High-yield bacterial expression and structural characterization of recombinant human insulin-like growth factor binding protein-2

The diverse biological activities of the insulin-like growth factors (IGF-1 and IGF-2) are mediated by the IGF-1 receptor (IGF-1R). These actions are modulated by a family of six IGF-binding proteins (ICFBP-1-6; 22-31 kDa) that via high affinity binding to the IGFs (K-D similar to 300-700 pM) both protect the IGFs in the circulation and attenuate IGF action by blocking their receptor access In recent years, IGFBPs have been implicated in a variety of cancers However, the structural basis of their interaction with IGFs and/or other proteins is not completely understood A critical challenge in the structural characterization of full-length IGFBPs has been the difficulty in expressing these proteins at levels suitable for NMR/X-ray crystallography analysis Here we describe the high-yield expression of full-length recombinant human IGFBP-2 (rhIGFBP-2) in Eschericha coli Using a single step purification protocol, rhIGFBP-2 was obtained with >95% purity and structurally characterized using NMR spectroscopy. The protein was found to exist as a monomer at the high concentrations required for structural studies and to exist in a single conformation exhibiting a unique intra-molecular disulfide-bonding pattern The protein retained full biologic activity. This study represents the first high-yield expression of wild-type recombinant human IGFBP-2 in E coli and first structural characterization of a full-length IGFBP (C) 2010 Elsevier Inc. All rights reserved

A convenient and high-yield synthesis for pyridinium and metal hexafluorocomplexes of vanadium, chromium, iron and cobalt

Pyridinium poly(hydrogen fluoride) reacts with the oxide of vanadium(V) and chlorides of chromium(III), iron (III) and Co(II) at room temperature forming the pyridinium salts of hexafluoro vanadate(V), hexafluorochromate(III), hexafluoroferrate(III) and hexafluorocobaltate(II) in near quantitative yields (80%). These pyridinium salts are the precursors for the preparation of the alkali metal hexafluorometallates by metathetic reactions in acetonitrile medium with the corresponding metal chlorides. The prepared salts have been identified by their infrared spectral data and elemental analysis.

A Convenient High-Yield Room-Temerature Synthesis of Mixed Tri(Amino) Silanes by Transmination of Tris(Dicyclohexylamino) Silane And-Their Characterization

Tris(dicyclohexylamino)silane. (DCA)3SiH. is prepared by the reaction of trichlorosilane with dicyclohexylamine. This is found to undergo transamination reactions with other secondary amines (R2NH). such as pyrrolidine, piperidine, hexamethyleneimine. morpholine. N-methylpiperazine and diethylamine to yield mixed tri(amino)silanes of the formula (DCA)(R2N)2SiH in quantitative yields. These new derivatives are found to be moisture sensitive and hydrolyze to yield their respective amines, hydrogen and silica. They are found to be stable in an inert atmosphere. They have been characterized by IR, NMR (H-1, Si-29), mass spectroscopy and CHN analysis. N-15 NMR for one of the compounds has been done.

High Yield Room Temperature Synthesis And Spectral Studies Of Tri(Amino)Silanes: (R2n)3SIH

Tri(amino)silanes were prepared by the condensation of trichlorosilane with secondary amines in 1:6 molar ratio. Reactions of trichlorosilane with pyrrolidine, piperidine, hexamethyleneimine, morpholine, N-methylpiperazine and diethylamine afford the tri(amino)silanes in nearly quantitative yields. Their physical and spectroscopic properties are discussed. All these compounds are highly sensitive to moisture and hydrolyse to silica and the respective amine with the evolution of hydrogen. The compounds have been characterised by IR, 1H NMR, [1H]29Si NMR spectroscopic methods and CHN elemental analysis.

