Cloning and expression of ophc2, a new organphosphorus hydrolase gene

The amino acid sequences of N-terminal and internal peptide of OPHC2, purified from Pseudomonas pseudoalcaligenes strain C2-1 in our lab, are determined. The full-length organphosphorus hydrolase gene ophc2 is cloned by PCR using the degenerate primers designed according to the sequences and future inverse PCR. The ophc2 gene is 975 bp long with G C content of 63%, comprising one open reading frame encoding a polypeptide of 324 amino acids with a molecular weight of 36 kD. The nucleotide sequence of ophc2 shows low homologies with those organphosphorus hydrolase genes deposited in GenBank, one of which exhibits the highest homology of 46.4% with ophc2. The organphosphorus hydrolase protein expressed in E. coli bears normal bioactivity.     

THz time-domain spectroscopy of amino acids

The optical characteristics of four kinds of amino acids （tyrosine, arginine, histidine and glutamine） filled with nitrogen at room temperature were studied by THz time-domain spectroscopy （THz-TDS）. Well-resolved absorption and refractive spectrums between 0.I and 2.8 THz were obtained based on the physical model for extracting the optical parameters of materials in THz range. The results not only fill up the spectra gap of amino acids in far-infrared range, supply data for amino acid molecular identification and conformation analysis, but also demonstrate significantly potential to promote the research and application of biological materials in bio-chemical and medical fields by THz-TDS.     

Extraction of Amino Acids from Excess Activated Sludge by Enzymatic Hydrolysis

A study was undertaken to investigate the production of amino acids from excess activated sludge (EAS) by enzymatic hydrolysis. Firstly, the protein was extracted from EAS. Secondly, the protein solution was further hydrolyzed under free enzyme or immobilized enzyme. The reversed phase high performance liquid chromatography (RP-HPLC) and inductively coupled plasma emission spectrometer (ICP) were applied to determine the contents of amino acids and heavy metals, respectively. The effects of enzyme/substrate(E/S), pH, temperature, and reaction time were investigated in detail. The results indicated that, the optimum conditions for protein hydrolysis were temperature 55℃, pH 10, E/S 9 g/L, and reaction time 8 h, and the highest yield of amino acids was more than 10 g/100 g dry sludge (DS) under free enzyme. Moreover, the security and nutrition were taken into consideration. There were seven kinds of essential amino acids and ten non-essential amino acids in the raw amino acid (RAA) solution, and the contents of heavy metals were lower, living up to Hygienical standard for feeds (China). This technology widens the source of amino acids and makes the extraction of amino acids from EAS more economic and effective.     

Characterization of the separate kinase domain of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

The bifunctional enzvme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase consists of two dis tinct domains which catalyze the synthesis and hydrolysis of fructose-2, 6-bisphosphate, respectively. In this work the properties of the separate 6-phosphofructo-2-kinase domain were investigated. Purification of the expressed separate do main or isolation of this domain from purified glutathione S-transferase (GST) fusion protein with thrombin cleavage led to the loss of its kinase activity. Thus the domain in the GST-tagged form was characterized. The two forms of the do main with different lengths (amino acids 1 ～ 249 and 1 ～ 286) were very similar in kinetic property and could catalyze the formation of fructose-2,6-bisphosphate with a kcat 4-fold lower than that of the full-length enzyme. In addition, the domain was much more sensitive to guanidine inactivation and heat denaturation, and less stable at pH values below 7 than the full-length enzyme. The results suggest that the separate kinase domain of the bifunctional enzyme is far less perfect in structure in the absence of the bisphosphatase domain, though it still possesses the 6-phosphofructo-2-kinase activity.     

