Group III PLA2 from the scorpion, Mesobuthus tamulus: cloning and recombinant expression in E. coli

Phospholipases A2 (PLA2) are enzymes that specifically hydrolyze the sn-2 fatty acid acyl bond of phospholipids, producing a free fatty acid and a lyso-phospholipid. We report the cloning and expression of a secretory phospholipase A2 (sPLA2) from Mesobuthus tamulus, Indian red scorpion. The nucleotide sequence codes for a 167 residue enzyme. The open reading frame codes for a 31 amino acid signal peptide followed by a mature portion of the protein. The primary structure shows the calcium binding motif, catalytic residues, 8 highly-conserved cysteines and C-terminal extension which classify it as a group III PLA2. The entire transcript was expressed in Escherichia coli and was purified by metal affinity chromatography under denaturing conditions. The protein was refolded by serial dilutions in the refolding buffer to its active form. Hemolytic assays indicate that the protein adopts a functional conformation. The functional requisites such as optimum pH of 8 and calcium dependency are shown. This report provides a simple but robust methodology for recombinant expression of toxic proteins

Inverse relationship between chitobiase and transglycosylation activities of chitinase-D from Serratia proteamaculans revealed by mutational and biophysical analyses

Serratia proteamaculans chitinase-D (SpChiD) has a unique combination of hydrolytic and transglycosylation (TG) activities. The TG activity of SpChiD can be used for large-scale production of chito-oligosaccharides (CHOS). The multiple activities (hydrolytic and/or chitobiase activities and TG) of SpChiD appear to be strongly influenced by the substrate-binding cleft. Here, we report the unique property of SpChiD substrate-binding cleft, wherein, the residues Tyr28, Val35 and Thr36 control chitobiase activity and the residues Trp160 and Trp290 are crucial for TG activity. Mutants with reduced (V35G and T36G/F) or no (SpChiDΔ30–42 and Y28A) chitobiase activity produced higher amounts of the quantifiable even-chain TG product with degree of polymerization (DP)-6, indicating that the chitobiase and TG activities are inversely related. In addition to its unprecedented catalytic properties, unlike other chitinases, the single modular SpChiD showed dual unfolding transitions. Ligand-induced thermal stability studies with the catalytically inactive mutant of SpChiD (E153A) showed that the transition temperature increased upon binding of CHOS with DP2–6. Isothermal titration calorimetry experiments revealed the exceptionally high binding affinities for E153A to CHOS with DP2–6. These observations strongly support that the architecture of SpChiD substrate-binding cleft adopted to control chitobiase and TG activities, in addition to usual chitinase-mediated hydrolysis

Assessment of Vaccine Wastage during a Pulse Polio Immunization Programme in India

A study to assess the wastage factor of oral polio vaccine (OPV) in the Pulse Polio Immunization (PPI) programme of the Government of India was undertaken by the Indian Council of Medical Research (ICMR) at approximately 31,000 immunization booths all over the country. The study was conducted through the network of 31 Human Reproduction Research Centres (HRRCs) and other ICMR institutes. Wastage at the point of administration of OPV was estimated to be 14.5% with a wastage factor of 1.17 which is well below the assumed wastage of 33% and the corresponding wastage factor of 1.5 in the PPI programme. The wastage and wastage factor as estimated in the present study were also less than the wastage of 25% and the wastage factor of 1.33 recommended by the World Health Organization. Minimum wastage (6.3%) at Kanchipuram and maximum wastage (22.1%) at Kanpur were observed. Further, the wastage of unopened vials and vials during use was also observed following colour changes on the vaccine vial monitor (VVM), indicating poor coldchain maintenance at the immunization site. In total, 13 booths reported wastage of nine or more unopened vials, whereas 19 booths reported wastage of nine or more vials during use because of colour changes on VVM. Other reasons for wastage of vaccine were also observed from a sample of booths. The technology of introducing VVM on OPV vials for monitoring the cold-chain proved useful in situations in which mass vaccination programmes such as PPI are carried out

Structural studies of analgesics and their interactions. Part 4. Crystal structures of phenylbutazone and a 2 : 1 complex between phenylbutazone and piperazine

The X-ray crystal structures of 4-butyl-1,2-diphenylpyrazolidine-3,5-dione (phenylbutazone)(I). and its 2 : 1 complex (II) with piperazine have been determined by direct methods and the structures refined to R 0.096 (2 300 observed reflections measured by diffractometer) and 0.074 (2 494 observed reflections visuallyestimated). Crystals are monoclinic, space group P21/c; for (I)a= 21.695(4), b= 5.823(2), c= 27.881(4)Å, = 108.06 (10)°, Z= 8, and for (II)a= 8.048(4), b= 15.081(4), c= 15.583(7)Å, = 95.9(3)°, Z= 2. The two crystallographically independant molecules in the structure of (I) are similar except for the conformation of the butyl group, which is disordered in one of the molecules. In the pyrazolidinedione group, the two C–C bonds are single and the two C–O bonds double. The two nitrogen atoms in the five-membered ring are pyramidal with the attached phenyl groups lying on the opposite sides of the mean plane of the ring. The phenylbutazone molecule in (II) exists as a negative ion owing to deprotonation of C-4. C-4 is therefore trigonal and the orientation of the Bu group with respect to the pyrazolidinedione group is considerably different from that in (I); there is also considerable electron delocalization along the C–O and C–C bonds. These changes in geometry and electronic structure may relate to biological activity. The doubly charged cationic piperazine molecule exists in the chair form with the nitrogen atoms at the apices. The crystal structure of (II) is stabilized by ionic interactions and N–H O hydrogen bonds