Use of a specific aromatase inhibitor for determining whether there is a role for oestrogen in follicle/oocyte maturation, ovulation and preimplantation embryo development

The specific role of oestrogen in follicular maturation, ovulation and early embryonic development was investigated using Fadrozole (CGS 16949A), a non-steroidal aromatase inhibitor, to block oestrogen synthesis specifically and effectively in experimental animals. Induced and normal cyclical follicular maturation as well as normal and hCG/LH-induced ovulation were relatively unaffected by significantly depleting oestrogen in all animals (hamsters, rabbits, monkeys) studied other than rats. Fadrozole treatment significantly reduced the number of healthy antral follicles produced and the ovulatory response to exogenous hCG of immature rats primed with pregnant mares' serum gonadotrophin. The effect was specific, in that exogenously administered oestrogen reversed the blockade. Depletion of oestrogen, starting early in pro-oestrus in hamsters, had no effect on ovulation, oocyte maturation and fertilization, as normal implantation sites were seen on day 6 after coitus. In rabbits, oestrogen depletion during the periovulatory phase affected oviductal morphology and function. Although fertilization was not impaired, early embryo development did not appear to be normal. In monkeys, oestrogen depletion during the follicular phase did not lead to a block of follicular maturation or ovulation but resulted in a significant reduction in secretion of cervical mucus. Administration of either Fadrozole or Tamoxifen during the early luteal phase in cyclic monkeys that were allowed to mate prevented implantation and this appears to be due to impaired fertilization or faulty embryo development. These results suggest that, although there is a clear requirement for oestrogen to support the reproductive cycle in the female, the need for oestrogen in regulating specific events is species dependent.

Secretion of endocrine signals by the primate embryo during the peri-implantation period

Development of preimplantation embryos and blastocyst implantation are critical early events in the establishment of pregnancy. In primates, embryonic signals, secreted during the peri-implantation period, are believed to play a major role in the regulation of embryonic differentiation and implantation. However, only limited progress has been made in the molecular and functional characterization of embryonic signals, partly due to severe paucity of primate embryos and the lack of optimal culture conditions to obtain viable embryo development. Two embryonic (endocrine) secretions, i.e. chorionic gonadotrophin (CG) and gonadotrophin releasing hormone (GnRH) are being studied. This article reviews the current status of knowledge on the recovery and culture of embryos, their secretion of CG, GnRH and other potential endocrine signals and their regulation and physiological role(s) during the peri-implantation period in primates, including humans.

Shear banding in a yield stress bearing Langmuir monolayer

We examine the shear-thinning behaviour of a two dimensional yield stress bearing monolayer of sorbitan tristearate at air/water interface. The flow curve consists of a linear region at low shear stresses/shear rates, followed by a stress plateau at higher values. The velocity profile obtained from particle imaging velocimetry indicates that shear banding occurs, showing coexistence of the fluidized region near the rotor and solid region with vanishing shear-rate away from the rotor. In the fluidized region, the velocity profile, which is linear at low shear rates, becomes exponential at the onset of shear-thinning, followed by a time varying velocity profile in the plateau region. At low values of constant applied shear rates, the viscosity of the film increases with time, thus showing aging behaviour like in soft glassy three-dimensional (3D) systems. Further, at the low values of the applied stress in the yield stress regime, the shear-rate fluctuations in time show both positive and negative values, similar to that observed in sheared 3D jammed systems. By carrying out a statistical analysis of these shear-rate fluctuations, we estimate the effective temperature of the soft glassy monolayer using the Galavatti-Cohen steady state fluctuation relation.

Heterologous expression of P5CS gene in chickpea enhances salt tolerance without affecting yield

Vigna Delta(1)-pyrroline-5-carboxylate synthetase (P5CS) cDNA was transferred to chickpea (Cicer arietinum L.) cultivar Annigeri via Agrobacterium tumefaciens mediated transformation. Following selection on hygromycin and regeneration, 60 hygromycin-resistant plants were recovered. Southern blot analysis of five fertile independent lines of T0 and T1 generation revealed single and multiple insertions of the transgene. RT-PCR and Western blot analysis of T0 and T1 progeny demonstrated that the P5CS gene is expressed and produced functional protein in chickpea. T1 transgenic lines accumulated higher amount of proline under 250 mM NaCl compared to untransformed controls. Higher accumulation of Na(+) was noticed in the older leaves but negligible accumulation in seeds of T1 transgenic lines as compared to the controls. Chlorophyll stability and electrolyte leakage indicated that proline overproduction helps in alleviating salt stress in transgenic chickpea plants. The T1 transgenics lines were grown to maturity and set normal viable seeds under continuous salinity stress (250 mM) without any reduction in plant yield in terms of seed mass.

