Peptide Synthesis India~ Indian Reserach Papers on Peptide Synthesis~November 2010 Page 1

A Mesorhizobium lipopolysaccharide (LPS) specific lectin (CRL) from the roots of nodulating host plant, Cicer arietinum.

Agrawal P, Kumar S, Jaiswal YK, Das HR, Das RH.

Proteomics and Structural Biology Division, Institute of Genomics and Integrative Biology, Delhi University Campus, Mall Road, Delhi 110 007, India; School of Studies in Biochemistry, Jiwaji University, Gwalior 474 011, India.
Abstract

A 30 kDa rabbit erythrocyte agglutinating glycoprotein isolated and characterized from the roots of Cicer arietinum and designated as cicer root lectin (CRL). Hemagglutination activity of CRL is strongly inhibited by cell surface LPS of nodulating cicer specific Rhizobium. CRL agglutinates mesorhizobial cells and not Escherichia coli or yeast cells. It binds to immobilized LPS of cicer specific Rhizobium only. The primary structure of CRL as predicted by peptide mass fingerprinting by MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) indicated ∼54% amino acid sequence homology with C. arietinum seedling lectin (Accession no. gi/3204123) and ∼26% with C. arietinum (Accession no. gi/110611256), and Pisum sativum (Accession nos. gi/230612, gi/6729956, gi/126148) lectins. These suggested CRL to be a member of vegetative tissue lectin. Circular dichroism analysis indicated that the secondary structure of CRL consists of 48% β-sheets, 26% random coils, and 11% α-helix. CRL has six isoforms of closely associated molecular mass with differential acidic pI of 5.30, 5.20, 5.15, 5.05, 5.00, 4.80. Identity of these isoforms was confirmed from their binding with cicer specific Rhizobium LPS. All the isoforms of CRL are differentially glycosylated as identified by deglycosylation and monosaccharide analysis using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). All these results suggest that unlike other plant lectins CRL is a LPS-binding lectin.
Copyright © 2010 Elsevier Masson SAS. All rights reserved.

PMID: 21055439 [PubMed - as supplied by publisher]

2.

Protein Pept Lett. 2010 Nov 8. [Epub ahead of print]
Characterization of LC-H(CC) Fusion Protein of Botulinum Neurotoxin Type A.

Singh MK, Dhaked RK, Singh P, Gupta P, Singh L.

Biotechnology Division, Defence Research & Development Establishment, Gwalior-474002, India. ramkumardhaked@hotmail.com.
Abstract

Botulinum neurotoxins (BoNTs) are highly potent toxins that inhibit neurotransmitter release from peripheral cholinergic synapses. The gene for encoding the full length light chain with H(CC) (binding) domain of Clostridium botulinum neurotoxin A was synthesized and cloned into a bacterial expression vector pQE30-UA and produced as an N-terminally six-histidine-tagged fusion protein (rBoNT/A LC-H(CC)). This protein was expressed in two different strains of Escherichia coli namely BL21(DE3) and SG13009. Expression at 37 ˚C revealed localization of rBoNT/A LC- H(CC) in inclusion body whereas it was expressed in soluble form at 21˚C. The recombinant fusion protein was purified by nickel affinity gel column chromatography and identified by monoclonal antibody and peptide mass fingerprinting. The recombinant protein was shown to bind with synaptic vesicles and gangliosides (GT1b) using enzyme-linked immunosorbent assay. The rBoNT/A LC-H(CC) was also found to be highly active on its substrate (SNAP-25) from rat brain, indicating that the expressed and purified rBoNT/A LC-H(CC) protein retains a functionally active conformation. Biologically active recombinant fusion protein was also evaluated for its immunological potential.

PMID: 21054265 [PubMed - as supplied by publisher]

3.

Biopolymers. 2010 Nov 4. [Epub ahead of print]
De novo design of α,β-didehydrophenylalanine containing peptides: From models to applications.

M G, V S C.

