Calcium Chloride Made E. coli Competent for Uptake of Extraneous DNA Through Overproduction of OmpC Protein

In the standard method of transformation of Escherichia coli with extraneous DNA, cells are made competent for DNA uptake by incubating in ice-cold 100?mM CaCl₂. Analysis of the whole protein profile of CaCl₂-treated E. coli cells by the techniques of one- and two-dimensional gel electrophoresis, MALDI-MS and immunoprecipitation revealed overproduction of outer membrane proteins OmpC, OmpA and heat-shock protein GroEL. In parity, transformation efficiency of E. coli ompC mutant by plasmid pUC19 DNA was found to be about 40?% lower than that of the wild type strain. Moreover, in E. coli cells containing groEL-bearing plasmid, induction of GroEL caused simultaneous overproduction of OmpC. On the other hand, less OmpC was synthesized in E. coli groEL mutant compared to its wild type counterpart, by CaCl₂-shock. From these results it can be suggested that in the process of CaCl₂-mediated generation of competence, the heat-shock chaperone GroEL has specific role in DNA entry into the cell, possibly through the overproduced OmpC and OmpA porins.     

A size dependent folding contour for cytochrome C

The paper describes an experimental construct of the folding route of the heme protein cytochrome-C. The construct highlights a slowing down near the nose of the folding funnel caused by the multiplicity of the energy traps near the native conformation created as a result of complex heme-peptide interaction. Interestingly the hydrodynamic size, the size heterogeneity and peroxidase activity serve as a triple measure of the distance of this near equilibrium departure from native conformation. Accordingly, the folding process is marked with a gradual and reversible reduction of mean hydrodynamic size, size heterogeneity and peroxidase activity (higher in unfolded state). The Dynamic Light Scattering based straightforward illustration of hydrodynamic size variation may serve as a model to slow folding observed in case of heme proteins, the heme itself serving as a natural facilitator for the native peptide conformation.     

An insight into the ribonucleolytic and antiangiogenic activity of buffalo lactoferrin

Lactoferrin (LF) has several biological effects ranging from ribonuclease activity to antiangiogenic activity. It thus serves as a potential target protein for studies related to ribonucleolytic activity in association with its antiangiogenic activity. We have isolated buffalo LF and checked the ribonucleolytic activity via an agarose gel-based assay and precipitation assay. The ribonucleolytic activity of LF is lower compared to RNase A and the pH profile is a bell-shaped curve, with a pK₁ value of 5.43 and pK₂ of 7.65. The ribonuclease inhibitor that inhibits many ribonuclease-type proteins by forming a tight complex is unable to inhibit the ribonucleolytic property of LF. Fe(III) behaves as a noncompetitive inhibitor for the ribonucleolytic activity of protein. The superoxide-scavenging activity of the protein has also been measured. Histidine modification by diethylpyrocarbonate was monitored by UV–Vis spectroscopy at pH 7 and pH 8 and the effect towards the ribonucleolytic activity was determined. The antiangiogenic property of LF was investigated by the chorioallantoic membrane assay. Finally, the possible active site was analyzed via docking studies and correlated with the experimental study.     

Side-chain peptide-synthetic polymer conjugates via tandem "ester-amide/thiol-ene" post-polymerization modification of poly(pentafluorophenyl methacrylate) obtained using ATRP

Herein the concept of tandem postpolymerization modification as a versatile route to synthesize well-defined, highly functionalized polymers is introduced. Poly(pentafluorophenyl methacrylate) obtained by atom transfer radical polymerization was first modified with allylamine, which displaces the active ester to give well-defined polymers with pendant alkene groups, which are difficult to obtain by direct (radical) polymerization of allylic-functional monomers. The produced poly(allylmethacrylamide) was modified by a second postpolymerization modification reaction with a thiol-terminated peptide (CVPGVG) using AIBN as the radical source. NMR, IR, and SEC demonstrated successful conjugation onto the polymer to give a polymer-peptide hybrid material. This versatile strategy should extend the scope of controlled radical polymerization and "click"-type reactions.     

