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In situ synthesized TiB–TiN reinforcedTi6Al4Valloy composite coatings: Microstructure, tribological and in-vitro biocompatibility
No full textWear resistant TiB–TiN reinforced Ti6Al4V alloy composite coatings were deposited on Ti substrate using laser based additive manufacturing technology. Ti6Al4V alloy powder premixed with 5wt% and 15wt% of boronnitride(BN)powder was used to synthesize TiB–TiN reinforcements in situ during laser deposition. Influences of laser power, scanning speed and concentration of BN on the microstructure, mechanical, in vitro tribological and biologicalproperties of the coatings were investigated
Disuccinyl Betulin Triggers Metacaspase-Dependent Endonuclease G-Mediated Cell Death in Unicellular Protozoan Parasite Leishmania donovani
No full textThe unicellular organism Leishmania undergoes apoptosis-like cell death in response to external stress or exposure to antileishmanial agents. Here, we showed that 3-O,28-O-disuccinyl betulin (DiSB), a potent topoisomerase type IB
inhibitor, induced parasitic cell death by generating oxidative stress. The characteristic feature of the death process resembled the programmed cell death (PCD) seen in higher eukaryotes. In the current study, the generation of reactive oxygen species (ROS), followed by the depolarization of mitochondrial membrane potential (��m), caused a loss in ATP production in Leishmania
parasites. This further gave positive feedback to produce a large amount of ROS, which in turn caused oxidative DNA
lesions and genomic DNA fragmentation. The treatment of promastigotes with DiSB induced high expression levels of metacaspase protein that led to cell death in this unicellular organism. The PCD was insensitive to benzyloxycarbonyl-Val-Ala-Asp( OMe)-fluoromethylketone (zVAD-fmk), suggesting that the death process was not associated with the activation of caspases.
DiSB treatment translocated Leishmania donovani endonuclease G (LdEndoG) from mitochondria to the nucleus, which was responsible for the DNA degradation process. Conditional antisense knockdown of L. donovani metacaspase (LdMC), as well as EndoG, -subverted death of the parasite and rescued cell cycle arrest in G1 phase. The present study on the effector molecules associated with the PCD pathway of the parasite should help to manifest the mechanisms of PCD and also might be exploited in
antileishmanial chemotherapy
Copper–phenanthroline catalysts for regioselective synthesis of pyrrolo[3′,4′:3,4]pyrrolo[1,2 a]furoquinolines/ phenanthrolines and of pyrrolo[1,2-a]phenanthrolines under mild conditions
No full textA new series of pyrrolo[3′,4′:3,4]pyrrolo[1,2-a]furoquinolines/phenanthrolines and pyrrolo[1,2-a]phenanthrolines were efficiently
built up from an 8-hydroxyquinoline derivative or phenanthroline via 1,3-dipolar cycloaddition reaction involving non-stabilized
azomethine ylides, generated in situ from the parent furo[3,2-h]quinoliniums/phenanthroliums, in presence of a copper(II)
chloride–phenanthroline catalytic system. The methodology combines general applicability with high yields
Studies of Early Events of Folding of a Predominately β‑Sheet Protein Using Fluorescence Correlation Spectroscopy and Other Biophysical Methods
No full textThe interplay between the early collapse of the
unfolded state and the formation of the secondary structure has been the subject of extensive research in protein chemistry. In this study, we used the intestinal fatty acid binding protein (IFABP), a small model protein with predominately β-sheet structure, to study the early events, including the early chain collapse and the formation of the secondary structure. We used a combination offluorescence correlation spectroscopy and far-UV circular dichroism
(CD) to understand how these early processes influence the late folding events like the stabilization of the secondary structure and aggregation. Acid-induced unfolded IFABP was found to collapse in the presence of low concentrations of added salt and aggregate at higher concentrations. Both the formation of the collapsed state and aggregation were conveniently probed by fluorescence correlation spectroscopy, a sensitive fluorescence technique with single-molecule resolution. In contrast, the
formation of the secondary structure was monitored by far-UV CD. The results suggested that backbone hydrogen bond
formation, not only the overall hydrophobicity of IFABP, may play crucial roles in the early collapse. Two mutant proteins positioned at a crucial nucleating site, namely, G80V and L64G, although being opposite in their overall hydrophobicity, collapsed relatively rapidly compared to the wild-type protein. The interconnection among the early collapse, the formation of the secondary structure, and aggregation was similar for these two mutants. Another mutant, G44V, which was identical in its overall hydrophobicity to G80V but situated in a region distant from the hydrophobic core, was found to be very different from G80V and L64G
Evaluation of bioactive components from natural products for prevention of toxicity and neurological disorders
No full textNature has created almost an inexhaustible array of molecular entities, which stands as
an infinite resource for novel therapeutics and scaffolds for designing of efficacious drugs
for a multitude of diseases. The synthetic products gained attention due to its cost and
time effectiveness, easy quality control, stringent regulation and quick effects, but their
safety and efficacy often remain questionable, resulting in the dependence on the natural
products by more than 80% of the total population in the developing world. This study
deals with the characterization of proteins, peptides and small organic molecules isolated
from natural sources like a medicinal plant Aristolochia indica, Russell’s viper (Daboia
russelli russelli) venom (RVV) and human placental extract towards possible
development of drugs against various illness.
