Identification of the active-site peptide of 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus oryzae

The non-oxidative decarboxylation of aromatic acids is a poorly understood reaction. The transformation of 2,3-dihydroxybenzoic acid to catechol in the fungal metabolism of indole is a prototype of such a reaction. 2,3-Dihydroxybenzoic acid decarboxylase (EC 4.1.1.46) which catalyzes this reaction was purified to homogeneity from anthranilate induced cultures of Aspergillus oryzae using affinity chromatography. The enzyme did not require cofactors like NAD(+), PLP, TPP or metal ions for its activity. There was no spectral evidence for the presence of enzyme bound cofactors. The preparation, which was adjudged homogeneous by the criteria of SDS-PAGE, sedimentation analysis and N-terminal analysis, was characterized for its physicochemical and kinetic parameters. The enzyme was inactivated by group-specific modifiers like diethyl pyrocarbonate (DEPC) and N-ethylmaleimide (NEM). The kinetics of inactivation by DEPC suggested the presence of a single class of essential histidine residues, the second order rate constant of inactivation for which was 12.5 M(-1) min(-1). A single class of cysteine residues was modified by NEM with a second order rate constant of 33 M(-1) min(-1). Substrate analogues protected the enzyme against inactivation by both DEPC and NEM, suggesting the Location of the essential histidine and cysteine to be at the active site of the enzyme. The incorporation of radiolabelled NEM in a differential labelling experiment was 0.73 mol per mol subunit confirming the presence of a single essential cysteine per active-site. Differentially labelled enzyme was enzymatically cleaved and the peptide bearing the label was purified and sequenced. The active-site peptide LLGLAETCK and the N-terminal sequence MLGKIALEEAFALPRFEEKT did not bear any similarity to sequences reported in the Swiss-Prot Protein Sequence Databank, a reflection probably of the unique primary structure of this novel enzyme. The sequences reported in this study will appear in the Swiss-Prot Protein Sequence Databank under the accession number P80402.

Effect of laser processing parameters on the structure of ductile iron

Laser processing of structure sensitive hypereutectic ductile iron, a cast alloy employed for dynamically loaded automative components, was experimentally investigated over a wide range of process parameters: from power (0.5-2.5 kW) and scan rate (7.5-25 mm s(-1)) leading to solid state transformation, all the way through to melting followed by rapid quenching. Superfine dendritic (at 10(5) degrees C s(-1)) or feathery (at 10(4) degrees C s(-1)) ledeburite of 0.2-0.25 mu m lamellar space, gamma-austenite and carbide in the laser melted and martensite in the transformed zone or heat-affected zone were observed, depending on the process parameters. Depth of geometric profiles of laser transformed or melt zone structures, parameters such as dendrile arm spacing, volume fraction of carbide and surface hardness bear a direct relationship with the energy intensity P/UDb2, (10-100 J mm(-3)). There is a minimum energy intensity threshold for solid state transformation hardening (0.2 J mm(-3)) and similarly for the initiation of superficial melting (9 J mm(-3)) and full melting (15 J mm(-3)) in the case of ductile iron. Simulation, modeling and thermal analysis of laser processing as a three-dimensional quasi-steady moving heat source problem by a finite difference method, considering temperature dependent energy absorptivity of the material to laser radiation, thermal and physical properties (kappa, rho, c(p)) and freezing under non-equilibrium conditions employing Scheil's equation to compute the proportion of the solid enabled determination of the thermal history of the laser treated zone. This includes assessment of the peak temperature attained at the surface, temperature gradients, the freezing time and rates as well as the geometric profile of the melted, transformed or heat-affected zone. Computed geometric profiles or depth are in close agreement with the experimental data, validating the numerical scheme.

First-order hyperpolarizabilities and pKa of weak organic acids in protic solvents are lineraly related

Second-order nonlinearities (beta) of five weak organic acids in protic solvents have been measured by the double-quantum Rayleigh scattering (DRS) technique. beta is found to bear a linear relationship to the pK(a) of these compounds in those solvents. A direct implication of this observation is that the DRS technique can be used to determine the pK(a) of weak organic acids in any solvent.