International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
Abstract

The de novo design of peptides and proteins has emerged as an approach for investigating protein structure and function. The success relies heavily on the ability to design relatively short peptides that can adopt stable secondary structures. To this end, substitution with α, β-dehydroamino acids, especially α,β-didehydrophenylalanine (ΔPhe or ΔF) has blossomed in manifold directions, providing a rich diversity of well-defined structural motifs. Introduction of α,β-didehydrophenylalanine induces β-bends in small and 3(10)-helices in longer peptide sequences. Most favorable conformation of ΔF residues are (φ,ψ) ∼ (60°, 30°), (-60°, -30°), (-60°, 150°) and (60°, -150°). These features have been exploited in designing helix-turn-helix, helical bundle arrangements, and glycine zipper type super secondary structural motifs. The unusual capability of α,β-didehydrophenylalanine ring to form a variety of multicentered interactions (N-H…O, C-H…O, C-H…π and N-H…π) suggests its possible exploitation for future de novo design of supramolecular structures. This work has now been extended to the de novo design of peptides with antibiotic, anti-fibrillization activity etc. More recently, self-assembling properties of small dehydropeptides have been explored. This review focuses primarily on the structural and functional behavior of α,β-didehydrophenylalanine containing peptides. © 2010 Wiley Periodicals, Inc. Biopolymers, 2010.

PMID: 21053260 [PubMed - as supplied by publisher]

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4.

Bioorg Med Chem. 2010 Oct 7. [Epub ahead of print]
Design, synthesis and inhibition activity of novel cyclic peptides against protein tyrosine phosphatase A from Mycobacterium tuberculosis.

Chandra K, Dutta D, Das AK, Basak A.

Department of Chemistry, Department of Biotechnology, Indian Institute of Technology, Kharagpur 721 302, India.
Abstract

Mycobacterium tuberculosis, the causative agent for tuberculosis has employed several signalling molecules to sense the host cellular environment and act accordingly. For example, protein tyrosine phosphatase A (MPtpA) of M. tuberculosis, a signalling protein belonging to the tyrosine phosphatase superfamily, is involved in phagocytosis and is active in virulent mycobacterial form. Starting from a β-lactam framework a new class of structure based cyclic peptide (CP) inhibitors was designed. The synthesis involves a crucial intramolecular transamidation via a ring opening reaction. All the compounds show moderate to good inhibitory activities against MPtpA in micromolar concentrations. The results of inhibition kinetics suggest mixed mode of inhibition. The binding constant determined from circular dichroism (CD) and fluorescence quenching studies shows strong binding of the hydrophilic side chain of CPs with the enzyme active site residues. All these are well supported by docking studies.
Copyright © 2010 Elsevier Ltd. All rights reserved.

PMID: 21050767 [PubMed - as supplied by publisher]

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5.

Org Lett. 2010 Nov 4. [Epub ahead of print]
Water-Induced Switching of β-Structure to Polyproline II Conformation in the 4S-Aminoproline Polypeptide via H-Bond Rearrangement.

Sonar MV, Ganesh KN.

Division of Organic Chemistry, National Chemical Laboratory, Dr Homi Bhabha Road, Pune 411008, India, and Indian Institute of Science Education and Research, 900, NCL Innovation Park, Dr Homi Bhabha Road, Pune 411008, India.
Abstract

4S-Aminoproline polypeptide 2 forms unusual β-structure in trifluoroethanol that switches to the polyproline II (PPII) form in aqueous medium, while 4R-aminoproline peptide 1 retains PPII form in both solvents. This first instance of a polyproline derivative showing a β-structure is attributed to competitive pH-dependent (4-NH(3)(+)/NH(2)) stereoelectronic effect (4R vs 4S) and the overriding importance of stereospecific intra/intermolecular H-bonding in (2,4)-cis-4S-aminoproline in contrast to (2,4)-trans-4R-aminoproline oligomers.

PMID: 21049969 [PubMed - as supplied by publisher]

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6.

Vet Immunol Immunopathol. 2010 Oct 13. [Epub ahead of print]
Identification and characterization of anti-microbial peptides from rabbit vaginal fluid.

Patgaonkar M, Aranha C, Bhonde G, Reddy KV.