Expression，reflolding and preliminary characterization of recombinant snake venom metalloproteinases：Implication for the hemorrhagic mechanism

Two cDNAs encoding hemorrhagic snake venom metalloproteinase acutolysin A and non-hemorrhagic metalloproteinase(BR)were cloned into the expression vector pET-22b,respectively,and the corresponding two recombinant proteins,A-22b and BR-22b,were produced in inclusion bodies in E.coli BL21(DE3).The reombinant proteins were then subjected to solubilization,purification and refolding in vitro.A-22b showed hemorrhagic activity and fibronectin.Natural autolysin A had both hemorrhagic activity and proteolytic activity toward these substrates.BR-22b showed the proteolytic activities toward fibrinogen,but no hemorrhagic activity.In addition,two chimeric genes,C1 and C2,were constructed and cloned into pET-22b,and the corresponding recombinant proteins,C1-22b and C2-22b,were also expressed in inclusion bodies.C1-22b involved N-terminal 110 amino acidos of BR and C-terminal 95 amino acids of acutolysin A,while C2-22b contained N-terminal 108 amino acids of acutolysin A and C-terminal 112 amino acids of BR. The biological activities of A-22b and BR-22b,respectively.Our results suggested that N-terminal major subdomain of a snake venom metalloproteinase might play a key role in hemorrhagic activity and have an appreciable effect on the selectivity for protein substrates.     

Branched chain amino acids and metabolic regulation

Amino acids are fundamental nutrients required for protein synthesis. The branched chain amino acids (BCAAs) leucine, isoleucine, and valine are the most abundant of the essential amino acids. BCAAs have recently been recognized as having functions in processes other than simple nutrition. For example, metabolic diseases are characterized by higher levels of circulating BCAAs. Moreover, supplementation with or deficiency in BCAAs is closely related to the regulation of metabolic homeostasis. Indeed, leucine deprivation induces increased lipolysis and thermogenesis, which result in fat loss, as well as suppressed lipogenesis and enhanced insulin sensitivity in the liver. Accumulating evidence has indicated that several amino acid sensors, including GCN2, ATF4, mTOR, and AMPK, play pivotal roles in the regulation of lipid metabolism, glucose metabolism, and energy homeostasis. Furthermore, the hypothalamus is critical for sensing amino acid levels and mediates the metabolic adaptation of the body upon limitation of essential amino acids (EAAs) through regulating expression of the S6K1, MC4R, and CRH. In this review, we highlight recent studies investigating the cellular mechanisms linking amino acids, amino acid sensors, metabolic regulation, and metabolic diseases. Amino acid sensing and metabolic regulation have become research hotspots in the metabolic field.     

Construction of Yeast Vectors with Resistance to Geneticin

IntroductionYeast Saccharomyces cerevisiae (S.cerevisiae) hasbeen widely used in molecular biology as well asinthe industrial production.Its genetic backgroundhas been well studied and a series of cloning andexpression vectors have been constructed for DNAtransformation into yeast[1 ] .The selection markersof these conventional vectors are usually H IS3 ,TRP1 ,LEU2 ,and URA3 [1 ,2 ] . These selectionmarkers are responsible for the biosynthesis ofsome amino acids and consequently the hos…     

Isolation, characterization and functional analysis of a cdc48 homologue from tobacco BY-2 cells

The fission yeast Schizosaccharomyces pombe was used to identify genes from tobacco BY-2 cells that may play roles in cell cycle regulation. A cDNA encoding a protein homologous to the yeast CDC48 was isolated and the gene was designated as NtCDC48. The cDNA contains an open reading frame coding for a predicted protein of 808 amino acids which comprises of two typical ATPase modules (aa 245?374 and aa 518?646). Overexpression of NtCDC48 in tobacco BY-2 cells led to an increase in the mitotic index as well as to the formation of diffused mitotic spindles. NtCDC48-GFP fusion proteins are distributed ubiquitously through G1 to M phases, yet their subcellular localization varied regularly along with the cell cycle progression. These results indicate that NtCDC48 may play an important role in the regulation of cell cycle in BY-2 cells.     