Catfish (Clarias batrachus) serum lectin recognizes polyvalent Tn [alpha-D-GalpNAc1-Ser/Thr], Talpha [beta-D-Galp-(1-->3)-alpha-D-GalpNAc1-Ser/Thr], and II [beta-D-Galp(1-->4)-beta-D-GlcpNAc1-] mammalian glycotopes

A new calcium dependent GalNAc/Gal specific lectin was isolated from the serum of Indian catfish, Clarias batrachus and designated as C. batrachus lectin (CBL). It is a disulfide-linked homodecameric lectin of 74.65kDa subunits and the oligomeric form is essential for its activity. Binding specificity of CBL was investigated by enzyme-linked lectin-sorbent assay using a series of simple sugars, polysaccharides, and glycoproteins. GalNAc was more potent inhibitor than Gal; and alpha glycosides of both were more inhibitory than their beta counterparts. CBL showed maximum affinity for human tumor-associated Tn-antigens (GalNAcalpha1-Ser/Thr) at the molecular level and was 3.5 times higher than GalNAc. CBL interacted strongly with polyvalent Tn and Talpha (Galbeta1,3GalNAcalpha1-) as well as multivalent-II (Galbeta1,4GlcNAcbeta1-) antigens containing glycoproteins and intensity of inhibition was 10(3)-10(5) times more than monovalent ones. The overall specificity of CBL lies in the order of polyvalent Tn, Talpha and II>monovalent TnMe-alphaGalNAc>monovalent Talpha> Me-betaGalNAc>Me-alphaGal>monovalent T>GalNAc>monovalent F>monovalent II>Me-betaGal>Gal.     

Rapamycin inhibits osteoblast proliferation and differentiation in MC3T3-E1 cells and primary mouse bone marrow stromal cells

While the roles of the mammalian target of rapamycin (mTOR) signaling in regulation of cell growth, proliferation, and survival have been well documented in various cell types, its actions in osteoblasts are poorly understood. In this study, we determined the effects of rapamycin, a specific inhibitor of mTOR, on osteoblast proliferation and differentiation using MC3T3-E1 preosteoblastic cells (MC-4) and primary mouse bone marrow stromal cells (BMSCs). Rapamycin significantly inhibited proliferation in both MC-4 cells and BMSCs at a concentration as low as 0.1 nM. Western blot analysis shows that rapamycin treatment markedly reduced levels of cyclin A and D1 protein in both cell types. In differentiating osteoblasts, rapamycin dramatically reduced osteoblast-specific osteocalcin (Ocn), bone sialoprotein (Bsp), and osterix (Osx) mRNA expression, ALP activity, and mineralization capacity. However, the drug treatment had no effect on osteoblast differentiation parameters when the cells were completely differentiated. Importantly, rapamycin markedly reduced levels of Runx2 protein in both proliferating and differentiating but not differentiated osteoblasts. Finally, overexpression of S6K in COS-7 cells significantly increased levels of Runx2 protein and Runx2 activity. Taken together, our studies demonstrate that mTOR signaling affects osteoblast functions by targeting osteoblast proliferation and the early stage of osteoblast differentiation.     

Protein translocase of mitochondrial inner membrane in Trypanosoma brucei

Translocases of mitochondrial inner membrane (TIMs) are multiprotein complexes. The only Tim component so far characterized in kinetoplastid parasites such as Trypanosoma brucei is Tim17 (TbTim17), which is essential for cell survival and mitochondrial protein import. Here, we report that TbTim17 is present in a protein complex of about 1,100 kDa, which is much larger than the TIM complexes found in fungi and mammals. Depletion of TbTim17 in T. brucei impairs the mitochondrial import of cytochrome oxidase subunit IV, an N-terminal signal-containing protein. Pretreatment of isolated mitoplasts with the anti-TbTim17 antibody inhibited import of cytochrome oxidase subunit IV, indicating a direct involvement of the TbTim17 in the import process. Purification of the TbTim17-containing protein complex from the mitochondrial membrane of T. brucei by tandem affinity chromatography revealed that TbTim17 associates with seven unique as well as a few known T. brucei mitochondrial proteins. Depletion of three of these novel proteins, i.e. TbTim47, TbTim54, and TbTim62, significantly decreased mitochondrial protein import in vitro. In vivo targeting of a newly synthesized mitochondrial matrix protein, MRP2, was also inhibited due to depletion of TbTim17, TbTim54, and TbTim62. Co-precipitation analysis confirmed the interaction of TbTim54 and TbTim62 with TbTim17 in vivo. Overall, our data reveal that TbTim17, the single homolog of Tim17/22/23 family proteins, is present in a unique TIM complex consisting of novel proteins in T. brucei and is critical for mitochondrial protein import.     

