Establishment and characterization of SV40 large T antigen-immortalized cell lines derived from fetal bovine brain tissues after prolonged cryopreservation

Bovine brain cell lines with specific characteristics are useful in vitro experimental systems for molecular and cellular investigation of the interactions between bovine specific neuropathogenic agents and the host. Here, we established two novel cell lines from cultures of cryopreserved fetal bovine brain tissue by the transfection of SV40 large T antigen. Both cell lines showed cobblestone morphology in DMEM/F12 medium supplemented with 10% fetal bovine serum, epidermal growth factor and basic fibroblast growth factor. They were immunostained with endothelial marker, Von Willebrand Factor. Endothelial properties, such as capillary-like tube formation on matrigel and the incorporation of DiI-AcLDL were confirmed with these cells. Removal of growth factors increased the number of cells expressing alpha-smooth muscle actin, suggesting the potential of these cell lines to differentiate into smooth muscle cells. This study suggests an efficient protocol to immortalize brain endothelial cell lines from fetal bovine brain tissue culture.     

Structural characteristics of oversulfated chondroitin/dermatan sulfates in the fibrous lesions of the liver with cirrhosis.

The structural characteristics of oversulfated chondroitin/dermatan sulfates (C/DSs) in the fibrous lesions of the rat liver with cirrhosis were examined. Long-Evans Cinnamon rats were subjected to the present study as the model animals with cirrhosis. The serial polyester wax sections of liver with cirrhosis were processed into the fibrous lesions and the nonfibrous lesions. The oversulfated C/DSs in the tissue sections on a glass slide were degraded to unsaturated disaccharides by chondroitinase ABC and ACII digestion in the presence of bacterial collagenase. Subsequently, the resulting unsaturated disaccharides were determined by the reversed-phase ion-pair high-performance liquid chromatography with fluorometric postcolumn derivatization using 2-cyanoacetamide as a reagent. Through these in situ investigations, we found some facts as follows: (i) in the fibrous lesion, the remarkable increase of the oversulfated C/DSs content and the decrease of the oversulfation degree of the C/DSs were observed compared with those in the nonfibrous lesion, (ii) the proportion of the iduronic acid content in the C/DSs in the fibrous lesion was significantly low compared with that in the nonfibrous lesion, and (iii) in the nonfibrous lesion close to the fibrous lesion, both quantitative and qualitative alterations of C/DSs were not observed at all. These findings indicate that the oversulfated C/DSs with low iduronic acid content are possible marker for the fibrogenesis of liver with cirrhosis.     

Thermal stabilization of Bacillus subtilis family-11 xylanase by directed evolution

We used directed evolution to enhance the thermostability of glycosyl hydrolase family-11 xylanase from Bacillus subtilis. By combining random point mutagenesis, saturation mutagenesis, and DNA shuffling, a thermostable variant, Xyl(st), was identified which contained three amino acid substitutions: Q7H, N8F, and S179C. The half-inactivation temperature (the midpoint of the melting curves) for the Xyl(st) variant compared with the wild-type enzyme after incubation for 10 min was elevated from 58 to 68 degrees C. At 60 degrees C the wild-type enzyme was inactivated within 5 min, but Xyl(st) retained full activity for at least 2 h. The stabilization was accompanied by evidence of thermophilicity; that is, an increase in the optimal reaction temperature from 55 to 65 degrees C and lower activity at low temperatures and higher activity at higher temperatures relative to wild type. To elucidate the mechanism of thermal stabilization, three-dimensional structures were determined for the wild-type and Xyl(st) enzymes. A cavity was identified around Gln-7/Asn-8 in wild type that was filled with bulky, hydrophobic residues in Xyl(st). This site was not identified by previous approaches, but directed evolution identified the region as a weak point. Formation of an intermolecular disulfide bridge via Cys-179 was observed between monomers in Xyl(st). However, the stability was essentially the same in the presence and absence of a reducing agent, indicating that the increased hydrophobicity around the Cys-179 accounted for the stability.     

An ATP-inhibited endonuclease from cauliflower (Brassica oleracea var. botrytis) inflorescence: purification and characterization

In our studies on the role of enzymes in plant DNA replication, recombination, and repair, we isolated from cauliflower (Brassica oleracea L. var. botrytis) inflorescences a single-stranded DNA-specific endonuclease that was inhibited by ATP. The endonuclease, designated cauliflower nuclease II, was purified to near homogeneity through six successive column chromatographies. The enzyme is a single polypeptide with a molecular mass of 70 kDa as judged by the results of sodium dodecyl sulfate-polyacry amide gel electrophoresis, activity gel, and gel-filtration column chromatography. The enzyme can cleave a linear or a circular single-stranded DNA but cannot cut or nick a double-stranded DNA. The mode of activity of the nuclease is endonucleolytic and non-processive. Interestingly, the endonuclease activity is strongly inhibited by less than 0.1 mM ATP, although the role of this inhibition is thus far unclear. While ATPγS and GTP can also inhibit the activity, other ribonucleoside triphosphates are much less effective. The optimum pH of the enzyme is 5.6. The enzyme requires an exceptionally high ionic strength, 0.2 M KCI for optimum activity, and without these ions no activity can be detected. The endonuclease activity is stimulated by Ca²⁺, which cannot be replaced by Mg²⁺ or Mn²⁺. The features of the enzyme and its relation to plant DNA metabolism are discussed. Received: 26 March 1998 / Accepted: 4 June 1998     

