Manoranjan Singh

Serotonin transporter promoter variants: Analysis in Indian autistic and control population.

Serotonin transporter (5-HTT) is a transmembrane protein belonging to Na+/Cl- dependent membrane transporter family and transports 5-HT across the membranes of presynaptic neurons. 5-HTT-linked polymorphic region (5-HTTLPR) gained much interest because of the differential regulation of expression and activity of 5-HTT by its various genotypes. A population-based study has been conducted on 5-HTTLPR with 358 individuals, which included 79 autistic probands, 136 parents, and 143 controls from two subpopulations of east and northeast regions of India. The genotypic frequencies of all the groups conform to Hardy-Weinberg equilibrium. With the finding of efficacy of serotonin reuptake inhibitors in ameliorating ritualistic behavior in autistic disorder, 5-HTT emerged as a putative candidate gene for autism and association studies have been carried out in different ethnic populations. But these studies were inconclusive due to conflicting results on association. Because such a study has never been performed in the Indian population, we have tested the possible involvement of 5-HTTLPR polymorphism with autism. The present study failed to establish any association or linkage of 5-HTTLPR with autism in the Indian population by case-control studies (chi2 = 1.314, P = 0.63) and family-based approaches (TDT chi2 = 0.22, P = 0.64 and HHRR-chi2 = 0.25, P = 0.61). However, when a meta-analysis of all the available TDT data, inclusive of the present study is carried out, we observed a significant preferential transmission of S-allele from parents to the affected offspring (chi2 = 7.51, P = 0.006) indicating an association of 5-HTTLPR with autism.

REFINED CRYSTAL STRUCTURE (2.3 A) OF A DOUBLE-HEADED WINGED BEAN ALPHA -CHYMOTRYPSIN INHIBITOR AND LOCATION OF ITS SECOND REACTIVE SITE

The crystal structure of a double‐headed α‐chymotrypsin inhibitor, WCI, from winged bean seeds has now been refined at 2.3 Å resolution to an R‐factor of 18.7% for 9,897 reflections. The crystals belong to the hexagonal space group P6122 with cell parameters a = b = 61.8 Å and c = 212.8 Å. The final model has a good stereochemistry and a root mean square deviation of 0.011 Å and 1.14° from ideality for bond length and bond angles, respectively. A total of 109 ordered solvent molecules were localized in the structure. This improved structure at 2.3 Å led to an understanding of the mechanism of inhibition of the protein against α‐chymotrypsin. An analysis of this higher resolution structure also helped us to predict the location of the second reactive site of the protein, about which no previous biochemical information was available. The inhibitor structure is spherical and has twelve anti‐parallel β‐strands with connecting loops arranged in a characteristic β‐trefoil fold common to other homologous serine protease inhibitors in the Kunitz (STI) family as well as to some non homologous functionally unrelated proteins. A wide variation in the surface loop regions is seen in the latter ones. Proteins 1999;35:321–331.

Reelin gene polymorphisms in the Indian population: a possible paternal 5'UTR-CGG-repeat-allele effect on autism.

Autism is a neurodevelopmental disorder with high heritability factor and the reelin gene, which codes for an extracellular matrix protein involved with neuronal migration and lamination is being investigated as a positional and functional candidate gene for autism. It is located on chromosome 7q22 within the autism susceptible locus (AUTS1); identified in earlier genome scans and several investigations have been carried out on various ethnic groups to assess possible association and linkage of the gene with autism. However, the findings are still inconclusive. In the present study which represents the first report of such a study on the Indian population, genotyping analyses of CGG repeat polymorphism at 5′UTR, two single nucleotide polymorphisms (SNP) at exon 6 and exon 50 were performed in 73 autistic subjects, 129 parents, and 80 controls. The allelic distributions of the repeat polymorphism and exon 50 T/C SNP were quite different from earlier reports in other populations. Allelic and genotypic distribution of the markers did not show any differences between the cases and controls. While our preliminary data on family‐based association studies on 58 trios showed no preferential transmission of any allele from the parents to the affected offspring, TDT and HHRR analyses revealed significant paternal transmission distortions for 10‐ and ≥11‐repeat alleles of CGG repeat polymorphism. Thus, the present study suggests that 5′UTR of reelin gene may have a role in the susceptibility towards autism with the paternal transmission and non‐transmission respectively of 10‐ and ≥11‐repeat alleles, to the affected offspring.

Design and synthesis of mannose analogues as inhibitors of α-mannosidase

Abstract A series of N-, C- and S- mannopyranosyl derivatives (4,9-16) have been synthesised and their inhibitory activity tested towards jackbean α-mannosidase (EC 3.2.1.24). These compounds are of mechanistic and synthetic interest in the design of new α-mannosidase inhibitors.

Psophocarpin B1, a storage protein of Psophocarpus tetragonolobus, has chymotrypsin inhibitory activity

Abstract Psophocarpin B 1 , a major storage protein of winged bean ( Psophocarpus tetragonolobus ) seeds, inhibits the activity of bovine pancreatic chymotrypsin. This inhibitory activity is abolished by heat treatment above 70°. In germinating seeds total chymotrypsin inhibitory activity (CIA) declines steadily as germination proceeds. By means of the Western blotting technique, it has been demonstrated that both psophocarpin B 1 and CIA disappear concurrently during germination.

