Folylpolyglutamate Derivatives of Pisum sativum L. Determination of Polyglutamate Chain Lengths by High Performance Liquid Chromatography Following Conversion to p-aminobenzoylpolyglutamates

The folypolyglutamate derivatives of pea seedlings (Pisum sativumL. cv. Homesteader) were extracted in the presence of 2-mercaptoethanoland cleaved to p-aminobenzoylpolyglutamates by treatment withZn-HCl. Azo dyes were formed by reaction with naphthylethylenediamine and purified by polyacrylamide gel chromatography. p-Aminobenzoylpolyglutamateswere regenerated from these dyes by Zn treatment and then concentratedin vacuo. These derivatives were separated according to glutamylchain length by high performance liquid chromatography on WhatmanPartisil SAX columns. The folylpolyglutamates of 4 day old peacotyledons, pea leaves and isolated chloroplasts were mainlytetra- and pentaglutamates. These and folates of shorter chainlength were labelled when seeds and aerial shoots were incubatedwith p-aminobenzoate-[¹⁴C]. Labelling of the pentaglutamatewas reduced in seeds that were imbibed in the presence of 0.1mM methotrexate. Studies of cotyledon folylpolyglutamate synthetaseshowed that polyglutamate chain length was affected by incubationtime and the concentration of tetra-hydrofolate monoglutamatein the reaction system. ¹Present address: Department of Biology, University of Lethbridge,Alberta, Canada T1K 3M4 ²Present address: Department of Horticulture, Xiong-yue AgriculturalCollege, Xiong-yue, Liaoning Province, China (Received August 4, 1989; Accepted December 5, 1989)     

Characterization of cultured insect cells selected by <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> crystal toxin

Summary Selection for resistance against Bacillus thuringiensis (Bt) Cry1Ac10 in the Trichoplusia ni (Hübner) cell line BTI-TN-5B1-4 (TnH5) was tested, and the development of resistance in the selected cells was like a S-form curve. Monitoring at the Cry1Ac10 50th challenge, the resistance ratio was 1, 294-fold as many as that of initial cells. But the resistance to Cry1Ac10 declined gradually when the selection was relaxed. The resistance declined rapidly at the low level of resistance and slowly at the high level of resistance. This resistant cell had high resistance to all the tested solubilized trypsin-treated mixture of crystal multitoxins of B. thuringiensis subsp. aizawai GC-91, an engineering bacterium of Bt, B. thuringiensis subsp. aizawai HD-133 and B. thuringiensis subsp. kurstaki HD-1, and low cross-resistance (19.7-fold) to activated Cry1C. Both N-acetyl-d-galactosamine (GalNAc) and tunicamycin did not inhibit the toxicity of Cry1Ac10 to the susceptible TnH5 cells. Comparison of the total proteins of the selected resistant cells with that of the nonselected susceptible cells by two-dimensional electrophoresis analysis showed that were obvious differences among the 11 protein expression. These results strongly suggest that there exists an unknown mechanism of resistance in the cell line that was different from the reported mechanisms in insects.     

High level secretory expression of murine OCIL by CHO cells and action of OCIL on osteoclast differentiation

Receptor activator of nuclear factor-kB ligand (RANKL), a well-known membrane-bound molecule expressed on osteoblasts and bone marrow stromal cells, is believed to induce osteoclast differentiation and activation by binding to the receptor activator of nuclear factor-kB (RANK), which is expressed on the surface of osteoclast lineage cells. This induction is inhibited by osteoprotegerin (OPG) that is secreted by osteoblast lineage and acts as a decoy receptor of RANKL. Currently the essential role of the OPG/RANKL/RANK system in the process of osteoclast maturation, as well as activation, has been well established, and the majority of bone resorption regulators control osteoclast formation and activation through their effects on this system and especially on the relative expression levels of RANKL and OPG [1].     

