Partial purification and properties of a transamidinase from Lathyrus sativus seedlings. Involvement in homoarginine metabolism and amine interconversions

A transamidinase was purified 463-fold from Lathyrus sativus seedlings by affinity chromatography on homoarginine--Sepharose. The enzyme exhibited a wide substrate specificity, and catalysed the reversible transfer of the amidino groups from donors such as arginine, homoarginine and canavanine to acceptors such as lysine, putrescine, agmatine, cadaverine and hydroxylamine. The enzyme could not be detected in the seeds, and attained the highest specific activity in the embryo axis on day 10 after seed germination. Its thiol nature was established by strong inhibition by several thiol blockers and thiol compounds in the presence of ferricyanide. In the absence of an exogenous acceptor, it exhibited weak hydrolytic activity towards arginine. It had apparent mol.wt. 210000, and exhibited Michaelis--Menten kinetics with Km 3.0 mM for arginine. Ornithine competitively inhibited the enzyme, with Ki 1.0 mM in the arginine--hydroxylamine amidino-transfer reaction. Conversion experiments with labelled compounds suggest that the enzyme is involved in homoarginine catabolism during the development of plant embryo to give rise to important amino acids and amine metabolites. Presumptive evidence is also provided for its involvement in the biosynthesis of the guanidino amino acid during seed development. The natural occurrence of arcain in L. sativus and mediation of its synthesis in vitro from agmatine by the transamidinase are demonstrated.

Preparation of 1-acyl-2-succinyl glycero-3-phosphorylcholine and evidence against its involvement in succinate dehydrogenase action

1-Acyl-2-succinyl glycero-3-phosphorylcholine (GPC) was synthesized and its properties described. Although 1-acyl-2-succinyl GPC is a good substrate for succinate dehydrogenase, experiments on the incorporation of [2,3-14C]succinate into mitochondrial lipids gave no evidence to indicate that it is an intermediate in the enzymic oxidation of succinate to fumarate, as has been suggested earlier.

Studies on the tryptophan residues of soybean agglutinin. Involvement in saccharide binding

Modification of tryptophan side chains of soybean agglutinin (SBA) with N-bromosuccinimide results in a loss of the hemagglutinating and carbohydrate binding activities of the protein. One residue/subunit is probably essential for the binding activity. Modification leads to a large decrease in the fluorescene of the protein accompained by a blue shift. Iodide ion quenching of the protein fluorescence shows that saccharide binding results in a decreased accessibility of some of the tryptophan side chains. These results strongly point towards the involvement of tryptophan residues in the active site of SBA.

Assessment of driver vision functions in relation to their crash involvement in India

Among the human factors that influence safe driving, visual skills of the driver can be considered fundamental. This study mainly focuses on investigating the effect of visual functions of drivers in India on their road crash involvement. Experiments were conducted to assess vision functions of Indian licensed drivers belonging to various organizations, age groups and driving experience. The test results were further related to the crash involvement histories of drivers through statistical tools. A generalized linear model was developed to ascertain the influence of these traits on propensity of crash involvement. Among the sampled drivers, colour vision, vertical field of vision, depth perception, contrast sensitivity, acuity and phoria were found to influence their crash involvement rates. In India, there are no efficient standards and testing methods to assess the visual capabilities of drivers during their licensing process and this study highlights the need for the same.

Studies on hypomethylation of liver DNA during early stages of chemical carcinogenesis in rat liver

