibitory activity in ten different varieties of Dolichos lablab perpureus. L. was determined. All the varieties tested exhibited appreciable level of proteinase inhibitory activity (PIA). The trypsin inhibitory activity (TIA) (Mean: 20170 TIU/g) was relatively higher than the chymotrypsin inhibitory activity (CIA) (Mean: 15380 CIU/g). Effect of temperature and cooking on PIA was studied. The nature of cooking medium and duration of cooking had profound effect on the PIA. The dry fried seeds lost their PIA very rapidly (91% in 20 min). Seeds cooked in slightly alkaline medium lost their PIA quickly (89% in 30 min) compared to those cooked in acidic (80% in 30 min) and neutral pH (83% in 30 min). The PIA in green pods was also determined and they had only one third of the PIA (8200 TIU/g and 8125 CIU/g) found in the dry seeds.