Division of Molecular Immunology, National Institute for Research in Reproductive Health (NIRRH), Indian Council of Medical Research (ICMR), Jehangir Merwanji Street, Parel, Mumbai 400 012, Maharashtra, India.
Abstract

Antimicrobial peptides (AMPs) serve as a first line of host defense and represent an important, though poorly understood components of the innate immune system. The present study was an attempt to identify and characterize the major molecules having anti-bacterial activities from the vaginal fluid of rabbit, Oryctologus cuniculus. AMPs from the rabbit vaginal fluid (RVF) were identified in the acid extracts of pooled RVF samples after RP-HPLC purification. The protein, RVFAMP was effective against Gram negative (Escherichia coli and Pseudomonas aeruginosa) and Gram positive (Staphylococcus aureus and Streptococcus pyogenes) bacteria. The results of acid urea-PAGE-gel overlay assay (AU-PAGE-GOA) demonstrated clear zone of growth inhibition of E. coli corresponding to 6 and 15kDa protein bands. LC-MS data of these proteins indicated that 15kDa protein consists of lysozyme, lipopolysaccharide binding protein (LBP), hemoglobin-α and β subunits (Hb-α/β), whereas 9kDa protein band consists of transthyretin and calcyclin while uteroglobulin and neutrophil antibacterial peptide-5 (NAMP-5) are present in the 6kDa protein band. Of the eight proteins, Hb-α derived protein was further characterized, as it showed the highest Probability Based Mowse Score (PBMS) of 288. A 25mer peptide, RVFHbαp was active against several clinical pathogens as demonstrated by minimum inhibitory concentration (MIC) and radial diffusion assays (RDA). The interaction of RVFHbαP with bacterial liposome membrane was assessed by calcein dye leakage assay. RVFHbαP did not show cytotoxicity against human endocervical cells (End1/E6E7) or erythrocytes. RT-PCR and immunofluorescence results revealed the expression of RVFHbαP mRNA and protein in rabbit vaginal tissue. To the best our knowledge, this is the first report describing the detection of AMPs in RVFs. In conclusion, these studies indicated that vaginal epithelial cells (VEC) derived RVFHbαP may have therapeutic potential in the management of reproductive well being of rabbits. The major reason for undertaking this study in rabbits is that, it forms an excellent in vivo model system for human's studies.
Copyright © 2010 Elsevier B.V. All rights reserved.

PMID: 21047689 [PubMed - as supplied by publisher]

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7.

Int J Nanomedicine. 2010 Oct 5;5:725-33.
Plasmid DNA delivery into MDA-MB-453 cells mediated by recombinant Her-NLS fusion protein.

Jeyarajan S, Xavier J, Rao NM, Gopal V.

Centre for Cellular and Molecular Biology, Council for Scientific and Industrial Research, Hyderabad, Andhra Pradesh, India.
Abstract

A major rate-limiting step in nonviral gene delivery is the entry of nucleic acids across various membrane barriers and eventually into the nucleus where it must be transcribed. Cell-penetrating peptides and proteins are employed to generate formulations that overcome these challenges to facilitate DNA delivery into cells efficiently. However, these are limited by their inability to deliver nucleic acids selectively due to lack of specificity because they deliver to both cancer and normal cells. In this study, through modular design, we generated a recombinant fusion protein designated as Her-nuclear localization sequence (Her-NLS), where heregulin-α (Her), a targeting moiety, was cloned in frame with cationic NLS peptide to obtain a cell-specific targeting biomolecule for nucleic acid delivery. The heregulin-α(1) isoform possesses the epidermal growth factor-like domain and binds to HER2/3 heterodimers which are overexpressed in certain breast cancers. Purified recombinant Her-NLS fusion protein binds plasmid DNA and specifically transfects MDA-MB-453 cells overexpressing the epidermal growth factor receptors HER2/3 in vitro. The approach described would also permit replacement of heregulin ligand with other targeting moieties that would be suited to cell-specific nucleic acid delivery mediated via receptor-ligand interactions.

PMID: 21042418 [PubMed - in process]PMCID: PMC2962268Free PMC Article

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8.

Cytotherapy. 2010 Nov 2. [Epub ahead of print]
Human bone marrow-derived mesenchymal cells differentiate and mature into endocrine pancreatic lineage in vivo.

Phadnis SM, Joglekar MV, Dalvi MP, Muthyala S, Nair PD, Ghaskadbi SM, Bhonde RR, Hardikar AA.