In vivo evidence of enhanced di-methylation of histone H3 K4 on upregulated genes in adipose tissue of diabetic db/db mice

Di-methylation of histone H3 lysine (K) 4, a component of the epigenetic memory, is associated with gene transactivation. In this study, we examined whether the development of diabetes induces di-methylation of histone H3 K4 on the upregulated genes. We searched for upregulated genes in mesenteric adipose tissue of insulin-resistant/diabetic db/db mice compared with non-diabetic db/m mice using microarray analysis. We also performed chromatin immunoprecipitation assays for di-methylation of histone H3 K4 in the upregulated genes in mesenteric adipose tissue of db/m and db/db mice. Di-methylation of histone H3 K4 was enhanced at the upstream and/or transcribed regions of upregulated genes including Atp6v0d2, Mmp12, Trem2 and Clec4d genes in mesenteric adipose tissue of db/db mice, as compared with db/m mice. These results suggest that di-methylation of histone H3 K4 is involved in the induction of Atp6v0d2, Mmp12, Trem2 and Clec4d in mesenteric adipose tissue in db/db mice.     

Characterization of three homoeologous cDNAs encoding chloroplast-targeted aminolevulinic acid dehydratase in common wheat

In the tetrapyrrole biosynthetic pathway of higher plants, 5-aminolevulinic acid (ALA) is metabolized by ALA dehydratase (ALAD). Here, we isolated ALAD1 cDNA from common wheat (Triticum aestivum L.) and its diploid progenitors, and produced transgenic tobacco plants expressing the wheat ALAD1 gene. The ALAD1 genes were highly conserved among wheat relatives, and three homoeologous loci of wheat ALAD1 (TaALAD1) were equally transcribed in common wheat. A transient expression assay of a TaALAD1-GFP (green fluorescent protein) fusion protein suggested that TaALAD1 is localized in chloroplasts. Overexpression of TaALAD1 in transgenic tobacco resulted in a significant increase in ALAD activity in leaves. Moreover, the transgenic tobacco showed vigorous growth and increased survival rate on medium containing ALA at herbicidal concentrations. These results indicate that wheat ALAD1 has catalytic activity in metabolizing ALA in plastids, and that ectopic expression of TaALAD1 in transgenic plants increases their tolerance to ALA application at high concentrations.     

Prion Replication Elicits Cytopathic Changes in Differentiated Neurosphere Cultures

The molecular mechanisms of prion-induced cytotoxicity remain largely obscure. Currently, only a few cell culture models have exhibited the cytopathic changes associated with prion infection. In this study, we introduced a cell culture model based on differentiated neurosphere cultures isolated from the brains of neonatal prion protein (PrP)-null mice and transgenic mice expressing murine PrP (dNP0 and dNP20 cultures). Upon exposure to mouse Chandler prions, dNP20 cultures supported the de novo formation of abnormal PrP and the resulting infectivity, as assessed by bioassays. Furthermore, this culture was susceptible to various prion strains, including mouse-adapted scrapie, bovine spongiform encephalopathy, and Gerstmann-Sträussler-Scheinker syndrome prions. Importantly, a subset of the cells in the infected culture that was mainly composed of astrocyte lineage cells consistently displayed late-occurring, progressive signs of cytotoxicity as evidenced by morphological alterations, decreased cell viability, and increased lactate dehydrogenase release. These signs of cytotoxicity were not observed in infected dNP0 cultures, suggesting the requirement of endogenous PrP expression for prion-induced cytotoxicity. Degenerated cells positive for glial fibrillary acidic protein accumulated abnormal PrP and exhibited features of apoptotic death as assessed by active caspase-3 and terminal deoxynucleotidyltransferase nick-end staining. Furthermore, caspase inhibition provided partial protection from prion-mediated cell death. These results suggest that differentiated neurosphere cultures can provide an in vitro bioassay for mouse prions and permit the study of the molecular basis for prion-induced cytotoxicity at the cellular level.     

Studies on Oxygen Transfer in Submerged Fermentations

This paper refers to the application of gas analyzers for the determination of oxygen transfer rate, showing examples in the studies and the performances of submerged fermentations. Oxygen and carbon dioxide analyzers were set to monitor the gas streams to and from the fermentor. Continuous data on the concentrations of oxygen and carbon dioxide in the air streams were thus provided throughout the fermentation. Distinctive characters of this method were applicability to fermentors in practice and ability of obtaining data directly relating to the fermentations.

The modification of sulfite oxidation method for the determination of oxygen transfer rate from air into liquid or of a measure of aeration effectiveness was made. The proposed method was the application of gas analyzers in the studies on submerged fermentation. Some comparative discussions were made between this and the conventional titrimetric method. This modified method could be applied to biological systems with no alteration, therefore, it was made possible to compare the sulfite solution with the biological systems in relation to the problems on oxygen transfer.