A new ten-membered lactone from <Emphasis Type="Italic">Tubercularia</Emphasis> sp<Emphasis Type="Italic">.</Emphasis> TF5, an endophytic fungus of <Emphasis Type="Italic">Taxus mairei</Emphasis>

One new ten-membered lactone (1) named (Z)-4,6,9-trihydroxy-10-nonyl-3,4,5,6,9,10-hexahydrooxecin-2-one along with 5-methoxycarbonylmellein (2) and cytochalasin D (3) were isolated from the culture of the endophytic fungus strain Tubercularia sp. TF5, originally separated from the inner bark of Taxus mairei obtained in Fujian Province, Southeast China. The structures of compounds 1–3 were elucidated by spectroscopic methods. The antimicrobial and cytotoxic activities of 1 were analyzed but it showed no significant activities.     

Analysis of <Emphasis Type="Italic">Escherichia coli</Emphasis> nicotinate mononucleotide adenylyltransferase mutants <Emphasis Type="Italic">in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

Background  

Adenylation of nicotinate mononucleotide to nicotinate adenine dinucleotide is the penultimate step in NAD⁺ synthesis. In Escherichia coli, the enzyme nicotinate mononucleotide adenylyltransferase is encoded by the nadD gene. We have earlier made an initial characterization in vivo of two mutant enzymes, NadD72 and NadD74. Strains with either mutation have decreased intracellular levels of NAD⁺, especially for one of the alleles, nadD72.     

Effects of <Emphasis Type="Italic">L</Emphasis>-Carnitine on Sucrose-Induced Hyperlipidaemia

Summary. L-Carnitine, L-(−)-β-hydroxy-γ-trimethylaminobutyrate, plays an important role as a factor necessary for the transport of long-chain fatty acids into the mitochondria. In order to investigate the influence of L-carnitine on hyperlipidaemias, the experimental model of the sucrose-induced hypertriglyceridaemia of the rat was used. In these experiments L-carnitine in the dose of 11 mg per day and 100 g body weight (over the period of 1 week) was able to antagonize the sucrose-induced hypertriglyceridaemia and the increase of serum free fatty acid level in female rats of the Wistar strain. Carnitine administration did not change the activities of lipogenic enzymes and fatty acid synthesis in the liver. However, L-carnitine increases the rate of hepatic fatty acid oxidation. Our results indicate a hypotriglyceridemic and free fatty acid lowering effect of L-carnitine, and suggest the use of this compound in the therapy of hyperlipidaemias.     

Extracellular Production and Characterization of Two <Emphasis Type="Italic">Streptomyces</Emphasis><Emphasis Type="SmallCaps">l</Emphasis>-Asparaginases

l-Asparaginase (ASNase) has proved its use in medical and food industries. Sequence-based screening showed the thermophilic Streptomyces strain Streptomyces thermoluteus subsp. fuscus NBRC 14270 (14270 ASNase) to positive against predicted ASNase primary sequences. The 14270 ASNase gene and four l-asparaginase genes from Streptomyces coelicolor, Streptomyces avermitilis, and Streptomyces griseus (SGR ASNase) were expressed in Streptomyces lividans using a hyperexpression vector: pTONA5a. Among those genes, only 14270 ASNase and SGR ASNase were successful for overexpression and detected in culture supernatants without an artificial signal peptide. Comparison of the two Streptomyces enzymes described above demonstrated that 14270 ASNase was superior to SGR ASNase in terms of optimum temperature, thermal stability, and pH stability.     

Antioxidant flavonoids from <Emphasis Type="Italic">Tamus communis</Emphasis> ssp. <Emphasis Type="Italic">cretica</Emphasis>

A new flavonoid, kaempferol-3,4′-di-O-α-L-rhamnopyranoside (1), and three known flavonoids (2–4) were isolated from the aerial parts of T. communis L. The structure of the new compound was elucidated on the basis of spectroscopic data. Compounds 1 and 2 showed significant antioxidant activity (IC₅₀ 187.151 ± 0.821 μM, and 92.079±0.513 μM, respectively), whereas compounds 3 and 4 showed moderate activity in DPPH free radical scavenging assays. Published in Khimiya Prirodnykh Soedinenii, No. 3, pp. 295–297, May–June, 2009.     