Stem Cells and Diabetes Section, National Centre for Cell Science, Pune, India.
Abstract

Abstract Background aims. The scarcity of human islets for transplantation remains a major limitation of cell replacement therapy for diabetes. Bone marrow-derived progenitor cells are of interest because they can be isolated, expanded and offered for such therapy under autologous/allogeneic settings. Methods. We characterized and compared human bone marrow-derived mesenchymal cells (hBMC) obtained from (second trimester), young (1-24 years) and adult (34-81 years) donors. We propose a novel protocol that involves assessment of paracrine factors from regenerating pancreas in differentiation and maturation of hBMC into endocrine pancreatic lineage in vivo. Results. We observed that donor age was inversely related to growth potential of hBMC. Following in vitro expansion and exposure to specific growth factors involved in pancreatic development, hBMC migrated and formed islet-like cell aggregates (ICA). ICA show increased abundance of pancreatic transcription factors (Ngn3, Brn4, Nkx6.1, Pax6 and Isl1). Although efficient differentiation was not achieved in vitro, we observed significant maturation and secretion of human c-peptide (insulin) upon transplantation into pancreactomized and Streptozotocin (STZ)-induced diabetic mice. Transplanted ICA responded to glucose and maintained normoglycemia in diabetic mice. Conclusions. Our data demonstrate that hBMC have tremendous in vitro expansion potential and can be differentiated into multiple lineages, including the endocrine pancreatic lineage. Paracrine factors secreted from regenerating pancreas help in efficient differentiation and maturation of hBMC, possibly via recruiting chromatin modulators, to generate glucose-responsive insulin-secreting cells.

PMID: 21039304 [PubMed - as supplied by publisher]

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9.

J Mol Recognit. 2010 Nov;23(6):577-82.
Proteolysis activity of IgM antibodies from rheumatoid arthritis patients' sera: evidence of atypical catalytic site.

Kamalanathan AS, Goulvestre C, Weill B, Vijayalakshmi MA.

Centre for BioSeparation Technology, VIT University, Vellore 632 014, Tamil Nadu, India.
Abstract

The IgM antibodies from rheumatoid arthritis (RA) patients' sera were screened for peptide hydrolyzing activity. Recovery of structurally intact IgM antibodies (Abs), in a single step, was achieved using a weak anion-exchange methacrylate monolith disk. The IgM Abs from patients' sera hydrolyzed the Pro-Phe-Arg-4-methyl-coumaryl-7-amide (PFR-MCA) substrate appreciably compared to the healthy donors. The apparent K(m) values of IgM Abs from patients' sera were between 0.4 and 0.7 mM. Furthermore, IgM Abs displayed 5 to 10-folds greater proteolysis activity than IgG Abs, recovered from the same pathological serum. The proteolysis activity, as a function, was found to be independent of IgM-RF titer value. Affinity labeling approach targeted at the catalytic site histidine was studied, using a specific irreversible inhibitor, N-α-tosyl-L-lysine chloromethyl ketone (TLCK). Despite modification of catalytic His, observation of serine protease like activity suggest presence of an atypical catalytic framework in a few pathological IgM Abs. Copyright © 2010 John Wiley & Sons, Ltd.

PMID: 21031477 [PubMed - in process]

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10.

World J Microbiol Biotechnol. 2010 Nov;26(11):2009-2018. Epub 2010 Mar 27.
Purification and characterization of two distinct acidic phytases with broad pH stability from Aspergillus niger NCIM 563.

Soni SK, Magdum A, Khire JM.

Division of Biochemical Sciences, National Chemical Laboratory, Pune, 411 008 India.
Abstract

Aspergillus niger NCIM 563 produced two different extracellular phytases (Phy I and Phy II) under submerged fermentation conditions at 30°C in medium containing dextrin-glucose-sodium nitrate-salts. Both the enzymes were purified to homogeneity using Rotavapor concentration, Phenyl-Sepharose column chromatography and Sephacryl S-200 gel filtration. The molecular mass of Phy I and II as determined by SDS-PAGE and gel filtration were 66, 264, 150 and 148 kDa respectively, indicating that Phy I consists of four identical subunits and Phy II is a monomer. The pI values of Phy I and II were 3.55 and 3.91, respectively. Phy I was highly acidic with optimum pH of 2.5 and was stable over a broad pH range (1.5-9.0) while Phy II showed a pH optimum of 5.0 with stability in the range of pH 3.5-9.0. Phy I exhibited very broad substrate specificity while Phy II was more specific for sodium phytate. Similarly Phy II was strongly inhibited by Ag(+), Hg(2+) (1 mM) metal ions and Phy I was partially inhibited. Peptide analysis by Mass Spectrometry (MS) MALDI-TOF also indicated that both the proteins were totally different. The K(m) for Phy I and II for sodium phytate was 2.01 and 0.145 mM while V(max) was 5,018 and 1,671 μmol min(-1) mg(-1), respectively. The N-terminal amino acid sequences of Phy I and Phy II were FSYGAAIPQQ and GVDERFPYTG, respectively. Phy II showed no homology with Phy I and any other known phytases from the literature suggesting its unique nature. This, according to us, is the first report of two distinct novel phytases from Aspergillus niger.