The investigation was initiated on the aqueous extract of the root of A. indica, a wellknown
medicinal plant used against snake envenomation in many parts of rural India.
Our aim was to evaluate the root extract of this ethnomedicine from its biochemical
characters. The aristolochic acid (AA) content of the extract is considerably low and the
solution does not exert any detectable toxicity in animals. It contains a large number of
proteins that appear to be clustered under native condition. It does not show nonspecific
protease activity but strong gelatinolytic, collagenase, nuclease and peroxidase activities.
It interacts with the components of RVV and partially inhibits proteolytic and L-amino
acid oxidase (LAAO) activities of the venom. Collectively, the properties of the extract
explain, at least in parts, neutralization of RVV-induced toxicity and also its application
in wound healing and inflammatory diseases
Modulation of Immune Responses by Small Regulatory RNA in Mammalian Macrophages
No full textInnate immune response provides the first line of defence in mammalian immune system. The innate immune response can be of two types: pro-inflammatory and anti-inflammatory. Activation with lipopolysaccharide (LPS) induces expression of pro-inflammatory cytokines in mammalian macrophages whereas infection with protozoan parasite Leishmania induces anti-inflammatory immune response. miRNAs are the tiny regulators of gene expression in metazoan. In monocytes and macrophages, miRNAs play a key regulatory role in controlling immune responses as expression of a number of cytokines is regulated by miRNAs. Upon macrophage activation, the pro-inflammatory cytokine mRNAs are rapidly translated, although expression of most of the miRNAs targeting these mRNAs remains largely unaltered. Therefore, to be translated in activated cells, the cytokine mRNAs must be protected from miRNA-mediated repression. But how these mRNAs become immune to miRNAs is unknown. We show that there is a transient reversal of miRNA-mediated repression during the early phase of pro-inflammatory response in macrophages, which leads to protection of cytokine mRNAs from miRNA-mediated repression. This derepression is caused by impaired miRNA binding to Ago2 protein, a key component of miRNP complex. In activated macrophage, Ago2 protein gets phosphorylated at Y529 position in a reversible manner affecting miRNA as well as target mRNA binding. Macrophages expressing mutant Ago2, that cannot be phosphorylated and thus remains bound to miRNAs and target mRNAs, showed weaken pro-inflammatory response and failed to prevent parasite invasion. We have also found that during longer incubation of LPS or during Leishmania donovani infection, a phosphatase PP2A dephosphorylates Ago2 to keep the miRISC active.The active miRISC then represses the pro-inflammatory cytokines. This helps activated macrophage to fine tune cytokine response to avoid hyper responsiveness at later stage of LPS stimulation. In infection model this helps Leishmania to evade pro-inflammatory immune response and to survive inside the macrophages
POTENTIAL OF THE [99mTc(CO)3(H2O)3] MOIETY FOR THE LABELING OF BIOMOLECULES FOR INFECTION DIAGNOSIS
No full textTreatment of visceral leishmaniasis: anomalous pricing and distribution of AmBisome and emergence of an indigenous liposomal amphotericin B, FUNGISOME
No full textVisceral leishmaniasis (VL) is one of the
severest forms of parasite borne diseases worldwide with a
mortality rate second only to malaria. Treatment of VL
patients with currently available chemotherapeutic agents
poses problems of large scale failure, toxicity, prolonged
hospitalization time, high treatment cost and drug resistance.