Enhanced Production of <Emphasis Type="SmallCaps">l</Emphasis>-Arginine by Expression of <Emphasis Type="Italic">Vitreoscilla</Emphasis> Hemoglobin Using a Novel Expression System in <Emphasis Type="Italic">Corynebacterium crenatum</Emphasis>

Corynebacterium crenatum SYPA 5-5 is an aerobic and industrial l-arginine producer. It was proved that the Corynebacterium glutamicum/Escherichia coli shuttle vector pJC1 could be extended in C. crenatum efficiently when using the chloramphenicol acetyltransferase gene (cat) as a reporter under the control of promoter tac. The expression system was applied to over-express the gene vgb coding Vitreoscilla hemoglobin (VHb) to further increase the dissolved oxygen in C. crenatum. As a result, the recombinant C. crenatum containing the pJC-tac-vgb plasmid expressed VHb at a level of 3.4 nmol g⁻¹, and the oxygen uptake rates reached 0.25 mg A₅₆₂⁻¹ h⁻¹ which enhanced 38.8% compared to the wild-type strain. Thus, the final l-arginine concentration of the batch fermentation reached a high level of 35.9 g L⁻¹, and the biomass was largely increased to 6.45 g L⁻¹, which were 17.3% and 10.5% higher than those obtained by the wild-type strain, respectively. To our knowledge, this is the first report that the efficient expression system was constructed to introduce vgb gene increasing the oxygen and energy supply for l-arginine production in C. crenatum, which supplies a good strategy for the improvement of amino acid products.     

Liquid chromatography/mass spectrometry characterization of <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Shigella</Emphasis> species

Liquid chromatography/quadrupole time of flight mass spectrometry (LC/QTOF MS) utilizing electrospray ionization was employed to monitor protein expression in Escherichia coli and Shigella organisms. Comparison with MALDI/TOF-MS revealed more proteins, particularly above 15 kDa. A combination of automated charge state deconvolution, spectral mirroring, and spectral subtraction was used to reveal subtle differences in the LC/MS data. Reproducible intact protein biomarker candidates were discovered based on their unique mass, retention time, and relative intensity. These marker candidates were implemented to differentiate closely related strain types, (e.g., two distinct isolates of E. coli O157:H7) and to correctly identify unknown pathogens. This LC/MS approach is less labor-intensive than pulsed-field gel electrophoresis, affords greater specificity than real-time PCR, and requires no primers or antibodies. Additionally, this approach would be beneficial during outbreaks of foodborne disease or bioterrorism investigations by complementing methods typically used in diagnostic microbiology laboratories.     

Overexpression of an Endochitinase Gene (<Emphasis Type="Italic">ThEn-42</Emphasis>) in <Emphasis Type="Italic">Trichoderma atroviride</Emphasis> for Increased Production of Antifungal Enzymes and Enhanced Antagonist Action Against Pathogenic Fungi

Trichoderma is one of the most promising biocontrol agents against plant fungal diseases. In this study, a transgenic strain of Trichoderma atroviride was characterized. The transgenic strain contains an endochitinase gene (ThEn-42) driven by the cellulase promoter cbh1 of T. reesei for overexpression of ThEn-42. The culture filtrates of the transformant and the parental strain grown in eight different media were evaluated for chitinase and antifungal enzyme production based on activity gels, protein profiles, and antifungal activities. Results demonstrated that chitinases are important components and synergistic interactions play a key role in the antagonistic action of T. atroviride. Moreover, altering medium nutrient concentration and composition led to enhanced production of antifungal enzymes, a potential strategy for mass production. Two of the culture filtrates contained almost pure endochitinase, and could be excellent commercial sources for this enzyme. Several culture filtrates were highly antifungal. Two filtrates were so effective in biocontrol of a fungal pathogen, Penicillium digitatum, that they not only inhibited spore germination but destroyed the spores completely when 20 μl of culture filtrate (corresponding to approximately 104 μg of total protein) was applied in a total volume of 150 μl (approximately 0.7 mg protein ml⁻¹).     