Structural characteristics and biological functions of the HIV-1 gp120 V3 region

Recent studies demonstrate that the V3 loop of HIV-1 gp120 plays an important role in the attachment of HIV-1 to the target cells. Several amino acids in this domain are involved in the interaction of gp120 with the co-receptors. The V3 loop elicits one of the earliest antiviral antibody responses in HIV-1 infection and has been identified as the principal neutralizing determinant (PND). A subset of antibodies to V3 loop show a broad range of neutralizing activity. Unfortunately, this loop undergoes broad mutation and is one of the hypervariable regions. Mutations of some amino acids in this PND could affect syncytium formation, virus infectivity and neutralization. Knowing the structural characteristics and biological functions of the V3 region could help us to understand mechanism of HIV infection and to develop new strategy against HIV-1. In this review, the structural characteristics, variation and biological functions of the V3 loop as well as immunological responses to the V3 loop are discussed.     

Nonlinear optical properties of Eu<Subscript>2</Subscript>O<Subscript>3</Subscript> doped 5ZnO-20Nb<Subscript>2</Subscript>O<Subscript>5</Subscript>-75TeO<Subscript>2</Subscript> glasses

The third-order optical nonlinearities, including third-order nonlinear susceptibility X^(3), nonlinear refractive index (n2) and temporal response, were measured with forward DFWM using Nd:YAG mode-locked pulse laser. The results show that Eu203 doped 5ZnO-20Nb2O5-75TeO2 glasses have large n2 and ultra-fast temporal response. Raman spectra show that Eu2O3 dopant induces the changes in the local structure of glasses. The higher the dopant concentration, the larger the nonlinear refractive n2 and the faster the temporal response. The enhancement on the third-order optical nonlinearities can be attributed to the deformation of the electronic clouds in [TeO4] enhanced by Eu2O3 dopant.     

Rapid solidification mechanism of Ag60Sb34Cu6 ternary alloy in drop tube

Ternary eutectic growth involves competitive nu-cleation and growth of three solids from one liquid. Thesolidification behavior of ternary eutectic alloy is morecomplex than that of binary eutectic alloy due to the addi-tion of the third component[1—4]. Up to now, most scientificinvestigations on ternary eutectic alloy focus on the influ-ence of changing the component or adding a fourth even afifth element on the performance of the alloy[5—8]. How-ever, the information on crystal growth char…     

Effect of proton irradiation on structure relaxation of Zr<Subscript>41.5</Subscript>Ti<Subscript>14.9</Subscript>Cu<Subscript>12.6</Subscript>Ni<Subscript>10.5</Subscript>Be<Subscript>20.4</Subscript> bulk metallic glass

Effects of proton irradiation on structure re-laxation of Zr_(41.5)Ti_(14.9)Cu_(12.6)Ni_(10.5)Be_(20.4) bulk metallic glass areinvestigated by means of X-ray diffraction, differential scan-ning calorimetric and electronic resistance measurements.The results show that, at 203 K, the structure ofZr_(41.5)Ti_(14.9)Cu_(12.6)Ni_(10.5)Be_(20.4) bulk metal glass is relaxed obvi-ously by proton irradiation with the energy and the dosebeing of 160 keV and 1.65×10~(16) ions/cm~2, respectively. How-ever, Zr_(41 .5)Ti_(14.9)Cu_(12.6)Ni_(10.5)Be_(20.4) bulk metallic glass becomesmore steady amorphous state under two conditions, one isthat the energy and the dose are 160 keV and 1.1×10~(15)ions/cm~2, respectively. Another is that the energy and thedose are 120 keV and 6.5×10~(15) ions/cm~2, respectively.     

Structural stability of infinite-layer CaCuCO<Subscript>2</Subscript> under high pressure

Since the discovery of high temperature supercon-ductivity by Bednorz et al. in 1986[1], many series of cu- prate superconductors have been synthesized. Critical temperature has been improved continuously. But no verdict has been given upon what mechanism causes su- perconductivity. CaCuO2 conductive layers are the parent structure of the high Tc cuprate superconductors, such as Cu-12 (n-1)n[2], Tl-12(n-1)n[3] and Hg-12(n-1)n[4,5] super- conductor families. In 1988, infinite-layer (Ca0.86S…     

Agrobacterium tumefaciens-mediated Transformation of CryⅠA(b) gene to Trichoderma harzianum