Expression of modified xynA gene fragments from Bacillus subtilis BE-91

Expression of modified xynA gene fragments in Escherichia coli BL21 was studied, using the complete xynA gene from Bacillus subtilis BE-91 as the positive control. The technical workflow consisted of the following steps: (1) predicting protein structures relative to the xynA gene; (2) designing primers for modifiers; (3) amplifying the modifiers; (4) integrating the modifiers with the pET-28a(+) vector; (5) transferring the recombinant plasmids into E. coli BL21; (6) evaluating and analyzing the expression of modified cells. The results were: (1) the xynA gene from BE-91 with the untranslated region deleted on both ends was able to promote XynA activity by 28.9 %; (2) deletion of the 1- to 16-amino acid (AA) coding sequence in the open reading frame on the 5′-end, deletion of the 209- to 213-AA fragment on the 3′-end and deletion of the 20 AA on both ends could promote XynA activity by 27.2, 27.7 and 24.0 %,respectively; (3) deletion of the 1- to 29-AA fragment on the 5′-end and deletion of the 197- to 213-AA fragment on the 3′-end could reduce XynA activity dramatically by 95.6 and 74.8 %, respectively; (4) inactivation factors of XynA would be either the first β-fold and the hydrophilic structure domain or the last two α-screws and the seventeenth turn region. The results mean that any deletion in the catalytic domain would lead to a decline or inactivation in XynA activity while the deletion of any sequence outside the catalytic domain could effectively promote XynA activity, as such sequences are unnecessary for XynA function.     

All-atom structural investigation of kinesin-microtubule complex constrained by high-quality cryo-electron-microscopy maps

In this study, we have performed a comprehensive structural investigation of three major biochemical states of a kinesin complexed with microtubule under the constraint of high-quality cryo-electron-microscopy (EM) maps. In addition to the ADP and ATP state which were captured by X-ray crystallography, we have also modeled the nucleotide-free or APO state for which no crystal structure is available. We have combined flexible fitting of EM maps with regular molecular dynamics simulations, hydrogen-bond analysis, and free energy calculation. Our APO-state models feature a subdomain rotation involving loop L2 and α6 helix of kinesin, and local structural changes in active site similar to a related motor protein, myosin. We have identified a list of hydrogen bonds involving key residues in the active site and the binding interface between kinesin and microtubule. Some of these hydrogen bonds may play an important role in coupling microtubule binding to ATPase activities in kinesin. We have validated our models by calculating the binding free energy between kinesin and microtubule, which quantitatively accounts for the observation of strong binding in the APO and ATP state and weak binding in the ADP state. This study will offer promising targets for future mutational and functional studies to investigate the mechanism of kinesin motors.     

Simultaneous measurement of plasma vitamin D(3) metabolites, including 4β,25-dihydroxyvitamin D(3), using liquid chromatography-tandem mass spectrometry

Simultaneous and accurate measurement of circulating vitamin D metabolites is critical to studies of the metabolic regulation of vitamin D and its impact on health and disease. To that end, we have developed a specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method that permits the quantification of major circulating vitamin D₃ metabolites in human plasma. Plasma samples were subjected to a protein precipitation, liquid–liquid extraction, and Diels–Alder derivatization procedure prior to LC–MS/MS analysis. Importantly, in all human plasma samples tested, we identified a significant dihydroxyvitamin D₃ peak that could potentially interfere with the determination of 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] concentrations. This interfering metabolite has been identified as 4β,25-dihydroxyvitamin D₃ [4β,25(OH)₂D₃] and was found at concentrations comparable to 1α,25(OH)₂D₃. Quantification of 1α,25(OH)₂D₃ in plasma required complete chromatographic separation of 1α,25(OH)₂D₃ from 4β,25(OH)₂D₃. An assay incorporating this feature was used to simultaneously determine the plasma concentrations of 25OHD₃, 24R,25(OH)₂D₃, 1α,25(OH)₂D₃, and 4β,25(OH)₂D₃ in healthy individuals. The LC–MS/MS method developed and described here could result in considerable improvement in quantifying 1α,25(OH)₂D₃ as well as monitoring the newly identified circulating metabolite, 4β,25(OH)₂D₃.     