DNA binding properties of novel dansylated distamycin analogues in which the fluorophore is directly conjugated to the N-methyl-pyrrole carboxamide backbone

Polyamides that are structural analogues of the naturally occurring DNA minor groove binding antibiotic distamycin (Dst) are promising candidates as gene modulators. Developing strategies for the large scale screening and monitoring of the cellular distribution of such ligands would aid the faster discovery of molecules, which would have eventual utility in molecular biology and medicine. Attachment of fluorescent tags would be a useful step towards this end. A fundamental question in this connection is whether the tag modifies the DNA binding affinity of the parent compounds. Towards answering this question, we have developed two oligopeptides that bear the dansyl (N, N-dimethylaminonaphthalene sulfonamido fluorophore) coupled directly to the N-terminus of the conjugated N-methylpyrrole carboxamide network, and possess three or four N-methyl pyrrole carboxamide units (abbreviated as Dn3 and Dn4 respectively). DNA binding abilities of these molecules were assessed from fluorescence titration experiments, duplex-DNA T-m analysis (employing both UV and fluorescence spectroscopy), induced circular dichroism measurements (ICD), salt dependence of ICD and apparent binding constant measurements (K-app) employing ethidium bromide (EtBr) displacement assay. Both these molecules 'reported' DNA binding in the form of an enhanced fluorescence emission. As judged from the ICD measurements, salt dependence of ICD, T-m analysis and K-app measurements, the binding affinities of the molecules that possessed dansyl group at their N-termini were lower than the ones with equivalent number of amide units, but possessed N-methylpyrrole carboxamide unit at their N-termini. These results would have implications in the future design of fluorescent polyamides.

Ethnobotany of the Monpa ethnic group at Arunachal Pradesh, India

Background: The present paper documents the uses of plants in traditional herbal medicine for human and veterinary ailments, and those used for dietary supplements, religious purpose, local beverage, and plants used to poison fish and wild animals. Traditional botanical medicine is the primary mode of healthcare for most of the rural population in Arunachal Pradesh. Materials and methods: Field research was conducted between April 2006 and March 2009 with randomly selected 124 key informants using semi-structured questionnaire. The data obtained was analyzed through informant consensus factor (F(IC)) to determine the homogeneity of informant's knowledge on medicinal plants. Results: We documented 50 plants species belonging to 29 families used for treating 22 human and 4 veterinary ailments. Of the medicinal plants reported, the most common growth form was herbs (40%) followed by shrubs, trees, and climbers. Leaves were most frequently used plant parts. The consensus analysis revealed that the dermatological ailments have the highest F(IC) (0.56) and the gastro-intestinal diseases have F(IC) (0.43). F(IC) values indicated that there was high agreement in the use of plants in dermatological and gastro-intestinal ailments category among the users. Gymnocladus assamicus is a critically rare and endangered species used as disinfectant for cleaning wounds and parasites like leeches and lice on livestocks. Two plant species (Illicium griffithii and Rubia cordifolia) are commonly used for traditional dyeing of clothes and food items. Some of the edible plants recorded in this study were known for their treatment against high blood pressure (Clerodendron colebrookianum), diabetes mellitus (Momordica charantia), and intestinal parasitic worms like round and tape worms (Lindera neesiana, Solanum etiopicum, and Solanum indicum). The Monpas of Arunachal Pradesh have traditionally been using Daphne papyracea for preparing hand-made paper for painting and writing religious scripts in Buddhist monasteries. Three plant species (Derris scandens, Aesculus assamica, and Polygonum hydropiper) were frequently used to poison fish during the month of June-July every year and the underground tuber of Aconitum ferrox is widely used in arrow poisoning to kill ferocious animals like bear, wild pigs, gaur and deer. The most frequently cited plant species; Buddleja asiatica and Hedyotis scandens were used as common growth supplements during the preparation of fermentation starter cultures. Conclusion: The traditional pharmacopoeia of the Monpa ethnic group incorporates a myriad of diverse botanical flora. Traditional knowledge of the remedies is passed down through oral traditions without any written document. This traditional knowledge is however, currently threatened mainly due to acculturation and deforestation due to continuing traditional shifting cultivation. This study reveals that the rural populations in Arunachal Pradesh have a rich knowledge of forest-based natural resources and consumption of wild edible plants is still an integral part of their socio-cultural life. Findings of this documentation study can be used as an ethnopharmacological basis for selecting plants for future phytochemical and pharmaceutical studies.

