Glutamine Synthetase of the Human Brain: Purification and Characterization

Glutamine synthetase (GS) isolated from human brain formed a single band on sodium dodecyl sulfate-polyacrylamide gel with a molecular weight of 44,000. The enzyme had a specific activity of 179.2 U/mg protein when assayed by measuring the rate of the formation of gamma-glutamylhydroxamate using hydroxylamine as a substrate. In the presence of manganese ions, the relative activity of human brain GS was much lower than that of the sheep brain enzyme. The suppression of activity by increasing the ADP concentration, however, was less marked in the human enzyme than that in the sheep enzyme. Antibodies were raised in rabbits against the purified enzyme. The double-immunodiffusion technique disclosed cross-reactivities among GSs isolated from human, sheep, and rat brains, but the enzymes were not immunologically identical. Immunohistochemically, GS was localized in the cytoplasm of astrocytes in the human and rat brains and in pericentral hepatocytes of the liver.     

Effect of soybean oil on the enhanced expression of hirudin gene in Hansenula polymorpha

Summary A heterologous gene from bloodsucking leech for anticoagulant, hirudin, has been expressed in the methylotrophic yeast Hansenula polymorpha. The addition of 1%(v/v) soybean oil to the medium as a stabilizer enhanced the expression of the hirudin gene in H. polymorpha with MOX promoter. The production of hirudin in the medium with soybean oil was 320mg/L in a 5L fermenter.     

Tegumental ultrastructure of juvenile and adult Echinostoma cinetorchis]

The tegumental ultrastructure of juvenile and adult Echinostoma cinetorchis (Trematoda: Echinostomatidae) was observed by scanning electron microscopy. Three-day (juvenile) and 16-day (adult) worms were harvested from rats (Sprague-Dawley) experimentally fed the metacercariae from the laboratory-infected fresh water snail, Hippeutis cantori. The worms were fixed with 2.5% glutaraldehyde, processed routinely, and observed by an ISI Korea DS-130 scanning electron microscope. The 3-day old juvenile worms were elongated and ventrally curved, with their ventral sucker near the anterior two-fifths of the body. The head crown was bearing 37-38 collar spines arranged in a zigzag pattern. The lips of the oral and ventral suckers had 8 and 5 type II sensory papillae respectively, and between the spines, a few type III papillae were observed. Tongue or spade-shape spines were distributed anteriorly to the ventral sucker, whereas peg-like spines were distributed posteriorly and became sparse toward the posterior body. The spines of the dorsal surface were similar to those of the ventral surface. The 16-day old adults were leaf-like, and their oral and ventral suckers were located very closely. Aspinous head crown, oral and ventral suckers had type II and type III sensory papillae, and numerous type I papillae were distributed on the tegument anterior to the ventral sucker. Scale-like spines, with broad base and round tip, were distributed densely on the tegument anterior to the ventral sucker but they became sparse posteriorly. At the dorsal surface, spines were observed at times only at the anterior body. The results showed that the tegument of E. cinetorchis is similar to that of other echinostomes, but differs in the number and arrangement of collar spines, shape and distribution of tegumenal spines, and type and distribution of sensory papillae.     

Design and application of highly responsive fluorescence resonance energy transfer biosensors for detection of sugar in living Saccharomyces cerevisiae cells

A protein sensor with a highly responsive fluorescence resonance energy transfer (FRET) signal for sensing sugars in living Saccharomyces cerevisiae cells was developed by combinatorial engineering of the domain linker and the binding protein moiety. Although FRET sensors based on microbial binding proteins have previously been created for visualizing various sugars in vivo, such sensors are limited due to a weak signal intensity and a narrow dynamic range. In the present study, the length and composition of the linker moiety of a FRET-based sensor consisting of CFP-linker(1)-maltose-binding protein-linker(2)-YFP were redesigned, which resulted in a 10-fold-higher signal intensity. Molecular modeling of the composite linker moieties, including the connecting peptide and terminal regions of the flanking proteins, suggested that an ordered helical structure was preferable for tighter coupling of the conformational change of the binding proteins to the FRET response. When the binding site residue Trp62 of the maltose-binding protein was diversified by saturation mutagenesis, the Leu mutant exhibited an increased binding constant (82 microM) accompanied by further improvement in the signal intensity. Finally, the maltose sensor with optimized linkers was redesigned to create a sugar sensor with a new specificity and a wide dynamic range. When the optimized maltose sensors were employed as in vivo sensors, highly responsive FRET images were generated from real-time analysis of maltose uptake of Saccharomyces cerevisiae (baker's yeast).     