However, most of these problems can be overcome
by the use of liposomal formulations of Amphotericin B
(L-AmB). Of the two L-AmBs currently available in Indian
market, AmBisome is imported and FUNGISOME is
indigenous. Initially AmBisome remained exorbitantly
costly and therefore inaccessible to most of the VL
patients. However, with the launch of FUNGISOME in
India, Gilead in agreement with WHO started a donation
program of AmBisome in developing countries through a
slashed price of US 18 per
vial. FUNGISOME, on the other hand, produced consistently
good results against VL both clinically and experimentally.
In the context of unavailability and price
anomaly of AmBisome, successful emergence of FUNGISOME
could mark it as the major L-AmB against VL
Enzymatic and Regulatory Attributes of Trehalose-6- Phosphate Phosphatase from Candida utilis and its Role During Thermal Stress
No full textTrehalose-6-phosphate phosphatase (TPP) catalyzes the final step in the biosynthesis of the anti-stress sugar trehalose. An 82 kDa TPP enzyme was isolated from Candida utilis with 61% yield and 43-fold purification. The protein sequence, determined by N-terminal sequencing and MALDI-TOF analysis, showed significant homology with known TPP sequences from related organisms. The full length
gene sequence of TPP of C. utilis was identified using rapid amplification of cDNA ends-PCR reaction (RACE-PCR). The gene was cloned and expressed in Escherichia coli BL21. Recombinant TPP enzyme was isolated using affinity chromatography. CD spectroscopy and steadystate
fluorescence revealed that the structural and conformational aspects were identical in both native and recombinant forms. The biochemical properties of the two forms were also similar. Km was determined to be ~0.8 mM. Optimum temperature and pH were found to be 30 °C and 8.5, respectively. Activity was dependent on the presence of divalent cations and inhibited by metal chelators. Methylationmediated regulation of TPP enzyme and its effect on the overall survival of the organism under stress were investigated. The results indicated that enhancement of TPP activity by methylation at the Cysteine residues increased resistance of Candida cells against thermal stress. This work involves extensive investigations toward understanding the physico-chemical properties of the first TPP enzyme from any yeast strain. The mechanism by which methylation regulates its activity has also been studied. A correlation between regulation of trehalosesynthesis and survivability of the organism under thermal stress was established
Association of purine asymmetry, strand-biased gene distribution and PolC within Firmicutes and beyond: a new appraisal
No full textstrands of replication, Strand-biased Gene Distribution (SGD) and presence of two isoforms of DNA polymerase III alpha subunit, PolC and DnaE. Despite considerable research efforts, it is not clear whether the co-existence of PAS, PolC and/or SGD is an essential and exclusive characteristic of the Firmicutes. The nature of
correlations, if any, between these three features within and beyond the lineages of Firmicutes has also remained
elusive. The present study has been designed to address these issues. A large-scale analysis of diverse bacterial genomes indicates that PAS, PolC and SGD are neither essentialnor exclusive features of the Firmicutes. PolC prevails in four bacterial phyla: Firmicutes, Fusobacteria, Tenericutes and Thermotogae, while PAS occurs only in subsets of Firmicutes, Fusobacteria and Tenericutes. There are five major compositional trends in Firmicutes: (I) an explicit PAS or G + A-dominance along the entire leading strand (II) only G-dominance in the leading strand, (III) alternate stretches of purine-rich and pyrimidine-rich sequences, (IV) G + T dominance along the leading strand, and (V) no identifiable patterns in base usage. Presence of strong SGD has been observed not only in genomes having PAS, but also in genomes with G-dominance along their leading strands – an observation that defies the notion of co-occurrence of PAS and SGD in Firmicutes. The PolC-containing non-Firmicutes organisms often have alternate stretches of R-dominant and Y-dominant sequences along their
genomes and most of them show relatively weak, but significant SGD. Firmicutes having G + A-dominance or
G-dominance along LeS usually show distinct base usage patterns in three codon sites of genes. Probable
molecular mechanisms that might have incurred such usage patterns have been proposed.Co-occurrence of PAS, strong SGD and PolC should not be regarded as a genome signature of the Firmicutes. Presence of PAS in a species may warrant PolC and strong SGD, but PolC and/or SGD not necessarily
implies PAS