Genetic engineering of a Ca(2+) dependent chemical switch into the linear biomotor kinesin

Kinesin is a linear motor protein driven by energy released by ATP hydrolysis. In the present work, we genetically installed an M13 peptide sequence into Loop 12 of kinesin, which is one of the major microtubule binding regions of the protein. Because the M13 sequence has high affinity for Ca(2+)-calmodulin, the association of the engineered kinesin with microtubules showed a steep Ca(2+)-dependency in ATPase activity at Ca(2+) concentrations of pCa 6.5-8. The calmodulin-binding domain of plant kinesin-like calmodulin-binding protein is also known to confer Ca(2+)-calmodulin regulation to kinesins. Unlike this plant kinesin, however, our novel engineered kinesin achieves this regulation while maintaining the interaction between kinesin and microtubules. The engineered kinesin is switched on/off reversibly by an external signal (i.e., Ca(2+)-calmodulin) and, thus, can be used as a model system for a bio/nano-actuator.     

Caffeine biosynthesis in young leaves of Camellia sinensis: In vitro studies on N-methyltransferase activity involved in the conversion of xanthosine to caffeine

The aim of this study was to investigate the S -adenosylmethionine dependent N -methyltransferase(s) (NMT) associated with the three methylation steps in the caffeine biosynthesis pathway in tea ( Camellia sinensis L.). NMT activity in cell-free preparations from young leaves was purified by anion-exchange and gel-filtration column chromatography. In both systems, a single zone of NMT activity, with broad substrate specificity was detected. The N-3 position of dimethylxanthine and monomethylxanthines was methylated more readily than N-1 while comparatively little substitution occurred at the N-7 locus. When xanthosine was used as a substrate only the N-7 position was methylated. These results indicate that a single NMT may participate in the conversion of xanthosine to caffeine. The apparent M_r of the NMT, estimated by gel filtration chromatography, was 61 000. The substrate specificity of the NMT is compatible with the operation of a xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine pathway as the main biosynthetic route to caffeine in young tea leaves. The data also indicate that the conversion of 7-methylxanthine → paraxanthine → caffeine may function as one of a number of minor pathways that also contribute to the production of caffeine.     

tRNA Modifying Enzymes,NSUN2 and METTL1, Determine Sensitivity to 5-Fluorouracil in HeLa Cells

Nonessential tRNA modifications by methyltransferases are evolutionarily conserved and have been reported to stabilize mature tRNA molecules and prevent rapid tRNA decay (RTD). The tRNA modifying enzymes, NSUN2 and METTL1, are mammalian orthologs of yeast Trm4 and Trm8, which are required for protecting tRNA against RTD. A simultaneous overexpression of NSUN2 and METTL1 is widely observed among human cancers suggesting that targeting of both proteins provides a novel powerful strategy for cancer chemotherapy. Here, we show that combined knockdown of NSUN2 and METTL1 in HeLa cells drastically potentiate sensitivity of cells to 5-fluorouracil (5-FU) whereas heat stress of cells revealed no effects. Since NSUN2 and METTL1 are phosphorylated by Aurora-B and Akt, respectively, and their tRNA modifying activities are suppressed by phosphorylation, overexpression of constitutively dephosphorylated forms of both methyltransferases is able to suppress 5-FU sensitivity. Thus, NSUN2 and METTL1 are implicated in 5-FU sensitivity in HeLa cells. Interfering with methylation of tRNAs might provide a promising rationale to improve 5-FU chemotherapy of cancer.     

