Anti-monocyte chemoattractant protein-1 gene therapy attenuates pulmonary hypertension in rats

Monocyte/macrophage chemoattractant protein-1 (MCP-1), a potent chemoattractant chemokine and an activator for mononuclear cells, may play a role in the initiation and/or progression of pulmonary hypertension (PH). To determine whether blockade of a systemic MCP-1 signal pathway in vivo may prevent PH, we intramuscularly transduced a naked plasmid encoding a 7-NH(2) terminus-deleted dominant negative inhibitor of the MCP-1 (7ND MCP-1) gene in monocrotaline-induced PH. We also simultaneously gave a duplicate transfection at 2-wk intervals or skeletal muscle-directed in vivo electroporation (EP) to evaluate whether a longer or higher expression might be more effective. The intramuscular reporter gene expression was enhanced 10 times over that by EP than by simple injection, and a significant 7ND MCP-1 protein in plasma was detected only in the EP group. 7ND MCP-1 gene transfer significantly inhibited the progression of MCT-induced PH as evaluated by right ventricular systolic pressure, right ventricular hypertrophy, medial hypertrophy of pulmonary arterioles, and mononuclear cell infiltration into the lung. Differential effects of longer or higher transgene expression were not apparent. Although the in vivo kinetics of 7ND MCP-1 gene therapy should be studied further, these encouraging results suggest that an anti-inflammatory strategy via blockade of the MCP-1 signal pathway may be an alternative approach to treat subjects with PH.     

Selective production of two diastereomers of disaccharide sugar alcohol, mannosylerythritol by Pseudozyma yeasts

Mannosylerythritol (ME) is the hydrophilic backbone of mannosylerythritol lipids as the most promising biosurfactants produced by different Pseudozyma yeasts, and has been receiving attention as a new sugar alcohol. Different Pseudozyma yeasts were examined for the sugar alcohol production using glucose as the sole carbon source. P. hubeiensis KM-59 highly produced a conventional type of ME, i.e., 4-O-β-d-mannopyranosyl-d-erythritol (4-ME). Interestingly, P. tsukubaensis KM-160 produced a diastereomer of 4-ME, i.e., 1-O-β-d-mannopyranosyl-d-erythritol (1-ME). In shake flask culture with 200 g/l of glucose, strain KM-59 produced 4-ME at a yield of 33.2 g/l (2.2 g/l/day of the productivity), while strain KM-160 produced 1-ME at 30.0 g/l (2.0 g/l/day). Moreover, the two strains were found to produce ME from glycerol; the maximum yields of 4-ME and 1-ME from 200 g/l of glycerol were 16.1 g/l (1.1 g/l/day) and 15.8 g/l (1.1 g/l/day), respectively. The production of 1-ME as the new diastereomer was further investigated in fed batch culture using a 5-l jar-fermenter. Compared to the flask culture, strain KM-160 gave three times higher productivity of 1-ME at 38.0 g/l (6.3 g/l/day) from glucose and at 31.1 g/l (3.5 g/l/day) from glycerol, respectively. This is the first report on the selective production of two diastereomers of ME, and should thus facilitate the functional development and application of the disaccharide sugar alcohol in the food and relative industries.     

Biochemical synthesis of novel,self-assembling glycolipids from ricinoleic acid by a recombinant α-glucosidase from <Emphasis Type="Italic">Geobacillus</Emphasis> sp.

Purpose of work  

To explore a novel glycolipid, we performed biochemical reactions using a recombinant α-glucosidase from Geobacillus sp. which shows excellent transglycosylation reaction to hydroxyl groups in a variety of compounds.     

Contribution Ratios of amyA, amyB, amyC Genes to High-Level α-Amylase Expression in Aspergillus oryzae

Aspergillus oryzae strains express α-amylases abundantly, and the genome reference strain RIB40 has three α-amylase genes (amyA, amyB, and amyC). However, there is no information on the contribution ratios of individual α-amylase genes to total expression. In this study, we generated single, double, and triple disruptants of α-amylase genes by employing a strain (ΔligD) with high gene-targeting efficiency and pyrG marker recycling in A. oryzae. All the disruptants showed reduced activities of α-amylases, and the triple disruptant completely lost activity. Comparative analyses of the activities and mRNA amounts of the α-amylases suggest that the contribution of amyA to the α-amylase expression is smaller than those of amyB and amyC. The present study suggests that the ability to express a large amount of α-amylases in A. oryzae is attributed to gene duplication of genes such as amyB and amyC.     