A touch of history and a peep into the future of the lipid-quinone known as coenzyme Q and ubiquinone

Coenzyme Q (ubiquinone), a fully substituted benzoquinone with polyprenyl side chain, participates in many cellular redox activities. Paradoxically it was discovered only in 1957, albeit being ubiquitous. It required a person, F. L. Crane, a place, Enzyme Institute, Madison, USA, and a time when D. E. Green was directing vigorous research on mitochondria. Located at the transition of 2-electron flavoproteins and 1-electron cytochrome carriers, it facilitates electron transfer through the elegant Q-cycle in mitochondria to reduce O-2 to H2O, and to H2O2, now a significant signal-transducing agent, as a minor activity in shunt pathway (animals) and alternative oxidase (plants). The ability to form Q-radical by losing an electron and a proton was ingeniously used by Mitchell to explain the formation of the proton gradient, considered the core of energy transduction, and also in acidification in vacuoles. Known to be a mobile membrane constituent (microsomes, plasma membrane and Golgi apparatus), allowing it to reach multiple sites, coenzyme Q is expected to have other activities. Coenzyme Q protects circulating lipoproteins being a better lipid antioxidant than even vitamin E. Binding to proteins such as QPS, QPN, QPC and uncoupling protein in mitochondria, QA and QB in the reaction centre in R. sphaeroides, and disulfide bond-forming protein in E. coli (possibly also in Golgi), coenzyme Q acquires selective functions. A characteristic of orally dosed coenzyme Q is its exclusive absorption into the liver, but not the other tissues. This enrichment of Q is accompanied by significant decrease of blood pressure and of serum cholesterol. Inhibition of formation of mevalonate, the common precursor in the branched isoprene pathway, by the minor product, coenzyme Q, decreases the major product, cholesterol. Relaxation of contracted arterial smooth muscle by a side-chain truncated product of coenzyme Q explains its effect of decreasing blood pressure. Extensive clinical studies carried out on oral supplements of coenzyine Q, initially by K. Folkers and Y. Yamamura and followed many others, revealed a large number of beneficial effects, significantly in cardiovascular diseases. Such a variety of effects by this lipid quinone cannot depend on redox activity alone. The fat-soluble vitamins (A, D, E and K) that bear structural relationship with coenzyme Q are known to be active in their polar forms. A vignette of modified forms of coenzyme Q taking active role in its multiple effects is emerging.

Melting and solidification behavior of Pb-Sn embedded alloy nano-particles

Nano-sized bimetallic dispersoids consisting of (Pb) and beta-(Sn) phases of eutectic composition (Pb26.1Sn73.9) embedded in aluminum and Al-Cu-Fe quasicrystalline matrices have been prepared by rapid solidification processing. The two phases, face centered cubic (Pb) and body center tetragonal, beta-(Sn) solid solution co-exist in all the embedded nanoparticles at room temperature. The phases bear crystallographic orientation relationship with the matrix. In situ TEM study has been carried out for the alloy particles to study the melting and the solidification behavior. The detailed microscopic observations indicate formation of a single-phase metastable fcc (Pb) in the nano-particles prior to the melting during heating. Solidification of these particles begins with nucleation of fcc (Pb), which phase separates into fcc (Pb) and beta-(Sn) lamellae in the solid state. In situ X-ray diffraction study is carried out to obtain lattice parameter of metastable fcc (Pb) and thereby an estimate of amount of Sn dissolved in the metastable (Pb) prior to the melting. The results are discussed in terms of a metastable phase diagram between fcc Pb and fcc Sn and invoking the size effect on the metastable phase diagram. The size factor is found to play a critical role in deciding the pathway of phase transformation as well as the extension of solid solubility of Sn in fcc (Pb) in the nano-particles.