Improvement of morpho-physiological traits in 'F₄' generation of Chickpea for increasing productivity

30-33India occupies first rank in the world by producing more than 25% of world food legume and largest producer of chickpea (Cicer arietinum L.) in the world. Chickpea is the third most important pulse among pulses in the world. Contribution of India is 64% in global chickpea production and productivity rank of the India is 29th. In India, chickpea crop ranks in the first position among pulses, occupying about 30% at total cultivated area of pulses and its contribution is 40% of the total pulse production. Observation were recorded on various morphophysiological traits in six parents and their six (F4) crosses. The results indicated that crosses between Pusa 256×F6-936 and Pusa 362 × Saki 9516 showed significantly lower membrane injury index (MII) i.e. 33.9 and 28.5% which were significantly less than their parents. Pusa 256 (47.9%), F6-936 (56.8%), Pusa 362 (34.1%) and Saki 9516 (55.9%). On the basis of results obtained, it is emphasized that parental lines having desirable physiological traits like relative water content (RWC), chlorophyll content and MII might have contributed directly for increasing productivity of new varieties under rainfed conditions

Structural studies of analgesics and their interactions. Part 4. Crystal structures of phenylbutazone and a 2 : 1 complex between phenylbutazone and piperazine

The X-ray crystal structures of 4-butyl-1,2-diphenylpyrazolidine-3,5-dione (phenylbutazone)(I). and its 2 : 1 complex (II) with piperazine have been determined by direct methods and the structures refined to R 0.096 (2 300 observed reflections measured by diffractometer) and 0.074 (2 494 observed reflections visuallyestimated). Crystals are monoclinic, space group P21/c; for (I)a= 21.695(4), b= 5.823(2), c= 27.881(4)Å, = 108.06 (10)°, Z= 8, and for (II)a= 8.048(4), b= 15.081(4), c= 15.583(7)Å, = 95.9(3)°, Z= 2. The two crystallographically independant molecules in the structure of (I) are similar except for the conformation of the butyl group, which is disordered in one of the molecules. In the pyrazolidinedione group, the two C–C bonds are single and the two C–O bonds double. The two nitrogen atoms in the five-membered ring are pyramidal with the attached phenyl groups lying on the opposite sides of the mean plane of the ring. The phenylbutazone molecule in (II) exists as a negative ion owing to deprotonation of C-4. C-4 is therefore trigonal and the orientation of the Bu group with respect to the pyrazolidinedione group is considerably different from that in (I); there is also considerable electron delocalization along the C–O and C–C bonds. These changes in geometry and electronic structure may relate to biological activity. The doubly charged cationic piperazine molecule exists in the chair form with the nitrogen atoms at the apices. The crystal structure of (II) is stabilized by ionic interactions and N–H O hydrogen bonds

Effect of sowing time of chickpea on its yield and plant growth in North –Western part of India

31-35The cultivation of chickpea (Cicer arietinum L.) is done on 35% area of the total area under pulses in India. Chickpea contributes 45% towards production of pulses. India’s share of chickpea in world level is about 67%. Chickpea is third most important pulse crop of the world. It is an excellent crop from diversification point of view. With limited resources, chickpea is more renumerative crop in north-western parts of India than wheat because it requires comparatively less inputs. More than 85% of chickpea is grown under rainfed areas and also grown on conserved soil moisture after the harvest of kharif crop . An experiment was conducted at I.A.R.I. New Delhi with three dates of sowing i.e. 15 November, 30 November and 15 December. It was observed that 30th November was better date of sowing than 15th November and 15ThDecember . Due to comparatively low temperature experienced by the crop in which growth of flowers, pod formation and seed development were better in perforamance. Late planting reduces the biomass production but increases harvest index. In early planting due to low temperature at flowering stage flowers and pods dropping was more resulting in poor yield. On the other hand in advance lines the harvest index of the genotypes was high. Though the biomass was poor resulting into the low seed yield. Therefore, date of sowing 30th  November was more appropriate for chickpea planting under north western parts of India. Seed yield per plant and number of seed per pod were higher on this date of sowing in comparison to other two dates of sowing i.e. 15 Nov. and 15 Dec. The yield of the crop sown on 30th November was 10% more than the crop sown on 15th November and 22%more than the crop sown on 15th December. The yield of advance lines was much more in comparison to the released genotypes.</sup

Response of different sowing dates on seed protein content and productiviQ of chickpea (Cicer arietirzum) genotgrpes

A field experiment was conducted during 2002-03 with 6 chickpea genotypes including, 3 released varieties, viz 'Pusa 256; "Pusa 372', 'BGD 72' and 3 advance lines, vk 'DG 36', 'DG 46', and 'DG 51' under different planting dates : normal (15 November), late (30 Nov.) and very late (1 5 December) were grown . The seed yield and seed protein content were significantly higher in late sowing, higher percentage of protein (25.55%) was recorded in late planting followed by very late (24.85%) and normal (23.83%) plantings. Among the genotypes DG 46' and 'DG 5 1 ' showed higher mean values of protein content, ie 25.88 and 25.05% respectively. In general advance breeding lines showed hgher mean values of protein content in comparison to the released varieties. The higher seed yield was recorded when plants were grown in late planting with 10 % more yield than normal and 22 %higher than very late planting. On the basis of data of heat-use efficiency advance line 'DG 36' was found superior as compared to other advance lines and released genotypes, so such material may be included in breeding programme to improve these desirable traits