Positional isotope exchange analysis of the pantothenate synthetase reaction

Pantothenate synthetase from Mycobacterium tuberculosis catalyzes the formation of pantothenate from ATP, D-pantoate, and beta-alanine. The formation of a kinetically competent pantoyl-adenylate intermediate was established by the observation of a positional isotope exchange (PIX) reaction within (18)O-labeled ATP in the presence of d-pantoate. When [betagamma-(18)O(6)]-ATP was incubated with pantothenate synthetase in the presence of d-pantoate, an (18)O label gradually appeared in the alphabeta-bridge position from both the beta- and the gamma-nonbridge positions. The rates of these two PIX reactions were followed by (31)P NMR spectroscopy and found to be identical. These results are consistent with the formation of enzyme-bound pantoyl-adenylate and pyrophosphate upon the mixing of ATP, D-pantoate, and enzyme. In addition, these results require the complete torsional scrambling of the two phosphoryl groups of the labeled pyrophosphate product. The rate of the PIX reaction increased as the D-pantoate concentration was elevated and then decreased to zero at saturating levels of D-pantoate. These inhibition results support the ordered binding of ATP and D-pantoate to the enzyme active site. The PIX reaction was abolished with the addition of pyrophosphatase; thus, PP(i) must be free to dissociate from the active site upon formation of the pantoyl-adenylate intermediate. The PIX reaction rate diminished when the concentrations of ATP and D-pantoate were held constant and the concentration of the third substrate, beta-alanine, was increased. This observation is consistent with a kinetic mechanism that requires the binding of beta-alanine after the release of pyrophosphate from the active site of pantothenate synthetase. Positional isotope exchange reactions have therefore demonstrated that pantothenate synthetase catalyzes the formation of a pantoyl-adenylate intermediate upon the ordered addition of ATP and pantoate.     

Hypoxia induces the activation of hepatic stellate cells through the PVT1-miR-152-ATG14 signaling pathway

Molecular and Cellular Biochemistry - Shrm4 is a protein that is exclusively expressed in polarized tissues. The physiological function of Shrm4 in the brain was required to be elucidated. Thus, we...     

Comparative genomics of methicillin-resistant Staphylococcus aureus ST239: distinct geographical variants in Beijing and Hong Kong

Background

The ST239 lineage is a globally disseminated, multiply drug-resistant hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA). We performed whole-genome sequencing of representative HA-MRSA isolates of the ST239 lineage from bacteremic patients in hospitals in Hong Kong (HK) and Beijing (BJ) and compared them with three published complete genomes of ST239, namely T0131, TW20 and JKD6008. Orthologous gene group (OGG) analyses of the Hong Kong and Beijing cluster strains were also undertaken.

Results

Homology analysis, based on highest-percentage nucleotide identity, indicated that HK isolates were closely related to TW20, whereas BJ isolates were more closely related to T0131 from Tianjin. Phylogenetic analysis, incorporating a total of 30 isolates from different continents, revealed that strains from HK clustered with TW20 into the ‘Asian clade’, whereas BJ isolates and T0131 clustered closely with strains of the ‘Turkish clade’ from Eastern Europe. HK isolates contained the typical φSPβ-like prophage with the SasX gene similar to TW20. In contrast, BJ isolates contained a unique 15 kb PT1028-like prophage but lacked φSPβ-like and φSA1 prophages. Besides distinct mobile genetic elements (MGE) in the two clusters, OGG analyses and whole-genome alignment of these clusters highlighted differences in genes located in the core genome, including the identification of single nucleotide deletions in several genes, resulting in frameshift mutations and the subsequent predicted truncation of encoded proteins involved in metabolism and antimicrobial resistance.

Conclusions

Comparative genomics, based on de novo assembly and deep sequencing of HK and BJ strains, revealed different origins of the ST239 lineage in northern and southern China and identified differences between the two clades at single nucleotide polymorphism (SNP), core gene and MGE levels. The results suggest that ST239 strains isolated in Hong Kong since the 1990s belong to the Asian clade, present mainly in southern Asia, whereas those that emerged in northern China were of a distinct origin, reflecting the complexity of dissemination and the dynamic evolution of this ST239 lineage.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-529) contains supplementary material, which is available to authorized users.