Functional Expression of <Emphasis Type="Italic">Bacillus anthracis</Emphasis> Protective Antigen in <Emphasis Type="Italic">E. coli</Emphasis>

The protective antigen (PA) of Bacillus anthracis is a potent immunogen and an important candidate vaccine. In addition, it is used in monitoring systems like enzyme-linked immunosorbent assay to assess antibodies against PA in immunized subjects. The low level of PA production in B. anthracis and the difficulty of separating it from other bacterial components have made the researchers do different studies with the aim of producing recombinant PA (rPA). In this study, to produce rPA as a recombinant protein vaccine, the partial sequence of protective antigen of B. anthracis, amino acids 175–764, as a potent immunogenic target was inserted in pET21b(+). This is a prokaryotic plasmid that carries an N-terminal T7.tag sequence. The integrity of constructed plasmid was confirmed using restriction enzyme mapping. rPA was expressed after induction with isopropyl β-d-1-thiogalactopyranoside in Escherichia coli BL21. Purification of rPA was done with an affinity system using anti T7.tag antibody. Electrophoresis and Western blotting confirmed the specificity of the expressed protein. BALB/c mice were immunized with obtained PA protein and evaluation of specific immunoglobulin G antibodies against PA in sera using Western blotting method and showed that rPA is immunogenic. The challenge of immunized mice with virulent strain of B. anthracis showed that rPA is functional to protect against pathogenic strain.     

Fibrinolytic Serine Protease Isolation from <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> An6 Grown on <Emphasis Type="Italic">Mirabilis jalapa</Emphasis> Tuber Powders

In this study, Mirabilis jalapa tuber powder (MJTP) was used as a new complex organic substrate for the growth and production of fibrinolytic enzymes by a newly isolated Bacillus amyloliquefaciens An6. Maximum protease activity (1,057 U/ml) with casein as a substrate was obtained when the strain was grown in medium containing (grams per liter) MJTP 30, yeast extract 6, CaCl₂ 1, K₂HPO₄ 0.1, and K₂HPO₄ 0.1. The strain was also found to grow and produce extracellular proteases in a medium containing only MJTP, indicating that it can obtain its carbon, nitrogen, and salts requirements directly from MJTP. The B. amyloliquefaciens An6 fibrinase (BAF1) was partially purified, and fibrinolytic activity was assayed in a test tube with an artificial fibrin clot. The molecular weight of the partially purified BAF1 fibrinolytic protease was estimated to be 30 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration. The optimum temperature and pH for the caseinolytic activity were 60 °C and 9.0, respectively. The enzyme was highly stable from pH 6.0 to 11.0 and retained 62% of its initial activity after 1 h incubation at 50 °C. However, the enzyme was inactivated at higher temperatures. The activity of the enzyme was totally lost in the presence of phenylmethylsulfonyl fluoride, suggesting that BAF1 is a serine protease.     

Antitumor sesquiterpene polyol esters from <Emphasis Type="Italic">Celastrus angulatus</Emphasis>

Two new antitumor sesquiterpene polyol esters with the β-dihydroagarofuran skeleton, 1 and 2, and three known compounds 3–5 were isolated from the high-polar MeOH extracts of the root bark of Celastrus angulatus. Their chemical structures were elucidated mainly by analyses of MS and NMR spectral data. Preliminary antitumor and insecticidal activities of these compounds were evaluated. Compounds 1 and 2 exhibited moderate antitumor activity against human breast cancer cell line (Bcap-37); the IC50 were 54.08 and 61.35 μM, respectively, and all the tested compounds were shown to possess minimal or no inhibitory activities against human colon (HT-29) and lung (NCI-H460) cancer cell lines. In addition, compounds 1–5 displayed mild insecticidal activities against the 4th instar larvae Mythimna separate, the KD50 were 610.5, 308.8, 917.8, 510.6 and 1120.5, respectively.     

