• Volume 21, Issue 5

      September 1996,   pages  577-734

    • Molecular dynamics study of interaction of dimyristoyl phosphotidyl choline with water

      V Kothekar

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      We present here results of molecular dynamics (MD) simulations on hydrated bilayers of 40 molecules of 1-2-dimyristoyl-sn-glycero-3-phosphatidyl choline (DMPC) in liquid crystalline (Lα) phase using two different models (i) with same (A) conformation for all DMPC molecules, (ii) with alternate rows having different (A and B reported in crystallographic studies on DMPC) conformations. The bilayers were hydrated using 776 and 1064 water molecules. Simulations have been carried out at 310K with AMBER 4.0 program, using united atom force field for 200 pico seconds (ps) after equilibration. During heating and equilibration constant pressure temperature (PT) conditions were maintained while in simulation of equillibrated bilayers constant volume temperature (VT) conditions were used. Subaveraged atomic coordinates were used to calculate geometric parameters of lipid molecules and lipid water interaction. Our results show larger flexibility of polar head group and glycerol region in Lα phase compared to gel or non-hydrated bilayers. Chain disorder was more towards end. Sn-2 chains were more disordered. Use of two types of starting conformations increased disorder. Trans fraction of chain torsional angle was higher in non-hydrated bilayer. However it was more disordered due to ‘swing’ movement of chains because of distortion in torsional angles α2 and 03 due to absence of water molecules. Trans fraction of the chains, order parameter and water penetration showed general agreement with the available experimental results. On the whole MD technique was found to be quite useful for depicting microscopic behaviour of liquid crystalline system and correlating the same with macroscopic changes observed experimentally.

    • Pharmacophores of the dual acting α, β-blockers as deduced, from molecular dynamics simulations

      Shantaram Kamath Evans Coutinho

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      The dual acting α, β-blockers have an important place in the management of hypertension. Molecular dynamics simulations have been carried out on all stereoisomers of seven dual acting α, β-blockers namely adimolol, amosulalol, bucindolol, carvedilol, labetalol, medroxalol and primidolol. Three families of conformations have been identified for the group of compounds. The pharmacophores for α and β-activity have been constructed for two of these families.

    • Alignment and phylogenetic analysis of type II DNA topoisomerases

      K Madhusudan V Nagaraja

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      DNA topoisomerases have been evolved to solve the topological problems of DNA during replication, transcription, recombination and segregation. Discovery of several new enzymes and their characterization has necessitated this compilation. This analysis shows the distinct evolutionary relatedness of type II DNA topoisomerases. A striking feature is the absence of a contiguous stretch of about 160 amino acids in one of the subunits of prokaryotic type II enzymes, which might have important implications to their structure and function.

    • Regulation of HSP70 in excitatory neurons: Possible implications for neuronal functioning

      AnoopKumar Thekkuveettil S C Lakhotia

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      Neurons maintain an intricate organization of cytoplasmic and membrane proteins for their integrity, quick communication across synapses and for other complex activities. Molecular chaperones such as members of the 70 kDa heat shock protein (HSP70) family may play very important roles in these functions. However, in spite of a recent report suggesting the presence of HSP70 related proteins in the synaptic vesicle docking complex at presynaptic sites and the known significant roles for HSP70 in excitotoxicity, there are remarkably few studies that have explored the potential role of HSP70 family proteins in physiological functions of neurons. Here we bring together direct and indirect evidences which suggest that several different pathways involved in long-term potentiation can influence the HSP70 levels at the synapse and hypothesize on possible physiological significance of this family of proteins in neuronal functions.

    • Determination of kinetic parameters of epitope-paratope interaction based on solid phase binding: An inexpensive alternate to biospecific interaction analysis

      G S Murthy N Venkatesh

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      A method has been developed for biospecific interaction analysis between antigen and antibody using solid phase binding approach. Real time kinetics between monoclonal antibody and human chorionic gonadotropin have been studied. Kinetic constants of the bimolecular reaction are determined. Affinity constants measured by several independent methods have been found to be relatively consistent. Convenient and simple procedures to determine affinity constant, Konand Koff of monoclonal antibody-human chorionic gonadotropin interaction using binding of [125I]hCG to immobilized monoclonal antibody are presented. Values obtained compare well with those obtained using surface plasmon resonance technology, making this method a viable alternative.