Designing copper-zirconium based nanowires for improving yield strength and plasticity by configuring surface atoms

Epitaxial-Bain-Path and Uniaxial-Bain-Path studies reveal that a B2-CuZr nanowire with Zr atoms on the surface is energetically more stable compared to a B2-CuZr nanowire with Cu atoms on the surface. Nanowires of cross-sectional dimensions in the range of similar to 20-50 are considered. Such stability is also correlated with the initial state of stress in the nanowires. It is also demonstrated here that a more stable structure, i.e., B2-CuZr nanowire with Zr atoms at surface shows improved yield strength compared to B2-CuZr nanowire with Cu atoms at surface site, over range of temperature under both the tensile and the compressive loadings. Nearly 18% increase in the average yield strength under tensile loading and nearly 26% increase in the averaged yield strength under compressive loading are observed for nanowires with various cross-sectional dimensions and temperatures. It is also observed that the B2-CuZr nanowire with Cu atom at the surface site shows a decrease in failure/plastic strain with an increase in temperature. On the other hand, B2-CuZr nanowires with Zr at the surface site shows an improvement in failure/plastic strain, specially at higher temperature as compared to the B2-CuZr nanowires which are having Cu atoms at the surface site. Finally, a possible design methodology for an energetically stable nano-structure with improved thermo-mechanical properties via manipulating the surface atom configuration is proposed.

Cobalt(II), Nickel(II) and Copper(II) complexes of a tetradentate Schiff base as photosensitizers: Quantum yield of O-1(2) generation and its promising role in anti-tumor activity

In the present investigation, a Schiff base N'(1),N'(3)-bis(E)-(5-bromo-2-hydroxyphenyl)methylidene]benzene-1,3-d icarbohydrazide and its metal complexes have been synthesized and characterized. The DNA-binding studies were performed using absorption spectroscopy, emission spectra, viscosity measurements and thermal denatuaration studies. The experimental evidence indicated that, the Co(II), Ni(II) and Cu(II) complexes interact with calf thymus DNA through intercalation with an intrinsic binding constant K-b of 2.6 x 10(4) M-1, 5.7 x 10(4) M-1 and 4.5 x 10(4) M-1, respectively and they exhibited potent photo-damage abilities on pUC19 DNA, through singlet oxygen generation with quantum yields of 0.32, 0.27 and 0.30 respectively. The cytotoxic activity of the complexes resulted that they act as a potent photosensitizers for photochemical reactions. (C) 2012 Elsevier B.V. All rights reserved.

NMR studies of preimplantation embryo metabolism in human assisted reproductive techniques: a new biomarker for assessment of embryo implantation potential

There has been growing interest in understanding energy metabolism in human embryos generated using assisted reproductive techniques (ART) for improving the overall success rate of the method. Using NMR spectroscopy as a noninvasive tool, we studied human embryo metabolism to identify specific biomarkers to assess the quality of embryos for their implantation potential. The study was based on estimation of pyruvate, lactate and alanine levels in the growth medium, ISM1, used in the culture of embryos. An NMR study involving 127 embryos from 48 couples revealed that embryos transferred on Day 3 (after 72 h in vitro culture) with successful implantation (pregnancy) exhibited significantly (p < 10(-5)) lower pyruvate/alanine ratios compared to those that failed to implant. Lactate levels in media were similar for all embryos. This implies that in addition to lactate production, successfully implanted embryos use pyruvate to produce alanine and other cellular functions. While pyruvate and alanine individually have been used as biomarkers, the present study highlights the potential of combining them to provide a single parameter that correlates strongly with implantation potential. Copyright (C) 2012 John Wiley & Sons, Ltd.