Molecular properties of the copolymers of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-diallyl-<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethylammonium chloride and maleic acid

The hydrodynamic and conformational properties of molecules of poly(N,N-diallyl-N,N-dimethylammonium chloride) and N,N-diallyl-N,N-dimethylammonium chloride-maleic acid copolymers of different compositions in solutions with various ionic-strength and pH values, as well as of the polyelectrolyte complex based on the copolymer with dodecyl sulfate anions in chloroform, are studied. For poly(N,N-diallyl-N,N-dimethylammonium chloride) molecules in a 1 M NaCl solution, the Kuhn segment length and the hydrodynamic diameter of the chain are estimated as A = 3.9 nm and d = 0.48 nm, respectively. In acidic solutions with pH 3.5, the copolymers demonstrate behavior typical for polyelectrolytes. In an alkaline solution with pH 13, when 1 M NaCl is added to the solution of the copolymer containing 29 mol % maleic acid units, there is an antipolyelectrolyte effect that manifests itself as an increase in the intrinsic viscosity of the copolymer and in the hydrodynamic radius of its molecules. It is found that an increase in the fraction of maleic acid units in the copolymer from 12 to 42 mol % brings about a reduction in the equilibrium rigidity of its macromolecules from 4.1 to 2.2 nm. The equilibrium rigidity of polyelectrolyte-complex molecules is higher than that of initial copolymer molecules owing to steric interactions arising between the aliphatic chains of dodecyl sulfate anions. In an electric field, the molecules of the complex are oriented owing to the induced dipole moment resulting from the displacement of dodecyl sulfate anions along the chain contour.     

Isolation and Characterization of a Novel Plasma Membrane Intrinsic Protein Gene, <Emphasis Type="Italic">LcPIP1</Emphasis>, in <Emphasis Type="Italic">Leymus chinensis</Emphasis> that Enhances Salt Stress Tolerance in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A novel plasma membrane intrinsic, LcPIP1, was isolated from Leymus chinensis using RACE method. The LcPIP1 has 288 amino acids with an estimated molecular mass of 30.6 kDa. Semi RT-PCR analysis indicated that the expression level of LcPIP1 was obviously higher in leaf than root. The LcPIP1 was also found to be induced by salt stress. In addition, transformed with the LcPIP1, Saccharomyces cerevisiae could increase tolerance to salt stress. These results indicate that the LcPIP1 gene seems to play a role in resistance against salt stress.     

<Emphasis Type="Italic">Z</Emphasis>-and <Emphasis Type="Italic">E</Emphasis>-conformers of <Emphasis Type="Italic">N</Emphasis>-chloroacetylcytisine

N-Chloroacetylcytisine was synthesized by acylation of (–)-cytisine. Stable Z- and E-conformers with respect to rotational isomerism around the N-12–CO bond were found in PMR spectra at room temperature. The point at which PMR resonances of the Z- and E-conformers coalesced upon heating was measured. The transition barrier between the conformers was estimated.     

Fucoidans from Brown Seaweeds <Emphasis Type="Italic">Sargassum hornery</Emphasis>, <Emphasis Type="Italic">Eclonia cava</Emphasis>, <Emphasis Type="Italic">Costaria costata</Emphasis>: Structural Characteristics and Anticancer Activity

Fucoidans were isolated by water extraction and ion-exchange chromatography from brown algae Eclonia cava, Sargassum hornery, and Costaria costata collected near of Korean coasts. The structures of fucoidans were investigated. Fucoidan from E. cava was mixture of sulfated rhamnogalactofucan and galactofucan. Fucoidan from C. costata was a sulfated galactofucan. Fucoidan isolated from S. hornery was separated into three fractions: a homofucan sulfate, a homofucan but without sulfate groups, and a sulfated rhamnofucan. The results clearly showed that fucoidans play an inhibitory role in colony formation in human melanoma and colon cancer cells and may be effective antitumor agents.     