Pathological progression of genetic Creutzfeldt–Jakob disease with a PrP V180I mutation

In comparison to sporadic Creutzfeldt–Jakob disease (sCJD) with MM1-type and MM2- cortical (MM2C)-type, genetic CJD with a prion protein gene V180I mutation (V180I gCJD) is clinically characterized by onset at an older age, slower progress, and the absence of visual disturbances or cerebellar symptoms. In terms of pathological characteristics, gliosis and neuronal loss are generally milder in degree, and characteristic spongiform change can be observed at both the early and advanced stages. However, little is known on the progress of spongiform change over time or its mechanisms. In this study, to elucidate the pathological course of V180I gCJD, statistical analysis of the size and dispersion of the major diameters of vacuoles in six V180I gCJD cases was performed, with five MM1-type sCJD and MM2C-type sCJD cases as controls. As a result, V180I gCJD showed no significant difference in vacuolar diameter regardless of disease duration. In addition, the dispersion of the major diameters of vacuoles in V180I gCJD was larger than that in the MM1-type, which was smaller than that in the MM2C-type. We speculated that the absence of difference in the size of the vacuoles regardless of disease duration suggests that tissue rarefaction does not result from the expansion of vacuole size and increase in number of vacuoles in V180Ig CJD. These features were considered to be significant pathological findings of V180I gCJD.     

Molecular cloning,purification and characterization of two endo-1,4-β-glucanases from Aspergillus oryzae KBN616

Two endo-1,4-β-glucanase genes, designated celA and celB, from a shoyu koji mold Aspergillus oryzae KBN616, were cloned and characterized. The celA gene comprised 877 bp with two introns. The CelA protein consisted of 239 amino acids and was assigned to the cellulase family H. The celB gene comprised 1248 bp with no introns. The CelB protein consisted of 416 amino acids and was assigned to the cellulase family C. Both genes were overexpressed under the promoter of the A. oryzae taka-amylase A gene for purification and enzymatic characterization of CelA and CelB. CelA had a molecular mass of 31 kDa, a pH optimum of 5.0 and temperature optimum of 55 °C, whereas CelB had a molecular mass of 53 kDa, a pH optimum of 4.0 and temperature optimum of 45 °C. Received: 3 July 1996 / Accepted: 15 July 1996     

Localization of choline acetyltransferase-expresing neurons in the larval vissual system of Drosophila melanogaster

Choline acetyltransferease (ChAT) is the enzyme catalyzing the biosynthesis of acetylcholine and is considered to be a phenotypically specific marker for cholinergic neurons. We have examined the distribution of ChAT-expressing neurons in the larval nervous system of Drosophila melanogaster by three different but complementary techniques: in situ hybridization with a cRNA probe to ChAT messenger RNA, immunocytochemistry using a monoclonal anti-ChAT antibody, and X-gal staining of transformed animals carrying a reporter gene composed of 7.4 kb of 5 flanking DNA from the ChAT gene fused to a lacZ reporter gene. All three techniques demonstrated ChAT-expressing neurons in the larval visual system. In embryos, the photoreceptor organ (Bolwig's organ) exhibited strong cRNA hybridization signals. The optic lobe of late third-instar larvae displayed ChAT immunoreactivity in Bolwig's nerve and a neuron close to the insertion site of the optic stalk. This neuron's axon ran in parallel with Bolwig's nerve to the larval optic neuropil. This neuron is likely to be a first-order interneuron of the larval visual system. Expression of the lacZ reporter gene was also detected in Bolwig's organ and the neuron stained by anti-ChAT antibody. Our observations indicate that acetylcholine may be a neurotransmitter in the larval photoreceptor cells as well as in a first-order interneuron in the larval visual system of Drosophila melanogaster.This work was supported by a grant from the National Institute of Neurological Disorders and Stroke.     

Effect of cerulenin on the production of mannosyl-erythritol lipids as biosurfactants by Candida antarctica

Summary The effect of cerulenin, anti-lipogenic antibiotic, on the production and fatty-acid profiles of biosurfactants (mannosyl-erythritol lipids) of Candida antarctica were investigated by changing the chain-length of fatty acid as the carbon source. On longer-chain acid (C₁₄ to C₁₈), the production and fatty-acid profiles of the biosurfactants were not entirely affected by the antibiotic, indicating that the yeast is able to synthesize the biosurfactants from those substrates without the operation of de novo synthesis system of fatty acid. These results thus confirm our previous presumption that a new type of chain-shortening system (partial -oxidation system) mainly contributes to the formation of the extracellular glycolipids as biosurfactants.     

An Action Spectrum for Light-induced Primordium Formation in a Basidiomycete, Favolus arcularius (Fr) Ames

The action spectrum for the initiation of fruiting (primordium formation) in Favolus arcularius was determined on the equal response basis. The detectable effect of light was observed in the region between 350 to 560 nanometers, showing six distinct peaks at 374, 398, 424, 446, 480, and 514 nanometers. The half maximum response is reached with 1.8 × 10⁸ ergs per cm² at the most effective wavelength, 398 nanometers. Since the inhibitors, diphenylamine and quinacrine, had no consistent effect on the primordium formation, it is suggested that the possible photoreceptor pigment(s) may be neither carotenoid nor flavinoid.