Solid Phase and Solution Phase Synthesis of Gamma Amino Acid Homo-Oligomers and Mixed Oligomers

Abstract  

An efficient stepwise synthesis of homo-oligomers and mixed oligomers of gabapentin and pregabalin on solid support using Fmoc-protected derivatives and HBTU/HOBt/DIEA as coupling agent is described. The synthesis was also carried out using solution phase methodology. The Gpn/Pgn homo oligomers and mixed oligomers forms C₉ helix in solution as determined by NMR study. Chiral as well as achiral gamma amino acids were used for the synthesis of oligomers in order to investigate the secondary structural preferences.     

Neurotrophic Requirements of Human Motor Neurons Defined Using Amplified and Purified Stem Cell-Derived Cultures

Human motor neurons derived from embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are a potentially important tool for studying motor neuron survival and pathological cell death. However, their basic survival requirements remain poorly characterized. Here, we sought to optimize a robust survival assay and characterize their response to different neurotrophic factors. First, to increase motor neuron yield, we screened a small-molecule collection and found that the Rho-associated kinase (ROCK) inhibitor Y-27632 enhances motor neuron progenitor proliferation up to 4-fold in hESC and hiPSC cultures. Next, we FACS-purified motor neurons expressing the Hb9::GFP reporter from Y-27632-amplified embryoid bodies and cultured them in the presence of mitotic inhibitors to eliminate dividing progenitors. Survival of these purified motor neurons in the absence of any other cell type was strongly dependent on neurotrophic support. GDNF, BDNF and CNTF all showed potent survival effects (EC₅₀ 1–2 pM). The number of surviving motor neurons was further enhanced in the presence of forskolin and IBMX, agents that increase endogenous cAMP levels. As a demonstration of the ability of the assay to detect novel neurotrophic agents, Y-27632 itself was found to support human motor neuron survival. Thus, purified human stem cell-derived motor neurons show survival requirements similar to those of primary rodent motor neurons and can be used for rigorous cell-based screening.     

Edelfosine-induced metabolic changes in cancer cells that precede the overproduction of reactive oxygen species and apoptosis

Background  

Metabolic flux profiling based on the analysis of distribution of stable isotope tracer in metabolites is an important method widely used in cancer research to understand the regulation of cell metabolism and elaborate new therapeutic strategies. Recently, we developed software Isodyn, which extends the methodology of kinetic modeling to the analysis of isotopic isomer distribution for the evaluation of cellular metabolic flux profile under relevant conditions. This tool can be applied to reveal the metabolic effect of proapoptotic drug edelfosine in leukemia Jurkat cell line, uncovering the mechanisms of induction of apoptosis in cancer cells.     

Structural and Stereochemical Studies of Hydroxyanthraquinone Derivatives from the Endophytic Fungus Coniothyrium sp

Four known hydroxyanthraquinones ( 1–4 ) together with four new derivatives having a tetralone moiety, namely coniothyrinones A–D ( 5–8 ), were isolated from the culture of Coniothyrium sp., an endophytic fungus isolated from Salsola oppostifolia from Gomera in the Canary Islands. The structures of the new compounds were elucidated by detailed spectroscopic analysis and comparison with reported data. The absolute configurations of coniothyrinones A ( 5 ), B ( 6 ), and D ( 8 ) were determined by TDDFT calculations of CD spectra, allowing the determination of the absolute configuration of coniothyrinone C ( 7 ) as well. Coniothyrinones A ( 5 ), B ( 6 ), and D ( 8 ) could be used as ECD reference compounds in the determination of absolute configuration for related tetralone derivatives. This is the first report of anthraquinones and derivatives from an isolate of the genus Coniothyrium sp. These compounds showed inhibitory effects against the fungus Microbotryum violaceum, the alga Chlorella fusca, and the bacteria Escherichia coli and Bacillus megaterium. Chirality 25:141–148, 2013. © 2012 Wiley Periodicals, Inc.     

Analysis of synonymous codon usage in H5N1 virus and other influenza A viruses

In this study, we calculated the codon usage bias in H5N1 virus and performed a comparative analysis of synonymous codon usage patterns in H5N1 virus, five other evolutionary related influenza A viruses and a influenza B virus. Codon usage bias in H5N1 genome is a little slight, which is mainly determined by the base compositions on the third codon position. By comparing synonymous codon usage patterns in different viruses, we observed that the codon usage pattern of H5N1 virus is similar with other influenza A viruses, but not influenza B virus, and the synonymous codon usage in influenza A virus genes is phylogenetically conservative, but not strain-specific. Synonymous codon usage in genes encoded by different influenza A viruses is genus conservative. Compositional constraints could explain most of the variation of synonymous codon usage among these virus genes, while gene function is also correlated to synonymous codon usages to a certain extent. However, translational selection and gene length have no effect on the variations of synonymous codon usage in these virus genes.     