A Comparative Study of Interaction of Tetracycline with Several Proteins Using Time Resolved Anisotropy,Phosphorescence, Docking and FRET

A comparative study of the interaction of an antibiotic Tetracycline hydrochloride (TC) with two albumins, Human serum albumin (HSA) and Bovine serum albumin (BSA) along with Escherichia Coli Alkaline Phosphatase (AP) has been presented exploiting the enhanced emission and anisotropy of the bound drug. The association constant at 298 K is found to be two orders of magnitude lower in BSA/HSA compared to that in AP with number of binding site being one in each case. Fluorescence resonance energy transfer (FRET) and molecular docking studies have been employed for the systems containing HSA and BSA to find out the particular tryptophan (Trp) residue and the other residues in the proteins involved in the binding process. Rotational correlation time (θ_c) of the bound TC obtained from time resolved anisotropy of TC in all the protein-TC complexes has been compared to understand the binding mechanism. Low temperature (77 K) phosphorescence (LTP) spectra of Trp residues in the free proteins (HSA/BSA) and in the complexes of HSA/BSA have been used to specify the role of Trp residues in FRET and in the binding process. The results have been compared with those obtained for the complex of AP with TC. The photophysical behaviour (viz., emission maximum, quantum yield, lifetime and θ_c) of TC in various protic and aprotic polar solvents has been determined to address the nature of the microenvironment of TC in the protein-drug complexes.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Rhododendron wattii</Emphasis> Cowan—a critically endangered and endemic plant from India

Rhododendron wattii Cowan is a rare and endangered plant found in northeast India. In an effort to boost specimen numbers, experiments of in vitro seed germination, shoot induction on different media supplemented with the cytokinin isopentenyladenine (2iP), and root induction with auxins α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) in woody plant medium (WPM) were carried out. A maximum mean shoot number of 7.72 per explant were obtained from nodal explants cultured on WPM and 39.36 μM 2iP with a maximum mean shoot length of 2.30 cm per explant. Among the auxins investigated for root induction, IBA at 2.45 μM was found to produce the most and the longest roots, when compared to other treatments. However, WPM supplemented with 0.2% (w/v) activated charcoal also showed 100% root formation with shoots having broader leaves compared to auxin treatments. About 60% of in vitro rooted plantlets transferred from lab to greenhouse conditions survived. Sixty acclimatized plants were reintroduced in the vicinity of their natural habitat at Naga Heritage Village, Kisama, Nagaland, in May 2016 for ex situ conservation. Survival of the reintroduced plants was confirmed during the field visit conducted in November 2016.     

Multivalent II [beta-D-Galp-(1-->4)-beta-D-GlcpNAc] and Talpha [beta-D-Galp-(1-->3)-alpha-D-GalpNAc] specific Moraceae family plant lectin from the seeds of Ficus bengalensis fruits

A galactose specific lectin was isolated from the seeds of Ficus bengalensis (Moraceae) fruits and designated as F. bengalensis agglutinin (FBA). The lectin was purified by affinity repulsion chromatography on fetuin-agarose and was a monomer of molecular mass 33kDa. Like other Moraceae family lectins, carbohydrate-binding activity of FBA was independent of any divalent cation. FBA did not bind with simple saccharides, however sugar ligands with aromatic aglycons showed pronounced binding. The combining site of FBA recognized preferably Galbeta1,4GlcNAcbeta1-(II) followed by Galbeta1,3GalNAcalpha1-(Talpha) containing glycotopes. Interaction with saccharides revealed that the combining site of FBA could well accommodate a tetrasaccharide, asialo GM1 glycan (Galbeta1,3GalNAcbeta1,4Galbeta1,4Glcbeta1-), whereas polyvalent Tn (GalNAcalpha1-Ser/Thr), one of the well-recognized ligands of Moraceae family lectin, did not interact well with FBA.