Stability of long unsupported twin circular tunnels in soils

The stability of two long unsupported circular parallel tunnels aligned horizontally in fully cohesive and cohesive-frictional soils has been determined. An upper bound limit analysis in combination with finite elements and linear programming is employed to perform the analysis. For different clear spacing (S) between the tunnels, the stability of tunnels is expressed in terms of a non-dimensional stability number (gamma H-max/c); where H is tunnel cover, c refers to soil cohesion, and gamma(max) is maximum unit weight of soil mass which the tunnels can bear without any collapse. The variation of the stability number with tunnels' spacing has been established for different combinations of H/D, m and phi; where D refers to diameter of each tunnel, phi is the internal friction angle of soil and m accounts for the rate at which the cohesion increases linearly with depth. The stability number reduces continuously with a decrease in the spacing between the tunnels. The optimum spacing (S-opt) between the two tunnels required to eliminate the interference effect increases with (i) an increase in H/D and (ii) a decrease in the values of both m and phi. The value of S-opt lies approximately in a range of 1.5D-3.5D with H/D = 1 and 7D-12D with H/D = 7. The results from the analysis compare reasonably well with the different solutions reported in literature. (C) 2013 Elsevier Ltd. All rights reserved.

Is The Burden of Medical Education Affecting Ethics of Medical Treatment?

A substantial number of medical students in India have to bear an enormous financial burden for earning a bachelor's degree in medicine referred to as MBBS (bachelor of medicine and bachelor of surgery). This degree program lasts for four and one-half years followed by one year of internship. A postgraduate degree, such as MD, has to be pursued separately on completion of a MBBS. Every medical college in India is part of a hospital where the medical students get clinical exposure during the course of their study. All or at least a number of medical colleges in a given state are affiliated to a university that mainly plays a role of an overseeing authority. The medical colleges usually have no official interaction with other disciplines of education such as science and engineering, perhaps because of their independent location and absence of emphasis on medical research. However, many of the medical colleges are adept in imparting high-quality and sound training in medical practices including diagnostics and treatment. The medical colleges in India are generally of two types, i.e., government owned and private. Since only a limited number of seats are available across India in the former category of colleges, only a small fraction of aspiring candidates can find admission in these colleges after performing competitively in the relevant entrance tests. A major advantage of studying in these colleges is the nominal tuition fees that have to be paid. On the other hand, a large majority of would-be medical graduates have to seek admission in the privately run medical institutes in which the tuition and other related fees can be mind boggling when compared to their public counterparts. Except for candidates of exceptionally affluent background, the only alternative for fulfilling the dream of becoming a doctor is by financing one's study through hefty bank loans that may take years to pay back. It is often heard from patients that they are asked by doctors to undergo a plethora of diagnostic tests for apparently minor illnesses, which may financially benefit those prescribing the tests. The present paper attempts to throw light on the extent of disparity in cost of a medical education between state-funded and privately managed medical colleges in India; the average salary of a new medical graduate, which is often ridiculously low when compared to what is offered in entry-level engineering and business jobs; and the possible repercussions of this apparently unjust economic situation regarding the exploitation of patients.

`Universal' recession curves and their geomorphological interpretation

The study of recession flows offers fundamental insights into basin hydrological processes and, in particular, into the collective behavior of the governing dominant subsurface flows and properties. We use here an existing geomorphological interpretation of recession dynamics, which links the exponent in the classic recession curve -dQ/dt - kQ(alpha) to the geometric properties of the time-varying drainage network to study the general properties of recession curves across a wide variety of river basins. In particular, we show how the parameter k depends on the initial soil moisture state of the basin and can be made to explicitly depend on an index discharge, representative of initial sub-subsurface storage. Through this framework we obtain a non-dimensional, event-independent, recession curve. We subsequently quantify the variability of k across different basins on the basis of their geometry, and, by rescaling, collapse curves from different events and basins to obtain a generalized, or `universal', recession curve. Finally, we analyze the resulting normalized recession curves and explain their universal characteristics, lending further support to the notion that the statistical properties of observed recession curves bear the signature of the geomorphological structure of the networks producing them. (C) 2014 Elsevier Ltd. All rights reserved.

Identifying regions for conservation of sloth bears through occupancy modelling in north-eastern Karnataka, India

In the absence of information on species in decline with contracting ranges, management should emphasize remaining populations and protection of their habitats. Threatened by anthropogenic pressure including habitat degradation and loss, sloth bears (Melursus ursinus) in India have become limited in range, habitat, and population size. We identified ecological and anthropogenic determinants of occurrence within an occupancy framework to evaluate habitat suitability of non-protected regions (with sloth bears) in northeastern Karnataka, India. We employed a systematic sampling methodology to yield presence absence data to examine a priori hypotheses of determinants that affected occupancy. These covariates were broadly classified as habitat or anthropogenic factors. Mean number of termite mounds and trees positively influenced sloth bear occupancy, and grazing pressure expounded by mean number of livestock dung affected it negatively. Also, mean percentage of shrub coverage had no impact on bear inhabitance. The best fitting model further predicted habitats in Bukkasagara, Agoli, and Benakal reserved forests to have 38%, 75%, and 88%, respectively, of their sampled grid cells with high occupancies (>0.70) albeit little or no legal protection. We recommend a conservation strategy that includes protection of vegetation stand-structure, maintenance of soil moisture, and enrichment of habitat for the long-term welfare of this species.

