Ginkgolide B production in cultured cells derived from Ginkgo biloba L. leaves

Callus cultures and cell suspension cultures derived from Ginkgo biloba L. leaves produced ginkgolidc B. In cell suspension cultures, the production reached a maximum by the 13th day of subculture and followed by a sharp decrease. The medium of Murashige and Skoog induced the highest ginkgolide B content in cultures while the medium of Schenk and Hildebrandt promoted cell growth. For the maximal production of ginkgolide B, cells were cultured in Murashige and Skoog medium modified to contain 1.0 mg/l of -naphthaleneacetic acid, 0.1 mg/1 of kinetin, 30 g/1 sucrose and 1.25 mM potassium phosphate with a molar ratio of ammonium to nitrate ions of 1 3.Abbreviations B5 Gamborg et al (1968) medium - GKB Ginkgolide B - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic aicd - SH Schenk and Hildebrandt (1972) medium     

SNAREs support atlastin-mediated homotypic ER fusion in Saccharomyces cerevisiae

Dynamin-like GTPases of the atlastin family are thought to mediate homotypic endoplasmic reticulum (ER) membrane fusion; however, the underlying mechanism remains largely unclear. Here, we developed a simple and quantitative in vitro assay using isolated yeast microsomes for measuring yeast atlastin Sey1p-dependent ER fusion. Using this assay, we found that the ER SNAREs Sec22p and Sec20p were required for Sey1p-mediated ER fusion. Consistently, ER fusion was significantly reduced by inhibition of Sec18p and Sec17p, which regulate SNARE-mediated membrane fusion. The involvement of SNAREs in Sey1p-dependent ER fusion was further supported by the physical interaction of Sey1p with Sec22p and Ufe1p, another ER SNARE. Furthermore, our estimation of the concentration of Sey1p on isolated microsomes, together with the lack of fusion between Sey1p proteoliposomes even with a 25-fold excess of the physiological concentration of Sey1p, suggests that Sey1p requires additional factors to support ER fusion in vivo. Collectively, our data strongly suggest that SNARE-mediated membrane fusion is involved in atlastin-initiated homotypic ER fusion.     

Enhanced fungal resistance in Arabidopsis expressing wild rice PR-3 (OgChitIVa) encoding chitinase class IV

Oryza grandiglumis Chitinase IVa (OgChitIVa) cDNA encoding a class IV chitinase was cloned from wild rice (Oryza grandiglumis). OgChitIVa cDNA contains an open reading frame of 867 nucleotides encoding 288 amino acid residues with a predicted molecular weight of 30.4 kDa and isoelectric point of 8.48. Deduced amino acid sequences of OgChitIVa include the signal peptide and chitin-binding domain in the N-terminal domain and conserved catalytic domain. OgChitIVa showed significant similarity at the amino acid level with related monocotyledonous rice and maize chitinase, but low similarity with dicotyledoneous chitinase. Southern blot analysis showed that OgChitIVa genes are present as two copies in the wild rice genome. It was shown that RNA expression of OgChitIVa was induced by defense/stress signaling chemicals, such as jasmonic acid, salicylic acid, and ethephon or cantharidin and endothall or wounding, and yeast extract. It was demonstrated that overexpression of OgChitIVa in Arabidopsis resulted in mild resistance against the fungal pathogen, Botrytis cinerea, by lowering disease rate and necrosis size. RT-PCR analysis showed that PR-1 and PR-2 RNA expression was induced in the transgenic lines. Here, we suggest that a novel OgChitIVa gene may play a role in signal transduction process in defense response against B. cinerea in plants. J.-H. Pak and E.-S. Chung contributed equally to this work.     

Nitric oxide synthase and calcium-binding protein-containing neurons in the hamster visual cortex

The distribution and morphology of neurons containing neuronal nitric oxide synthase (NOS), and calcium-binding proteins calbindin D28K and calretinin in the hamster visual cortex were compared by immunocytochemistry. Staining for NOS, calbindin D28K and calretinin was seen both in the specific layers and in the selective cell types. The densest concentration of anti-NOS-immunoreactive (IR) neurons was found in layer VI. Most of the calbindin D28K-IR neurons were located in layers II/III and V while the calretinin-IR neurons were predominantly located in layers II/III. The labeled neurons varied in morphology. The large majority of NOS-IR neurons were round or oval cells with many dendrites coursing in all directions. The majority of the calbindin D28K-IR neurons were stellate and round or oval cells with multipolar dendrites. The majority of the calretinin-IR neurons were vertical fusiform cells with long processes traveling perpendicular to the pial surface. Our study showed that 14.7% and 27.5% of the NOS-IR cells in the hamster visual cortex contained calbindin D28K or calretinin, respectively. These results indicate that NOS, calbindin and calretinin are located in specific layers and specific cell types and the vast majority of NOS-containing neurons are limited to neurons that do not express calbindin D28K or calretinin.     