Bifunctional thrombin inhibitors based on the sequence of hirudin45-65

The interaction of alpha-thrombin with the hirudin (HV1) fragment N alpha-acetyl desulfo hirudin45-65 (P51) was investigated. Kinetic analysis revealed that P51 inhibits the proteolysis of a tripeptidyl substrate with Ki = 0.72 +/- 0.13 and 0.11 +/- 0.03 microM for bovine and human alpha-thrombins, respectively. The inhibition was partially competitive, affecting substrate binding to the enzyme-inhibitor complex by a factor alpha = 2 (bovine) and alpha = 4 (human) characteristic of hyperbolic inhibitors. P51 also inhibited thrombin-induced fibrin clot formation with IC50 values of 0.94 +/- 0.20 and 0.058 +/- 0.006 microM for bovine and human alpha-thrombins, respectively. The enhanced antithrombin activity for human thrombin could be attributed to species variations in the putative auxiliary "anion" exosite since N alpha-acetyl desulfo hirudin55-65 displayed the same rank order of potency shift in a clotting assay without inhibiting the amidolytic activity of either enzyme. From these observations, a potent thrombin inhibitor was designed having modified residues corresponding to the P1 and P3 recognition sites. N alpha-Acetyl[D-Phe45, Arg47] hirudin45-65 (P53) emerged as a pure competitive inhibitor with a Ki = 2.8 +/- 0.9 nM and IC50 = 4.0 +/- 0.8 nM (human alpha-thrombin) and is designated as a "bifunctional" inhibitor. Its enhanced potency could be explained by a cooperative intramolecular interaction between the COOH-terminal domain of the inhibitor and the auxiliary exosite of thrombin on the one hand, and the modified NH2-terminal residues with the catalytic site on the other.     

Preparation and characterization of the water-soluble heme-binding domain of cytochrome c1 from the Rhodobacter sphaeroides bc1 complex.

The ubiquinol:cytochrome c2 oxidoreductase (bc1 complex) of Rhodobacter sphaeroides consists of four subunits. One of these subunits, cytochrome c1, is the site of interaction with cytochrome c2, a periplasmic protein. In addition, the sequences of the fbcC gene and of the cytochrome c1 subunit that it encodes suggest that the protein should be located on the periplasmic side of the cytoplasmic membrane and that it is anchored to the membrane by a single membrane-spanning alpha-helix located at the carboxyl-terminal end of the polypeptide. Site-directed mutagenesis of the fbcC gene was used to alter the codon for Gln228 to a stop codon. This results in the production of a truncated version of the cytochrome c1 subunit that lacks the membrane anchor at the carboxyl terminus. The bc1 complex fails to assemble properly as a result of this mutation, but the Rb. sphaeroides cells expressing the altered gene contain a water-soluble form of cytochrome c1 in the periplasm. The water-soluble cytochrome c1 was purified and characterized. The amino-terminal sequence is identical with that of the membrane-bound subunit, indicating the signal sequence is properly processed. High pressure liquid chromatography gel filtration chromatography indicates it is monomeric (28 kDa). The heme content and electrochemical properties are similar to those of the intact subunit within the complex. Flash-induced electron transfer kinetics measured using whole cells demonstrated that the water-soluble cytochrome c1 is competent as a reductant for cytochrome c2 within the periplasmic space. These data show that the isolated water-soluble cytochrome c1 retains many of the properties of the membrane-bound subunit of the bc1 complex and, therefore, will be useful for further structural and functional characterization.     

Synthesis of Aromatic Carbonyl Compounds by Friedel-Crafts Reaction Using BF3 Catalyst

Friedel-Crafts reaction using BF₃ catalyst was investigated in order to synthesize various carbonyl compounds.     

Mode of Loss of Phosphatidyl-ethanolamine in Autoxidation

This report identifies and describes the chemical structure of granular material that accumulates in the cytoplasm of Selenomonas ruminantium grown in glucose or lactate medium. The granular material was identified to be glycogen. Its molecular weight was about 2 x 10⁷ daltons. Conversion into maltose with α-amylase, β-amylase, and isoamylase was 61%, 37%, and 15%, respectively. The glycogen was digested completely by joint action of β-amylase and isoamylase, and its conversion into maltose was 103%. The average chain length of the glycogen was 23.5. The maximum absorption of the iodine complex of the glycogen was at 520 nm. These results led us to conclude that the chemical structure of this glycogen was similar to that of plant amylopectin, unlike normal Microbiol or animal glycogen so far known. When S. ruminantium was grown in glucose medium, the amount of glycogen in cells reached about 260 µg/mg dry weight of cells during late exponential phase and early stationary phase.     