Bearing Capacity of Circular Footings on Reinforced Soils

A method is presented for determining the ultimate bearing capacity of a circular footing reinforced with a horizontal circular sheet of reinforcement placed over granular and cohesive-frictional soils. It was assumed that the reinforcement sheet could bear axial tension but not the bending moment. The analysis was performed based on the lower-bound theorem of the limit analysis in combination with finite elements and linear optimization. The present research is an extension of recent work with strip foundations reinforced with different layers of reinforcement. To incorporate the effect of the reinforcement, the efficiency factors eta(gamma) and eta(c), which need to be multiplied by the bearing capacity factors N-gamma and N-c, were established. Results were obtained for different values of the soil internal friction angle (phi). The optimal positions of the reinforcements, which would lead to a maximum improvement in the bearing capacity, were also determined. The variations of the axial tensile force in the reinforcement sheet at different radial distances from the center were also studied. The results of the analysis were compared with those available from literature. (C) 2014 American Society of Civil Engineers.

Effects of Withania somnifera and Tinospora cordifolia Extracts on the Side Population Phenotype of Human Epithelial Cancer Cells: Toward Targeting Multidrug Resistance in Cancer

Recent reports suggest the existence of a subpopulation of stem-like cancer cells, termed as cancer stem cells (CSCs), which bear functional and phenotypic resemblance with the adult, tissue-resident stem cells. Side population (SP) assay based on differential efflux of Hoechst 33342 has been effectively used for the isolation of CSCs. The drug resistance properties of SP cells are typically due to the increased expression of ABC transporters leading to drug efflux. Conventionally used chemotherapeutic drugs may often leads to an enrichment of SP, revealing their inability to target the drug-resistant SP and CSCs. Thus, identification of agents that can reduce the SP phenotype is currently in vogue in cancer therapeutics. Withania somnifera (WS) and Tinospora cordifolia (TC) have been used in Ayurveda for treating various diseases, including cancer. In the current study, we have investigated the effects of ethanolic (ET) extracts of WS and TC on the cancer SP phenotype. Interestingly, we found significant decrease in SP on treatment with TC-ET, but not with WS-ET. The SP-inhibitory TC-ET was further fractionated into petroleum ether (TC-PET), dichloromethane (TC-DCM), and n-butyl alcohol (TC-nBT) fractions using bioactivity-guided fractionation. Our data revealed that TC-PET and TC-DCM, but not TC-nBT, significantly inhibited SP in a dose-dependent manner. Furthermore, flow cytometry-based functional assays revealed that TC-PET and TC-DCM significantly inhibited ABC-B1 and ABC-G2 transporters and sensitized cancer cells toward chemotherapeutic drug-mediated cytotoxicity. Thus, the TC-PET and TC-DCM may harbor phytochemicals with the potential to reverse the drug-resistant phenotype, thus improving the efficacy of cancer chemotherapy.

Volume change behavior of lime treated gypseous soil - influence of mineralogy and microstructure

Series of oedometer tests and micro-analytical studies (XRD, SEM and EDAX) have been carried out to investigate the influence of varying gypsum content on swell, compressibility and permeability of lime treated montmorillonitic soil after curing for different period. Immediate swell is observed on inundation of compacted samples with water and continuously increased with gypsum content. However, changes in swell are found to be marginal with curing. This is attributed to the formation and growth of ettringite crystals by ionic reactions of aluminum calcium-sulfate in the presence of water which is confirmed through detailed micro-analysis. The higher swell in uncured specimens and gradual reduction in swell with increase in curing periods are due to relative dominance of formation and growth of ettringite and cementitious compounds, respectively. Also, the ionic reaction products are found to bear a significant influence on the compressibility and permeability behavior. (C) 2015 Elsevier B.V. All rights reserved.