    • Amylases of the thermophilic fungusThermomyces lanuginosus: Their purification, properties, action on starch and response to heat

      Ravi S Mishra Ramesh Maheshwari

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      A thermophilic fungus Thermomyces lanuginous strain IISc 91, secreted one form each of α-amylase and glucoamylase during growth. Both enzymes were purified to homogeneity by ion-exchange and gel-filtration chromatography and obtained in mg quantities. α-Amylase was considered to be a dimeric protein of ∼ 42 kDa and contained 5% (by mass) carbohydrate. It was maximally active at pH 5.6 and at 65°C. It had an activation energy of 44 kJ mol-1. The apparent Km for soluble starch was 2.5 mg ml-1. The enzyme produced exceptionally high levels of maltose from raw potato starch. At 50°C, the enzyme was stable for > 7h. At 65°C, α-amylase was nearly 8-times more stable in the presence of calcium. Addition of calcium increaed the melting temperature of α-amylase from 66°C to 73°C. Upon incubation at 94°C, α-amylase was progressively and irreversibly inactivated, and converted into an inactive 72 kDa trimeric species.

      Glucoamylase was a monomeric glycoprotein of ∼ 45 kDa with a carbohydrate content of 11% (by mass). It effected up to 76% conversion of starch in 24 h producing glucose as the sole product. Its apparent Km for soluble starch was 0.04 mg ml-1 and Vmax was 660 Mmol glucose min-1 mg protein-1. It also hydrolyzed maltose. Its activity on maltooligosaccharides increased with the chain length of the substrates. Glucoamylase was stable at 60°C for over 7h. Its activation energy was 61 kJ mol-1 Glucoamylase did not show synergistic effect with α-amylase. The properties of α-amylase and glucoamylase of Thermomyces lanuginosus strain IISc 91 suggest their usefulness in the commercial production of maltose and glucose syrups.

    • Cloning of acryIIIA endotoxin gene ofBacillus thuringiensis var.tenebrionis and its transient expression inindica rice

      Tony M Johnson A S Rishi Pritilata Nayak Soumitra K Sen

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      Damage caused to rice production by coleopteran insects like rice weevil (Sitophilus oryzae), a stored grain insect pest and rice hispa (Dicladispa armigera), a pest of the growing plant is quite high. In order to combat the damage, generation of insect resistant transgenic rice plant was considered desirable. CryIIIA endotoxin ofBacillus thuringiensis var.tenebrionis, a 65 kDa protein toxic to coleopteran insects, figured as the candidate gene product. Thus, the cryIIIA gene was isolated from a local isolate ofBacillus thuringiensis var.tenebrionis. The gene was tailored at the N-terminal end to its minimal size by using a synthetic ATG codon which replaced the first codon next to ATG of threonine to proline. This modification did not affect the functional property of the gene product. A chimeric construct of the modifiedcryIIIA gene was developed containing CaMV35S promoter andnos terminator for plant expression. The expressibility of thecryIIIA gene inindica rice was judged through test for transient expression in indica rice protoplasts.

    • Mutagenesis study to ameliorate the bacterial expression of phosphoprotein P of vesicular stomatitis virus

      Susanta Pahari D J Chattopadhyay

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      The phosphoprotein gene of vesicular stomatitis virus, a Rhabdovirus, has been inserted into bacterial expression plasmids containing the Escherichia coli tac promoter and ribosome binding site (RBS). A low level of expression of the protein was detected. Sequence analysis showed the presence of 15 nucleotides in the spacer region i.e., between the Shine-Dalgarno sequence and ATG. Alteration of the distance and the sequence in the spacer region by oligonucleotide-directed mutagenesis revealed a correlation among the expression levels, accessibility of the RBS and requirement for a minimum spacing of at least 7 nucleotides between the Shine-Dalgarno sequence and ATG for optimal gene expression.