Identification of DNA Segments Capable of Rescuing a Non-Mendelian Mutant in Paramecium

The non-Mendelian mutant d48 of Paramecium tetraurelia contains micronuclear wild type A genes, but at autogamy and conjugation proper processing fails and new macronuclei lack A genes. When cloned A genes are injected into the macronucleus of d48, proper processing is restored at the next autogamy; d48 is rescued, becoming permanently wild type. In the present study we have injected portions of the A gene into d48. We find that the ability to rescue extends over a large portion of the gene, with highest activity near a series of 221-bp repeat units in the middle of the gene. Regions outside the A gene are inactive.     

<Emphasis Type="Italic">In vitro</Emphasis> adventitious shoot organogenesis and plant regeneration from seedling explants of <Emphasis Type="Italic">Albizia odoratissima</Emphasis> L.f. (Benth.)

Epicotyl, petiole, and cotyledon explants derived from 14-d-old seedlings of Albizia odoratissima were cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of either 6-benzylaminopurine (BAP) solely or in combination with 0.5 μM naphthalene-3-acetic acid (NAA). The percentage of shoot regeneration and the number of shoots regenerated varied significantly depending on the type of explants used, the concentration of plant growth regulators, and the orientation of explants on the culture medium. The best response in terms of the percentage of shoot regeneration was obtained from epicotyls cultured horizontally on MS medium supplemented with 5 μM BAP, whereas the highest number of shoots per responding explant was recorded on medium containing 2.5 μM BAP and 0.5 μM NAA. Successful rooting was achieved by placing the microshoots onto MS medium containing 25 μM indole-3-butyric acid (IBA) for 24 h first, then transferring to the same medium without IBA. Of the various substrates tested, vermiculite was the best for plant acclimatization, as 75% of the plants survived and became established.     

Cloning and expression of thermophilic catechol 1,2-dioxygenase gene (catA) from Streptomyces setonii

Streptomyces setonii (ATCC 39116) degrades various single aromatic compounds such as phenol or benzoate via an ortho-cleavage pathway using catechol 1,2-dioxygenase (C12O). A PCR using degenerate primers based on the conserved regions of known C12O-encoding genes amplified a 0.45-kbp DNA fragment from S. setonii total DNA. A Southern hybridization analysis and size-selected DNA library screening using the 0.45-kbp PCR product as a probe led to the isolation of a 6.4-kbp S. setonii DNA fragment, from which the C12O-encoding genetic locus was found to be located within a 1.4-kbp DNA fragment. A complete nucleotide sequencing analysis of the 1.4-kbp DNA fragment revealed a 0.84-kbp open reading frame, which showed a strong overall amino acid similarity to the known high-G+C Gram-positive (but significantly less to the Gram-negative) bacterial mesophilic C12Os. The heterologous expression of the cloned 1.4-kbp DNA fragment in Escherichia coli demonstrated that this C12O possessed a thermophilic activity within a broad temperature range (up to 65 degrees C) and showed a higher activity against 3-methylcatechol than catechol or 4-methylcatechol, but no activity against protocatechuate.     

林可链霉菌黑色素生物合成基因的克隆与表达

以pIJ702的melCl－C2基因为探针杂交林可链霉菌（Streptomyceslincolnensis）78－11染色体DNA,呈现出3．2kb的BamHI片段和2．6kb的SphI片段等一系列阳性条带。构建了含3．0～3．5kbBamHI片段的林可链霉菌78-11基因文库,从中分离克隆了黑色素生物合成基因melCl和melC2,并测定了含有mel基因的重组子pRSB336插入片段的全部DNA顺序。3152bpBamHI片段含有5个开放阅读框架,其中melCl和melC2与链霉菌属三个种的相应基因具有较高的同源性。此外,林可链霉菌78－11的melC2基因产物与人和鼠的酪氨酸酶轻微同源,分别为17．3％和24．5％。种种迹象表明,melCl、melC2和orf3组成黑色素生物合成操纵子结构。为了进一步鉴定上述克隆的林可链霉菌78-11黑色素生物合成基因,构建了分别含有新霉素抗性基因启动子和正反方向mel基因的重组质粒pPZ518和pPZ519,并转化变铅青链霉菌TK23。随机挑选的12个pPZ518转化子在R2YE培养基上均能分泌淡褐色色素,而所有的pPZ519和pES1转化子则都呈白色。     

Imobilization of β-glucosidase using the cellulose-binding domain of Bacillus subtilis endo-β-1,4-glucanase

A recombinant plasmid pβCBD was constructed for immobilization of Cellulomonas fimi β-glucosidase (Cbg) using the cellulose-binding domain (CBD) of Bacillus subtilis BSE 616 endo-β-1,4-glucanase (Beg). The Cbg-CBD _Beg fusion protein, 80 kDa, was expressed in Escherichia coli and immobilized to Avicel. Cellobiose was completely hydrolyzed with the immobilized fusion protein. The fusion protein bound to Avicel retained full activity during continuous operation for 24 h at 4°C. This revised version was published online in November 2006 with corrections to the Cover Date.