Our finding that the inhibitors of DNA methylation, 5-azacytidine, 5-azadeoxycytidine or adenosine dialdehyde, given after a carcinogen all potentiated initiation suggested that hypomethylation of DNA during repair synthesis of DNA might play a role in the initiation of the carcinogenic process. To examine this aspect further, we have asked the question, do the nodules which develop from initiated cells after promotion with 1% orotic acid exhibit an altered methylation pattern in their DNA? The methylation status of the DNA from nodules has been examined using the restriction endonucleases HpaII/MspI and HhaI which distinguish between methylated and unmethylated cytosines in their nucleotide recognition DNA 5'-CCGG and 5'-GCGC respectively. The proto-oncogenes, c-myc, c-fos and c-Ha-ras, in the DNA were primarily studied in this investigation because of their possible involvement in cell proliferation and/or in cell transformation and tumorigenesis. The results indicate that in the nodule DNA, c-myc and c-fos are hypomethylated in the sequence of CCGG while the c-Ha-ras shows hypomethylation in the alternating GCGC sequence. This methylation pattern seen in the nodule DNA is not found in the DNA of the non-nodular surrounding liver or liver tissue after exposure to promoter or carcinogen alone. It is also not found in the DNA of regenerating liver. It is particularly significant that the methylation patterns in the c-myc and c-Ha-ras regions are similar to those found in several cancer tissues. The results suggest that this methylation pattern is acquired early in the carcinogenic process and raises the question whether it has any bearing on the process.

Sex-specific organisation of middle repetitive DNA sequences in the mealybug Planococcus lilacinus

Differential organisation of homologous chromosomes is related to both sex determination and genomic imprinting in coccid insects, the mealybugs. We report here the identification of two middle repetitive sequences that are differentially organised between the two sexes and also within the same diploid nucleus. These two sequences form a part of the male-specific nuclease-resistant chromatin (NRC) fraction of a mealybug Planococcus lilacinus. To understand the phenomenon of differential organisation we have analysed the components of NRC by cloning the DNA sequences present, deciphering their primary sequence, nucleosomal organisation, genomic distribution and cytological localisation, Our observations suggest that the middle repetitive sequences within NRC are functionally significant and we discuss their probable involvement in male-specific chromatin organisation.

The repertoire of protein kinases encoded in the draft version of the human genome: atypical variations and uncommon domain combinations

Background: Phosphorylation by protein kinases is central to cellular signal transduction. Abnormal functioning of kinases has been implicated in developmental disorders and malignancies. Their activity is regulated by second messengers and by the binding of associated domains, which are also influential in translocating the catalytic component to their substrate sites, in mediating interaction with other proteins and carrying out their biological roles. Results: Using sensitive profile-search methods and manual analysis, the human genome has been surveyed for protein kinases. A set of 448 sequences, which show significant similarity to protein kinases and contain the critical residues essential for kinase function, have been selected for an analysis of domain combinations after classifying the kinase domains into subfamilies. The unusual domain combinations in particular kinases suggest their involvement in ubiquitination pathways and alternative modes of regulation for mitogen-activated protein kinase kinases (MAPKKs) and cyclin-dependent kinase (CDK)-like kinases. Previously unexplored kinases have been implicated in osteoblast differentiation and embryonic development on the basis of homology with kinases of known functions from other organisms. Kinases potentially unique to vertebrates are involved in highly evolved processes such as apoptosis, protein translation and tyrosine kinase signaling. In addition to coevolution with the kinase domain, duplication and recruitment of non-catalytic domains is apparent in signaling domains such as the PH, DAG-PE, SH2 and SH3 domains. Conclusions: Expansion of the functional repertoire and possible existence of alternative modes of regulation of certain kinases is suggested by their uncommon domain combinations. Experimental verification of the predicted implications of these kinases could enhance our understanding of their biological roles.

Development of design charts for stress intensity factors at tips of multi-site cracks in unstiffened curved panels

Multi-site damage need to be addressed and evaluated in order to assess the integrity of aging aircraft structures. One of the problems recognized in the recent times is the effect of interaction between two or more cracks in the close neighborhood in such structures. The present paper deals with such a problem and presents numerical estimates of stress intensity factors at a crack tip in an un-stiffened curved panel with a secondary crack in the vicinity of a primary crack. The results are presented in the form of design charts. These results should be useful in evaluation in the damage tolerance evaluation of aircraft structures with multi-site damage. (C) 2014 Elsevier Ltd. All rights reserved.