In this study, Cry ⅠA(b) gene was successfully transferred into the biocontrol fungus Trichoderma harzianum with an efficiency of 60-180 transformants per 10^6 spores by using Agrobacterium tumefaciens-mediated transformation. Putative transformants were analyzed to test the presence of Cry ⅠA(b) gene by Southern blot. Most transformants contained a single T-DNA copy. RT-PCR analysis showed that the Cry ⅠA(b) gene was transcribed. Antifungal activities and insecticidal activities of the transformants were examined. There was no obvious difference in antifungal activities between the transformants and their wild strains. The modified mortalities of the transformants T1 and T2 were 69.57% and 91.30%, respectively. The tranformation system mediated by A. tumefaciens proved to be a powerful tool for the filamentous fungi transformation and functional genomic study with its high transformation frequency, simplicity of T-DNA integration, and genetic stability of transformants.     

Preparation of modified SiO2 colloidal spheres with succinic acid and the assembly of colloidal crystals

SiO2 colloidal spheres were synthesized by St-ber method. In order to enhance surface charge of the SiO2 spheres, they were modified with succinic acid. Scanning electron microscope (SEM) shows that the average size of modified SiO2 spheres is 473 nm, and its distribution standard deviation is less than 5%; Fourier-transform infrared spectra (FT-IR) and X-ray photoelectron spectrometer (XPS) results indicate that one end of succinic acid is chemically bonded to the SiO2 spheres through esterification; Zeta potential of the modified SiO2 spheres in water solution is improved from -53.72 to -67.46 mV, and surface charge density of the modified SiO2 spheres is enhanced from 0.19 to 0.94 μC/cm2. SiO2 colloidal crystal was fabricated from aqueous colloidal solution by the vertical deposition method at 40℃ and 60% relative humidity. SEM images show that the sample of SiO2 colloidal crystal is face-centered cubic (fcc) structure with its (111) planes parallel to the substrate. Transmission measurement shows the existence of photonic band gap at 1047 nm.     

Hydrothermal synthesis and characterization of new hybrid open-framework indium phosphate-oxalates

Crystalline open-framework inorganic materials have been widely studied because of the wide variety of the structures as well as their applications in heterogeneous catalysis, adsorption and ion exchange[1,2]. Of many open-framework solids, metal phosphates are an important family of materials, a large number of aluminum phos-phates[3,4], gallium phosphates[5—7], zinc phosphates[8—10], cobalt phosphate[11], beryllium phosphates[12—14] have been prepared and characterized. Recently, open-fra…     

Synthesis and crystal structure of the trimetallic erbium tetrahydride complex [Li(THF)4][{(ButCp)2Er(μ-H)} 3-(μ3-H)]

Reaction of [(Bu′tCp)2Er(μ-CI)]2 with Bu′Li in 1:1 molar ratio in THF at -78℃, after work-up, afforded the trimetallic erbium tetrahydride complex [Li(THF)4][{(Bu′Cp)2Er(μ-H)}3(μ3-H)] (1) by β-hydrogen elimination reaction. Crystal structure determination reveals that complex 1 has discrete ion pair structure. The anion is composed of three (Bu′Cp)2Er units to form a triangle array connected by three bridged hydrogen atoms, and the fourth hydrogen atom coordinated to three Er^3 ions. The coordination num-ber for each Er^3 ion is 9.     

Synthesis and characterization of two new types of oxovanadium complexes with pyrazole as ligand

Coordination chemistry of pyrazole-base ligands has been extensively studied since the poly(pyrazole)borate was invented by Trofimenko in 1966[1―4]. A variety of bidentate or multidentate pyrazole-derived ligands have been employed in coordination chemis…     

[Zn(bim)2]·(H2O)1.67: A metal-organic open-framework with sodalite topology

Considerable efforts have been focused on the crystal engineering of the supramolecular architectures with desired properties such as zeolitic, magnetic and electric conducting materials[1—3]. But crystal engineering is still a great challenge to the chemists since lack of control over the coordination modes of metal centers and ligands during assembly of extended structures[4]. Some successful synthetic strategies based on topologic control of building blocks have been developed especially i…