Purification of a storage protein of Psophocarpus tetragonolobus

Abstract Psophocarpin B 1 , one of the major seed storage proteins of Psophocarpus tetragonolobus (winged bean), has been purified to homogeneity and crystallized. It is an acidic protein with a pI of 5.5 and M r of 20 000, as determined by gel filtration in the absence of denaturating agents. Polyacrylaniide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE), indicates the absence of a quaternary structure of the protein. Using specific antibody raised in rabbits, a radio-immunoassay (RIA) method has been developed for its quantitation. It shows that 12% of the total soluble protein in the tuber of this plant is psophocarpin B 1 whereas it represents 9% of the seed protein. In the pods, it is only a minor component (1% of the total soluble protein) and it is not detectable in stems and leaves. Psophocarpin B 1 undergoes a time-dependent degradation in germinating seeds.

Purification and characterization of a protein phosphatase from winged bean

A protein phosphatase (WbPP) has been purified from the soluble fraction of the winged bean (Psophocarpus tetragonolobus) shoot extract. The preparation is essentially homogenous as shown by the constant specific activity of the enzyme across the peak fractions, eluted from the thiophosphorylated histone-Sepharose affinity column, the last step of purification and by single protein bands on polyacrylamide gel electrophoresis (PAGE) in the presence as well as absence of denaturating agents. The monomeric nature of WbPP is revealed by an M(r) of 92,000 and 85,000, respectively, as estimated by SDS-PAGE and gel permeation chromatography under non-denaturating conditions. Autophosphorylated calmodulin-like domain protein kinase (P-WbCDPKI) [Saha, P., & Singh, M. (1995). Biochem. J., 305, 205] and phosphohistone H1 (P-hisH1), prepared by using the other homologous CDPK, i.e. WbCDPKII [Ganguly, S., & Singh, M. (1998). Phytochemistry, 48(1), 61], are good substrates of the purified enzyme, while P-hisH1 and phosphocasein prepared by using heterologous cAMP-dependent protein kinase, are respectively very poor and totally inactive as substrate. WbPP is adjudged to be a protein phosphoserine phosphatase since phosphoserine is the only phosphorylated amino acid residue detected in our earlier analysis of P-WbCDPKI and P-hisH1. The enzyme is strongly stimulated by a combination of Mg2+ and Ca2+, without being dependent on either of them and is also unaffected by calmodulin and fluphenazine. Orthovanadate strongly inhibits the enzyme while okadaic acid is a poor inhibitor.

Characterization of a second calcium-dependent protein kinase from winged bean

In plants, Ca2+ has emerged as the predominant second messenger for signal transduction, as cyclic nucleotides are not known to play any significant role in this system. Earlier, we characterized an interesting Ca(2+)-dependent protein kinase, WbCDPK (winged bean calmodulin-like domain protein kinase), from the soluble fraction of winged bean (Psophocarpus tetragonolobus) shoot extract. Here an isoform of WbCDPK is purified to apparent homogeneity from the same winged bean shoot extract. It is a single polypeptide chain protein-serine kinase, having an M(r) of about 70,000 and like WbCDPK, its preferred substrates are histone H1, syntide 2 and MLC-peptide (a synthetic myosin light chain related peptide) and it is totally dependent on Ca2+ for its activity, but exogenous calmodulin (CaM) does not stimulate it. However, it is strongly inhibited by CaM antagonists, indicating the presence of a CaM-like domain, as in WbCDPK. The two enzymes do not cross react immunologically and the isoform differs significantly from WbCDPK in its apparent inability to catalyse the autophosphorylation reaction, which is known to cause down-regulation of substrate phosphorylation in the case of WbCDPK.

Crystallization and preliminary X-ray studies of psophocarpin B1, a chymotrypsin inhibitor from winged bean seeds.

Psophocarpin B1 is a 20,000 Mr protein of winged bean (Psophocarpus tetragonolobus) seeds having chymotrypsin inhibitory activity. Single crystals of this protein suitable for X-ray crystallographic studies have been obtained by the vapour diffusion method using ammonium sulphate. The crystals are hexagonal, space group P6(4)22 or P6(2)22, cell dimensions a = b = 61 A, c = 210 A. They are stable to irradiation with X-rays and diffract to at least 2.6 A resolution.

A concanavalin A-like lectin in the developing seed of Canavalia ensiformis

Abstract During the early stages of seed development (Stage A) in Canavalia ensiformis (jackbean), the presence of an α- d -mannoside specific lectin(s) is shown by hemagglutination assays, although the usual subunit of concanavalin A ( M r 26 000), is not detectable by SDS-polyacrylamide gel electrophoresis. By employing affinity chromatography on Sephadex G-50 we have identified a Con A-like lectin with specificity to α- d -mannoside and α- d -glucoside in the seeds at early stages of development. Its native M r has been estimated by gel filtration to be ca 122 000 with subunits of ca 28 500 as determined by SDS-polyacrylamide gel electrophoresis. It accounts for the hemagglutinating activity in the immature seeds of jackbean. In the later stage of seed development (Stage B), the high hemagglutination titre values are consistent with the presence of significant amounts of both this Con A-like lectin and Con A, whereas the equally high titre values in the dry mature seeds (Stage C) may be attributable essentially to Con A since the M r , 28 500 polypeptide is present only as a very minor component. Immunological and peptide mapping studies reveal a very close structural relationship between this lectin and Con A. These similarities and the inverse relationship in the relative abundance of these two proteins during different stages of seed development indicate an apparent precursor-product relationship between these two lectins.