PMID: 20976287 [PubMed]PMCID: PMC2949565Free PMC Article

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11.

Rapid Commun Mass Spectrom. 2010 Nov 30;24(22):3248-54.
Using mass spectrometry to detect buffalo salivary odorant-binding protein and its post-translational modifications.

Rajkumar R, Karthikeyan K, Archunan G, Huang PH, Chen YW, Ng WV, Liao CC.

Center for Pheromone Technology, Department of Animal Science, Bharathidasan University, Trichirappalli 620 024, Tamilnadu, India.
Abstract

A large number of mammalian odorant-binding proteins, which are lipocalins, have been studied. These proteins participate in peri-receptor events by selecting and carrying odorant molecules. The present study aimed at identifying the buffalo salivary odorant-binding protein (sOBP), and to determine its post-translational modification using mass spectrometry. The buffalo salivary 21  kDa protein was initially separated adopting sodium dodecyl sulfate-polyacrylamide gel electrophoresis and it was identified as sOBP with high statistical reliability using liquid chromatography/tandem mass spectrometry (LC/MS/MS) and SEQUEST, for the first time. Further, the post-translationally modified peptides were screened adopting MS/MS. A total of four post-translational modifications, namely glycation at lysine-(59), hydroxylation at lysine-(134), ubiquitination at lysine-(121), and dihydroxylation in lysine-(108), were recorded. Moreover, these modifications have not been identified in buffalo salivary odorant-binding protein.
2010 John Wiley & Sons, Ltd.

PMID: 20972998 [PubMed - in process]

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* Research Support, Non-U.S. Gov't

12.

Methods Mol Biol. 2011;684:159-70.
Proteomic analysis of thylakoid membranes.

Yadavalli V, Nellaepalli S, Subramanyam R.

Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, India.
Abstract

Chlamydomonas is a model organism to study photosynthesis. Thylakoid membranes comprise several proteins belonging to photosystems I and II. In this chapter, we show the accurate proteomic measurements in thylakoid membranes. The chlorophyll-containing membrane protein complexes were precipitated using chloroform/methanol solution. These complexes were separated using two-dimensional gel electrophoresis, and the resolved spots were exercised from the gel matrix and digested with trypsin. These peptide fragments were separated by MALDI-TOF, and the isotopic masses were blasted to a MASCOT server to obtain the protein sequence. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF). The method discussed here would be a useful method for the separation and identification of thylakoid membrane proteins.

PMID: 20960129 [PubMed - in process]

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* Research Support, Non-U.S. Gov't

13.

BMC Bioinformatics. 2010 Oct 18;11:519.
Statistical analysis and molecular dynamics simulations of ambivalent α -helices.

Bhattacharjee N, Biswas P.

Department of Chemistry, University of Delhi, Delhi - 110007, India. pbiswas@chemistry.du.ac.in.
Abstract

ABSTRACT:

BACKGROUND: Analysis of known protein structures reveals that identical sequence fragments in proteins can adopt different secondary structure conformations. The extent of this conformational diversity is influenced by various factors like the intrinsic sequence propensity, sequence context and other environmental factors such as pH, site directed mutations or alteration of the binding ligands. Understanding the mechanism by which the environment affects the structural ambivalence of these peptides has potential implications for protein design and reliable local structure prediction algorithms. Identification of the structurally ambivalent sequence fragments and determining the rules which dictate their conformational preferences play an important role in understanding the conformational changes observed in misfolding diseases. However, a systematic classification of their intrinsic sequence patterns or a statistical analysis of their properties and sequence context in relation to the origin of their structural diversity have largely remained unexplored.