A new 3,4-dihydroisocoumarin isolated from <Emphasis Type="Italic">Botryosphaeria</Emphasis> sp<Emphasis Type="Italic">.</Emphasis> F00741

A new 3,4-dihydroisocoumarin, named botryoisocoumarin A (1), was obtained from the fermentation culture of Botryosphaeria sp. F00741, together with two known ones, 3,8-dihydroxy-3-methylisochroman-1-one (2) and 4,8-dihydroxy-3-methylisochroman-1-one (3). Their structures were elucidated by spectroscopic analyses, including 1D and 2D NMR experiments and ESI mass spectrometry, and by comparison with those reported. Compound 1 was evaluated for its antitumor and antimicrobial activities in vitro.     

Cloning of Thermostable Cellulase Genes of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> and Their Secretive Expression in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Screening for the powerful cellulase genes with improved activities remains a challenge for the biorefinery research. In this study, five cellobiohydrolase genes and one endoglucanase gene sourced from Clostridium thermocellum DSM 1237, cbhA, celK, celO, cel48Y, cel48S, and celA were cloned into a newly established tool vector pP43JM2 and expressed in two Bacillus subtilis strains, B. subtilis WB600 and B. subtilis WB800, respectively. Most of the cellulases produced in the B. subtilis recombinants were efficiently secreted into the culture medium. These secreted soluble proteins showed distinct cellulase activities using phosphoric acid swollen cellulose (PASC) as the substrate and they also demonstrated strong synergistic effects for PASC, Avicel cellulose, and the dilute acid pretreated corn stover. The current work provided a quick secretive cloning method for screening cellulase genes and may provide a host strain for constructing a consolidated bioprocessing platform with the capacity of secretive expression of multiple cellulases.     

Expression of a <Emphasis Type="Italic">hemA</Emphasis> Gene from <Emphasis Type="Italic">Agrobacterium radiobacter</Emphasis> in a Rare Codon Optimizing <Emphasis Type="Italic">Escherichia coli</Emphasis> for Improving 5-aminolevulinate Production

The 5-aminolevulinate (ALA) synthase gene (hemA) from Agrobacterium radiobacter zju-0121, which was cloned previously in our laboratory, contains several rare codons. To enhance the expression of this gene, Escherichia coli Rosetta(DE3), which is a rare codon optimizer strain, was picked out as the host to construct an efficient recombinant strain. Cell extracts of the recombinant E. coli were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under the appropriate conditions. The results indicated that the activity of ALA synthase expressed in Rosetta(DE3)/pET-28a(+)-hemA was about 20% higher than that in E. coli BL21(DE3). Then the effects of precursors (glycine and succinate) and glucose, which is an inhibitor for ALA dehydratase as well as the carbon sources for cell growth, on the production of 5-aminolevulinate were investigated. Based on an optimal fed-batch culture system described in our previous work, up to 6.5 g/l (50 mM) ALA was produced in a 15-l fermenter.     

Comparative characteristics of bud meristem lipids from <Emphasis Type="Italic">Picea obovata</Emphasis> and <Emphasis Type="Italic">Pinus sylvestris</Emphasis>

It was shown that the lipid content of Pinus sylvestris L. was greater than that of Picea obovata L. The difference was 43–45% of total lipids in winter. Polar fractions dominated the lipid composition of both conifers. The phospholipid content was greater than the glycolipid content in winter. A significant amount of glycolipids that was equal to that of phospholipids was present simultaneously in meristem of wintering pine buds. Swelling buds of both species showed in spring an elevated content of glycolipids.