Enhanced resistance againstEscherichia coli infection by subcutaneous administration of the hot-water extract ofChlorella vulgaris in cyclophosphamide-treated mice

Summary The effects ofChlorella vulgaris extract (CVE-A) on the recovery of leukocyte number and the augmentation of resistance to bacterial infection were examined in CDF1 mice made neutropenic by cyclophosphamide (CY). They were treated intraperitoneally with CY (150 mg/kg) on day 0, and were given CVE-A (50 mg/kg) subcutaneously (s. c.) every other day from day 1 to day 13 after CY treatment. CVE-A accelerated the recovery of polymorphonuclear leukocytes (PMN) in the peripheral blood in CY-treated mice. The number of granulocyte/monocyte-progenitor cells (CFU-GM) in the spleen increased rapidly and highly after the administration of CVE-A in CY-treated mice, in contrast to the absence of change due to CVE-A in the number of bone marrow cells in CY-treated mice. Administration of CVE-A in CY-treated mice enhanced the accumulation of PMN in the inflammatory site and the activity of the accumulated leukocyte cells in luminol-dependent chemiluminescence. The mice became highly susceptible to an intraperitoneal infection withE.coli on day 4 after CY treatment, whereas the mice given CVE-A showed an enhanced resistance againstE.coli infection, irrespective of the timing of challenge. The bacterial number in CY-treated mice increased explosively after inoculation, resulting in death within 24 h. A progressive elimination of bacteria was observed from 6 h in the peritoneal cavity, spleen and liver of CY-treated mice given CVE-A s.c. These results indicate that CVE-A can be used as a potent stimulant of nonspecific resistance to infection in neutropenic mice.     

Identification and migration of primordial germ cells in Atlantic salmon,Salmo salar: Characterization of Vasa,Dead End,and Lymphocyte antigen 75 genes

No information exists on the identification of primordial germ cells (PGCs) in the super‐order Protacanthopterygii, which includes the Salmonidae family and Atlantic salmon (Salmo salar L.), one of the most commercially important aquatic animals worldwide. In order to identify salmon PGCs, we cloned the full‐length cDNA of vasa, dead end (dnd), and lymphocyte antigen 75 (ly75/CD205) genes as germ cell marker candidates, and analyzed their expression patterns in both adult and embryonic stages of Atlantic salmon. Semi‐quantitative RT‐PCR results showed that salmon vasa and dnd were specifically expressed in testis and ovary, and vasa, dnd, and ly75 mRNA were maternally deposited in the egg. vasa mRNA was consistently detected throughout embryogenesis while dnd and ly75 mRNA were gradually degraded during cleavages. In situ analysis revealed the localization of vasa and dnd mRNA and Ly75 protein in PGCs of hatched larvae. Whole‐mount in situ hybridization detected vasa mRNA during embryogenesis, showing a distribution pattern somewhat different to that of zebrafish; specifically, at mid‐blastula stage, vasa‐expressing cells were randomly distributed at the central part of blastodisc, and then they migrated to the presumptive region of embryonic shield. Therefore, the typical vasa localization pattern of four clusters during blastulation, as found in zebrafish, was not present in Atlantic salmon. In addition, salmon PGCs could be specifically labeled with a green fluorescence protein (GFP) using gfp‐rt‐vasa 3′‐UTR RNA microinjection for further applications. These findings may assist in understanding PGC development not only in Atlantic salmon but also in other salmonids. Mol. Reprod. Dev. © 2013 Wiley Periodicals, Inc.     

Stability of recombinant bovine interferon-γ antiviral activity in the absence of stabilizing additives

The stability of recombinant bovine interferon-γ (rbIFN-γ) produced by a baculovirus expression system was investigated under different storage conditions: freezing-thawing and storage for 30 days at temperatures of -80, 4, 25, and 37°C. Antiviral activity was not significantly decreased by freeze-thawing at least five times. Furthermore, although not statistically different, antiviral activity gradually decreased as temperature increased. These findings suggest that rbIFN-γ possesses high thermal and freeze-thaw stability.