RESULTS: In this work, the conformational variability of α-helices is studied by mapping sequences from the non-redundant database to identical sequences across all classes of the SCOP (Structural Classification of Proteins) database. Some helices retain their conformations when mapped in the SCOP database while others exhibit a complete/partial switch to non-helical conformations. The results clearly depict the differences in the propensities of amino acids for the variable and conserved helices. Sequences flanking these ambivalent sequence fragments have anisotropic propensities at the N- and C-termini. This structural variability is depicted by molecular dynamics simulations in explicit solvent, which show that the short conserved helices retain their conformations while their longer counterparts fray into two or more shorter helices. Variable helices in the non-redundant database exhibit a trend of retaining helical conformations while their corresponding non-helical conformations in SCOP database show large deviations from their respective initial structures by adopting partial or full helical conformations. Partially ambivalent helices are also found to retain their respective conformations.

CONCLUSIONS: All sequence fragments which show structural diversity in different proteins of the non-redundant database are investigated. The final conformation of these ambivalent sequences are dictated by a fine tuning of their intrinsic sequence propensity and the anisotropic amino acid propensity of the flanking sequences. This analysis may unravel the connection between diverse secondary structures, which conserve the overall structural fold of the protein thus determining its function.

PMID: 20955581 [PubMed - in process]PMCID: PMC2973962Free Article

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14.

Mol Biosyst. 2010 Oct 18. [Epub ahead of print]
Structure-based identification of MHC binding peptides: Benchmarking of prediction accuracy.

Kumar N, Mohanty D.

National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India. deb@nii.res.in.
Abstract

Identification of MHC binding peptides is essential for understanding the molecular mechanism of immune response. However, most of the prediction methods use motifs/profiles derived from experimental peptide binding data for specific MHC alleles, thus limiting their applicability only to those alleles for which such data is available. In this work we have developed a structure-based method which does not require experimental peptide binding data for training. Our method models MHC-peptide complexes using crystal structures of 170 MHC-peptide complexes and evaluates the binding energies using two well known residue based statistical pair potentials, namely Betancourt-Thirumalai (BT) and Miyazawa-Jernigan (MJ) matrices. Extensive benchmarking of prediction accuracy on a data set of 1654 epitopes from class I and class II alleles available in the SYFPEITHI database indicate that BT pair-potential can predict more than 60% of the known binders in case of 14 MHC alleles with AUC values for ROC curves ranging from 0.6 to 0.9. Similar benchmarking on 29 522 class I and class II MHC binding peptides with known IC(50) values in the IEDB database showed AUC values higher than 0.6 for 10 class I alleles and 9 class II alleles in predictions involving classification of a peptide to be binder or non-binder. Comparison with recently available benchmarking studies indicated that, the prediction accuracy of our method for many of the class I and class II MHC alleles was comparable to the sequence based methods, even if it does not use any experimental data for training. It is also encouraging to note that the ranks of true binding peptides could further be improved, when high scoring peptides obtained from pair potential were re-ranked using all atom forcefield and MM/PBSA method.

PMID: 20953500 [PubMed - as supplied by publisher]

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15.

Biotechnol Prog. 2010 Aug 25. [Epub ahead of print]
Development of a process to manufacture PEGylated orally bioavailable insulin.

Hazra P, Adhikary L, Dave N, Khedkar A, Manjunath HS, Anantharaman R, Iyer H.

Research and Development, Biocon Limited, Electronic City, Bangalore 560100, India.
Abstract

To make insulin orally bioavailable, insulin was modified by covalent attachment (conjugation) of a short-chain methoxy polyethylene glycol (mPEG) derivative to the ε-amino group of a specific amino acid residue (LysB(29)). During the conjugation process, activated PEG can react with any of the free amino groups, the N-terminal of the B chain (PheB(1))(,) the N-terminal of the A chain (GlyA(1)), and the ε-amino group of amino acid (LysB(29)), resulting in a heterogeneous mixture of conjugated products. The abundance of the desired product (Methoxy-PEG(3)-propionyl-insulin at LysB(29):IN-105) in the conjugation reaction can be controlled by changing the conjugation reaction conditions. Reaction conditions were optimized for maximal yield by varying the proportions of protein to mPEG molecule at various values of pH and different salt and solvent concentrations. The desired conjugated molecule (IN-105) was purified to homogeneity using RP-HPLC. The purified product, IN-105, was crystallized and lyophilized into powder form. The purified product was characterized using multiple analytical methods including ESI-TOF and peptide mapping to verify its chemical structure. In this work, we report the process development of new modified insulin prepared by covalent conjugation of short chain mPEG to the insulin molecule. The attachment of PEG to insulin resulted in a conjugated insulin derivative that was biologically active, orally bioavailable and that showed a dose-dependent glucose lowering effect in Type 2 diabetes patients. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010.