Plasmodium berghei glycine cleavage system T-protein is non-essential for parasite survival in vertebrate and invertebrate hosts

T-protein, an aminomethyltransferase, represents one of the four components of glycine cleavage system (GCS) and catalyzes the transfer of methylene group from H-protein intermediate to tetrahydrofolate (THF) forming N-5, N-10-methylene THF (CH2-THF) with the release of ammonia. The malaria parasite genome encodes T-, H- and L-proteins, but not P-protein which is a glycine decarboxylase generating the aminomethylene group. A putative GCS has been considered to be functional in the parasite mitochondrion despite the absence of a detectable P-protein homologue. In the present study, the mitochondrial localization of T-protein in the malaria parasite was confirmed by immunofluorescence and its essentiality in the entire parasite life cycle was studied by targeting the T-protein locus in Plasmodium berghei (Pb). PbT knock out parasites did not show any growth defect in asexual, sexual and liver stages indicating that the T-protein is dispensable for parasite survival in vertebrate and invertebrate hosts. The absence of P-protein homologue and the non-essentiality of T protein suggest the possible redundancy of GCS activity in the malaria parasite. Nevertheless, the H- and L-proteins of GCS could be essential for malaria parasite because of their involvement in alpha-lcetoacid dehydrogenase reactions. (C) 2014 Elsevier B.V. All rights reserved.

Calcium Permeable AMPA Receptor-Dependent Long Lasting Plasticity of Intrinsic Excitability in Fast Spiking Interneurons of the Dentate Gyrus Decreases Inhibition in the Granule Cell Layer

The local fast-spiking interneurons (FSINs) are considered to be crucial for the generation, maintenance, and modulation of neuronal network oscillations especially in the gamma frequency band. Gamma frequency oscillations have been associated with different aspects of behavior. But the prolonged effects of gamma frequency synaptic activity on the FSINs remain elusive. Using whole cell current clamp patch recordings, we observed a sustained decrease of intrinsic excitability in the FSINs of the dentate gyrus (DG) following repetitive stimulations of the mossy fibers at 30 Hz (gamma bursts). Surprisingly, the granule cells (GCs) did not express intrinsic plastic changes upon similar synaptic excitation of their apical dendritic inputs. Interestingly, pairing the gamma bursts with membrane hyperpolarization accentuated the plasticity in FSINs following the induction protocol, while the plasticity attenuated following gamma bursts paired with membrane depolarization. Paired pulse ratio measurement of the synaptic responses did not show significant changes during the experiments. However, the induction protocols were accompanied with postsynaptic calcium rise in FSINs. Interestingly, the maximum and the minimum increase occurred during gamma bursts with membrane hyperpolarization and depolarization respectively. Including a selective blocker of calcium-permeable AMPA receptors (CP-AMPARs) in the bath; significantly attenuated the calcium rise and blocked the membrane potential dependence of the calcium rise in the FSINs, suggesting their involvement in the observed phenomenon. Chelation of intracellular calcium, blocking HCN channel conductance or blocking CP-AMPARs during the experiment forbade the long lasting expression of the plasticity. Simultaneous dual patch recordings from FSINs and synaptically connected putative GCs confirmed the decreased inhibition in the GCs accompanying the decreased intrinsic excitability in the FSINs. Experimentally constrained network simulations using NEURON predicted increased spiking in the GC owing to decreased input resistance in the FSIN. We hypothesize that the selective plasticity in the FSINs induced by local network activity may serve to increase information throughput into the downstream hippocampal subfields besides providing neuroprotection to the FSINs. (c) 2014 Wiley Periodicals, Inc.

Markov chain splitting methods in structural reliability integral estimation

Monte Carlo simulation methods involving splitting of Markov chains have been used in evaluation of multi-fold integrals in different application areas. We examine in this paper the performance of these methods in the context of evaluation of reliability integrals from the point of view of characterizing the sampling fluctuations. The methods discussed include the Au-Beck subset simulation, Holmes-Diaconis-Ross method, and generalized splitting algorithm. A few improvisations based on first order reliability method are suggested to select algorithmic parameters of the latter two methods. The bias and sampling variance of the alternative estimators are discussed. Also, an approximation to the sampling distribution of some of these estimators is obtained. Illustrative examples involving component and series system reliability analyses are presented with a view to bring out the relative merits of alternative methods. (C) 2015 Elsevier Ltd. All rights reserved.