Characterization of ELP-fused ω-Transaminase and Its Application for the Biosynthesis of β-Amino Acid

Optically pure amines, β-amino acids and γ-amino acids are the valuable precursors to produce biologically active compounds. The ω-TAs are the class of enzymes which are widely used to produce such compounds. In this work (S)-ω-transaminase from the thermophilic eubacterium Sphaerobacter thermophilus (St-TA) was fused with Elastin-like polypeptides (ELPs) through the cloning process and expressed in E. coli cells. The characterization of this fusion complex was performed with respect to thermostability and effect of DMSO. Where in case of St-TA-ELP-V₆₀, major difference in the transition temperature (T_t) was observed, wherein a T_t of 38 and 70°C was observed at the increasing concentration of DMSO from 5 to 25% (v/v). Interestingly, these fusion proteins the activity was preserved even after the aggregation of fusion complex at Tt. The substrate specificity and product inhibition analysis showed that ω-TA-ELPs had comparable results as that of wild type ω-TA. Moreover, the fused ω-TA could be efficiently reused for up to 20 batches of transamination reaction. Furthermore, the applicability of the fusion protein for the production of a sitagliptin precursor (R)-3-amino-4-(2,4,5-triflurophenyl) butanoic acid (3-ATfBA) was evaluated, wherein 3-ATfBA was synthesized with good conversion (65%).     

Characterization of the cupin-type phosphoglucose isomerase from the hyperthermophilic archaeon Thermococcus litoralis

The gene encoding phosphoglucose isomerase was cloned from Thermococcus litoralis, and functionally expressed in Escherichia coli. The purified enzyme, a homodimer of 21.5 kDa subunits, was biochemically characterized. The inhibition constants for four competitive inhibitors were determined. The enzyme contained 1.25 mol Fe and 0.24 mol Zn per dimer. The activity was enhanced by the addition of Fe(2+), but inhibited by Zn(2+) and EDTA. Enzymes with mutations in conserved histidine and glutamate residues in their cupin motifs contained no metals, and showed large decreases in k(cat). The circular dichroism spectra of the mutant enzymes and the wild type enzyme were essentially the same but with slight differences.     

Voluntary exercise improves insulin sensitivity and adipose tissue inflammation in diet-induced obese mice

Exercise promotes weight loss and improves insulin sensitivity. However, the molecular mechanisms mediating its beneficial effects are not fully understood. Obesity correlates with increased production of inflammatory cytokines, which in turn, contributes to systemic insulin resistance. To test the hypothesis that exercise mitigates this inflammatory response, thereby improving insulin sensitivity, we developed a model of voluntary exercise in mice made obese by feeding of a high fat/high sucrose diet (HFD). Over four wk, mice fed chow gained 2.3 +/- 0.3 g, while HFD mice gained 6.8 +/- 0.5 g. After 4 wk, mice were subdivided into four groups: chow-no exercise, chow-exercise, HFD-no exercise, HFD-exercise and monitored for an additional 6 wk. Chow-no exercise and HFD-no exercise mice gained an additional 1.2 +/- 0.3 g and 3.3 +/- 0.5 g respectively. Exercising mice had higher food consumption, but did not gain additional weight. As expected, GTT and ITT showed impaired glucose tolerance and insulin resistance in HFD-no exercise mice. However, glucose tolerance improved significantly and insulin sensitivity was completely normalized in HFD-exercise animals. Furthermore, expression of TNF-alpha, MCP-1, PAI-1 and IKKbeta was increased in adipose tissue from HFD mice compared with chow mice, whereas exercise reversed the increased expression of these inflammatory cytokines. In contrast, expression of these cytokines in liver was unchanged among the four groups. These results suggest that exercise partially reduces adiposity, reverses insulin resistance and decreases adipose tissue inflammation in diet-induced obese mice, despite continued consumption of HFD.     

Extracellular production of a glycolipid biosurfactant, mannosylerythritol lipid, from Candida antarctica

Candida antarctica (sp. SY16) required avegetable oil as the carbon source to produce a biosurfactant, mannosylerythritol lipid (MEL-SY16). Biosurfactant production was 31 g l^–1 after 7 days in a batch culture and was not growth associated. In a two-stage culture, glycerol and oleic acid were used as an initial and a feeding carbon source, respectively, and 41 g l^–1 biosurfactant was produced after 8 days.