PMID: 20949602 [PubMed - as supplied by publisher]

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16.

Mar Drugs. 2010 Aug 19;8(8):2384-94.
Total synthesis and antimicrobial activity of a natural cycloheptapeptide of marine origin.

Dahiya R, Gautam H.

Department of Pharmaceutical Chemistry, NRI Institute of Pharmacy, Bhopal 462 021, Madhya Pradesh, India. rajivdahiya77@rediffmail.com
Abstract

The present study deals with the first total synthesis of the proline-rich cyclopolypeptide stylisin 2 via a solution phase technique by coupling of the Boc-L-Pro-L-Ile-L-Pro-OH tripeptide unit with the L-Phe-L-Pro-L-Pro-L-Tyr-OMe tetrapeptide unit, followed by cyclization of the resulting linear heptapeptide fragment. The chemical structure of the finally synthesized peptide was elucidated by FTIR, ¹H/¹³C-NMR and FAB MS spectral data, as well as elemental analyses. The newly synthesized peptide was subjected to antimicrobial screening against eight pathogenic microbes and found to exhibit potent antimicrobial activity against Pseudomonas aeruginosa, Klebsiella pneumoniae and Candida albicans, in addition to moderate antidermatophyte activity against pathogenic Trichophyton mentagrophytes and Microsporum audouinii when compared to standard drugs--gatifloxacin and griseofulvin.

PMID: 20948913 [PubMed - in process]PMCID: PMC2953409Free PMC Article

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17.

Chemistry. 2010 Oct 13. [Epub ahead of print]
Short-Peptide-Based Hydrogel: A Template for the In Situ Synthesis of Fluorescent Silver Nanoclusters by Using Sunlight.

Adhikari B, Banerjee A.

Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata, 700 032 (India), Fax: (+91) 332473-2805.
Abstract

N-terminally Fmoc-protected dipeptide, Fmoc-Val-Asp-OH, forms a transparent, stable hydrogel with a minimum gelation concentration of 0.2 % w/v. The gelation property of the hydrogel was investigated by using methods such as transmission electron microscopy, field-emission scanning electron microscopy, atomic force microscopy and Fourier transform infrared spectroscopy. The silver-ion-encapsulating hydrogel can efficiently and spontaneously produce fluorescent silver nanoclusters under sunlight at physiological pH (7.46) by using a green chemistry approach. Interestingly, in the absence of any conventional reducing agent but in the presence of sunlight, silver ions were reduced by the carboxylate group of a gelator peptide that contains an aspartic acid residue. These clusters were investigated by using UV/Vis spectroscopy, photoluminescence spectroscopy, high-resolution transmission electron microscopy (HR-TEM), atomic force microscopy (AFM) and X-ray diffraction (XRD) studies. Mass spectrometric analysis shows the presence of a few atoms in nanoclusters containing only Ag(2). The reported fluorescent Ag nanoclusters show excellent optical properties, including a very narrow emission profile and large Stokes shift (>100 nm). The reported fluorescent Ag nanoclusters within hydrogel are very stable even after 6 months storage in the dark at 4 °C. The as-prepared hydrogel-nanocluster conjugate could have applications in antibacterial preparations, bioimaging and other purposes.

PMID: 20945315 [PubMed - as supplied by publisher]

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18.

Fungal Biol. 2010 Sep;114(9):731-8. Epub 2010 Jun 19.
Molecular and structural characterization of GTP-cyclohydrolase II in Eremothecium ashbyi NRRL Y-1363: cDNA cloning, comparative sequence analysis and molecular modeling.

Sengupta S, Chandra TS.

Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600 036, India.
Abstract

GTP-cyclohydrolase II (GCH II) encoded by RIB1 gene catalyzes the first committed step in the riboflavin biosynthetic pathway. We report here the cloning and characterization of the entire RIB1 ORF (EaRIB1) of 942bp by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE-PCR) in Eremothecium ashbyi where it was found to be present as a single-copy gene. EaRIB1 sequence is available at the GenBank Accession Number EF565374. The putative peptide of 313-aa has a high similarity of 60-70% with GCH II sequences from other ascomycete fungi. Gene expression and alignment studies confirmed the functional annotation of this gene. Homology model was developed with Escherichia coli (PDB 2BZ1) as template to identify the catalytic domains and to explore its functional architecture. We report here the first three-dimensional model of any fungal GCH II which due to its absence in humans assumes significance for anti-fungal drug targeting.
Copyright © 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

PMID: 20943182 [PubMed - in process]

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Publication Types:

* Research Support, Non-U.S. Gov't

Secondary Source ID:

* GENBANK/EF565374
* PDB/PDB2BZ1

19.

Expert Opin Ther Targets. 2010 Nov;14(11):1177-97.
Novel drug targets based on metallobiology of Alzheimer's disease.

Bandyopadhyay S, Huang X, Lahiri DK, Rogers JT.

Indian Institute of Toxicology Research, Lucknow, India. sanghmitra@iitr.res.in
Abstract

IMPORTANCE OF THE FIELD: Increased localization of Zn, Fe, Cu and Al within the senile plaques (SP) exacerbates amyloid beta (Aβ)-mediated oxidative damage, and acts as catalyst for Aβ aggregation in Alzheimer's disease (AD). Thus, disruption of aberrant metal-peptide interactions via chelation therapy holds considerable promise as a rational therapeutic strategy against Alzheimer's amyloid pathogenesis.

AREAS COVERED IN THIS REVIEW: The complexities of metal-induced genesis of SP are reviewed. The recent advances in the molecular mechanism of action of metal chelating agents are discussed with critical assessment of their potential to become drugs.

WHAT THE READER WILL GAIN: Taking into consideration the interaction of metals with the metal-responsive elements on the Alzheimer's amyloid precursor protein (APP), readers will gain understanding of several points to bear in mind when developing a screening campaign for AD-therapeutics.

TAKE HOME MESSAGE: A functional iron-responsive element (IRE) RNA stem loop in the 5' untranslated region (UTR) of the APP transcript regulates neural APP translation. Desferrioxamine, clioquinol, tetrathiolmolybdate, dimercaptopropanol, VK-28, and natural antioxidants, such as curcumin and ginko biloba need critical evaluation as AD therapeutics. There is a necessity for novel screens (related to metallobiology) to identify therapeutics effective in AD.

PMID: 20942746 [PubMed - in process]

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20.

Folia Microbiol (Praha). 2010 Sep;55(5):520-7. Epub 2010 Oct 13.
Peptides representing the specific variable regions but not the full porin proteins can be useful for antibody based diagnosis of typhoid fever.

Kumar S, Kapil S, Gautam V, Verma SK, Ray P.

Defence Research and Development Establishment, Division of Microbiology, Gwalior 474 002, India. subodh@drde.drdo.in
Abstract

Antibody response to major porin proteins of S. Typhi (OmpC and OmpF) was evaluated in sera of typhoid patients (culture positive, n = 28; Widal positive, n = 16). Sera from fever patients (n = 6) having etiology other than Salmonella, and normal healthy human controls (n = 18) were also included. No significant difference between the anti-OmpC and anti-OmpF antibodies (Ab) of typhoid patients and controls was observed. The amino acid sequences of OmpC (and OmpF) porin of enterobacteria was aligned and searched for the variable regions specific to S. Typhi. Two regions, each representing one specific variable region of OmpC and OmpF, were selected (peptides for these regions were custom synthesized). The peptides were evaluated for Ab response of sera. A significantly higher level of Ab to both the peptides was observed in the sera of typhoid patients. The findings suggest that porins of S. Typhi are cross reactive and are not good markers for Ab-based diagnosis of typhoid fever, however, peptides representing the variable regions specific to S. Typhi may have greater diagnostic potential.

PMID: 20941590 [PubMed - in process]

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