    • Age related changes in ovarian follicular kinetics in the Indian skipper frogRana cyanophlyctis (Schn.)

      J T Kulkarni K Pancharatna

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      Changes in ovarian follicular kinetics were studied in relation to aging in the Indian skipper frogRana cyanophlyctis.Age was determined by skeletochronology, by counting the number of growth rings and lines of arrest of growth from the cross sections of 4th phalange of 4th toe. For follicular kinetics study oocytes were counted under binocular using 10% of Bouin’s fixed ovary and they were classified into first growth phase, medium-sized second growth phase, large-sized second growth phase and atretic follicles.

      Analysis of phalangeal cross sections indicated that frogs ranging 14–54 g in body weight and 4.9–8.9 cm in body size showed 1–7 year rings. Frogs that weighed 14–16 g showed 1 year ring, and contained immature ovaries; those with 18 g body weight had one to two year rings, in which second growth phase oocytes appeared for the first time in the primiparous ovary. Frogs with 20–54 g body weight showed 2–5 year rings in which ovary contained 5–24% of second growth phase oocytes. Further, body weight, body size, ovarian weight, number and size of second growth phase oocytes and total number of oocytes showed a significant (P < 0.05) positive correlation, while, the number of first growth phase and atretic follicles showed a poor correlation with age.

      The results suggest that in nature, the age ofRana cyanophlyctisranges between 1–7 years. Phalangeal growth rings are formed annually. Females attain sexual maturity in 2nd year. Frogs with 2–5 years of age may constitute breeding females. Body weight, body size, ovarian mass, number of second growth phase and total oocytes, and egg size increase with age up to 5 years.

    • Isolation and identification of yolk proteins in Indian major carp,Labeo rohita

      M Bhakta P Nath

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      Two yolk proteins (YP1 and YP2) from the ovaries of Indian major carp, Labeo rohita were isolated by gel filtration and partially characterized by the use of hydroxyapatite ultrogel column in conjunction with native PAGE. On native PAGE YP1 gave a single protein band, whereas YP2 of gel filtration revealed the contamination of YP1, which was removed by adsorption chromatography on hydroxyapatite ultrogel and then the YP2 was the purified one as judged by electrophoresis. Both YP1 and YP2 also stained for lipid and contained alkalilabile phosphorus. Therefore, both yolk proteins were lipophosphoprotein. The molecular weights of YP1 and YP2 were 620 kDa and 225 kDa respectively as determined by gel filtration on Sepharose 4B. When YP1 and YP2 were compared in relation to some physicochemical characteristics with yolk proteins of other oviparous vertebrates including fish, they were lipovitellin like.

      Antiserum to YP2 crossreacted with YP2 and vitellogenin suggesting that YP2 was the cleaved product of vitellogenin. Anti-YP2 antiserum was not crossreacted with native YP1, whereas reduced and/or denatured YP1 was crossreacted indicating the presence of antigenic determinants in the inner core region of YP1 polypeptide.

    • Stratification of density in dry deciduous forest using satellite remote sensing digital data—An approach based on spectral indices

      P S Roy K P Sharma A Jain

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      Forest density expressing the stocking status constitutes the major stand physiognomic parameter of Indian forest. Density and age are often taken as surrogate to structural and compositional changes that occur with the forest succession. Satellite remote sensing spectral response is reported to provide information on structure and composition of forest stands. The various vegetation indices are also correlated with forest canopy closure. The paper presents a three way crown density model utilizing the vegetation indices viz., advanced vegetation index, bare soil index and canopy shadow index for classification of forest crown density. The crop and water classes which could not be delineated by the model were finally masked from normalized difference vegetation index and TM band 7 respectively. The rule based approach has been implemented for land use and forest density classification. The broad land cover classification accuracy has been found to be 91.5%. In the higher forest density classes the classification accuracy ranged between 93 and 95%, whereas in the lower density classes it was found to be between 82 and 85%.

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