The role of YKL-40 in the pathogenesis of autoimmune diseases: a comprehensive review

YKL-40, a chitinase-3-like protein 1 (CHI3L1) or human cartilage glycoprotein 39 (HC gp-39), is expressed and secreted by various cell-types including macrophages, chondrocytes, fibroblast-like synovial cells and vascular smooth muscle cells. Its biological function is not well elucidated, but it is speculated to have some connection with inflammatory reactions and autoimmune diseases. Although having important biological roles in autoimmunity, there were only attempts to elucidate relationships of YKL-40 with a single or couple of diseases in the literature. Therefore, in order to analyze the relationship between YKL-40 and the overall diseases, we reviewed 51 articles that discussed the association of YKL-40 with rheumatoid arthritis, psoriasis, systemic lupus erythematosus, Behçet disease and inflammatory bowel disease. Several studies showed that YKL-40 could be assumed as a marker for disease diagnosis, prognosis, disease activity and severity. It is also shown to be involved in response to disease treatment. However, other studies showed controversial results particularly in the case of Behçet disease activity. Therefore, further studies are needed to elucidate the exact role of YKL-40 in autoimmunity and to investigate its potential in therapeutics.     

Changes in Cellular Fatty Acid Composition of Cephalosporium acremonium during Cephalosporin C Production

Cephalosporium acremonium was cultivated in fermentation medium containing sucrose or methyl oleate as a carbon source for cephalosporin C production. The level of antibiotic production was 48 g of cephalosporin C per liter under optimum conditions when methyl oleate was used. The C_18:1 (oleic acid) methyl ester appeared to be utilized faster than the C_18:2 (linoleic acid) methyl ester in fermentation broth. Physiological characteristics of C. acremonium were investigated by determining the fatty acid composition of the total cellular free lipid. Significant changes in cellular fatty acid composition occurred during inoculum cultivation and fermentation. The percentage of C_18:1 increased from 19.1 to 38.5%, but the percentage of C_18:2 decreased from 56.7 to 36.1%, and there was an increase in pH during inoculum cultivation. The cellular fatty acid composition of C. acremonium grown in fermentation medium containing methyl oleate (methyl oleate medium) was significantly different from that in fermentation medium containing sucrose (sucrose medium). The major fatty acids detected were C_16:0 (palmitic acid), C_18:1, and C_18:2. In methyl oleate medium, the ratio of C_18:1 to C_18:2 increased from 0.34 to 1.37, while the cell morphology changed from hyphae to arthrospores and conidia. In contrast, in sucrose medium, the ratio of C_18:1 to C_18:2 decreased from 0.70 to 0.43, and most of the cells remained hyphal at the end of fermentation. We observed that hyphae contained a higher proportion of C_18:2 but arthrospores and conidia contained a higher proportion of C_18:1.     

Plasmodium vivax: Comparison of the immune responses between oral and parenteral immunization of rPv54 in BALB/c mice

The merozoite surface protein-1 (MSP-1) from Plasmodium vivax was evaluated as an oral vaccine candidate by cloning and expressing the interspecies conserved block 10 (ICB10) of the MSP-1 from a Korean isolate in Escherichia coli. The expressed fusion protein contained ICB10 and a maltose-binding protein (MBP), rPv54, has a molecular weight of approximately 54 kDa as determined by SDS-PAGE analysis. IgG against rPv54 was successfully produced in BALB/c mice by oral immunization and sustained for more than 4 months. IgG2b was dominantly produced in both oral and parenteral immunizations. The rPv54 increased the frequency of NK, NKT, CD4+ T, CD8+ T, and B cells in both immunizations. IL-5 and TNF-α were increased in both significantly. In conclusion, rPv54 might be a valuable potential vaccine candidate for the oral and parenteral immunization against vivax malaria.     

Plasma and ovarian thyroxine levels in relation to sexual maturation and gestation in female Sebastes inermis

Plasma T₄ (L-thyroxine) concentrations in the viviparous rockfish Sebastes inermis showed a peak (119·3±27·8 ng ml⁻¹) during the development of embryos. Ovarian T₄ concentrations increased during vitellogenesis and final maturation and decreased during embryogenesis.However, total T₄ content within the ovary increased continuously up to the larval stages. Plasma oestradiol-17β, (E₂) concentrations peaked at the final maturation stage and then recovered to the level seen during the non-reproductive periods. Plasma testosterone (T) concentrations showed a peak (5·33±1·08 ng ml⁻¹) at the embryo stage and high levels were maintained throughout the gestation period. Plasma T₄ concentrations during the embryo stage and ovarian T₄ content during vitellogenesis were much higher than the levels reported in oviparous fishes. These data suggest that in viviparous rockfish, T₄ may be required for sustaining gestation or embryo development within the maternal body. In addition, the high content of total T₄ in the ovary implies that there probably does exist a maternal-embryo relationship during gestation as well as during vitellogenesis.