Chemo-enzymatic synthesis of 3-(2-naphthyl)- L-alanine by an aminotransferase from the extreme thermophile, Thermococcus profundus

Hyper-thermostable aminotransferase from Thermococcus profundus (MsAT) was used to synthesize 3-(2-naphthyl)-l-alanine (Nal) by transamination between its corresponding -keto acid, 3-(2-naphthyl)pyruvate (NPA) and l-glutamate (Glu) at 70 °C. Equilibrium of this reaction was shifted toward Nal production due to its low solubility, giving rise to Nal precipitate. Optically pure Nal (>99% ee) was synthesized with 93% (mol mol^–1) yield from 180 mM NPA and 360 mM Glu.     

Analysis of codon usage diversity of bacterial genes with a self-organizing map (SOM): characterization of horizontally transferred genes with emphasis on the E. coli O157 genome

With increases in the amounts of available DNA sequence data, it has become increasingly important to develop tools for comprehensive systematic analysis and comparison of species-specific characteristics of protein-coding sequences for a wide variety of genomes. In the present study, we used a novel neural-network algorithm, a self-organizing map (SOM), to efficiently and comprehensively analyze codon usage in approximately 60,000 genes from 29 bacterial species simultaneously. This SOM makes it possible to cluster and visualize genes of individual species separately at a much higher resolution than can be obtained with principal component analysis. The organization of the SOM can be explained by the genome G+C% and tRNA compositions of the individual species. We used SOM to examine codon usage heterogeneity in the E. coli O157 genome, which contains 'O157-unique segments' (O-islands), and showed that SOM is a powerful tool for characterization of horizontally transferred genes.     

Cytokine expression in rat molar gingival periodontal tissues after topical application of lipopolysaccharide

It is well known that proinflammatory cytokines produced by host cells play an important role in periodontal tissue destruction. However, the localization of the cytokines in in vivo periodontal tissues during development of periodontal disease has not been determined. Immunohistochemical expression of proinflammatory cytokines including IL-1!, IL-1#, and TNF-! was examined at 1 and 3 h, and 1, 2, 3, and 7 days after topical application of lipopolysaccharide (LPS; 5 mg/ml in physiological saline) from E. coli into the rat molar gingival sulcus. In the normal periodontal tissues, a small number of cytokine-positive epithelial cells were seen in the junctional epithelium (JE), oral sulcular and oral gingival epithelium, in addition to macrophages infiltrating in the subjunctional epithelial area and osteoblasts lining the alveolar bone surface. Epithelial remnants of Malassez existing throughout periodontal ligament were intensely positive for IL-1# but negative for the other two cytokines. At 3 h after the LPS treatment, almost all cells in the JE were strongly positive for the cytokines examined. In addition, several cytokine-positive cells, including neutrophils, macrophages, and fibroblasts, were seen in the subjunctional epithelial connective tissue. At day 2, expression of the cytokines in the JE gradually decreased, while cytokine-positive cells in the connective tissue increased in number. Positive staining of the cytokines was seen in osteoclasts and preosteoclasts which appeared along the alveolar bone margin in this period. The number of cytokine-positive cells decreased by day 7. These findings indicate that, in addition to macrophages, neutrophils, and fibroblasts, the JE cells are a potent source of TNF-!, IL-1!, and IL-1# reacting to LPS application, and suggest that JE cells may play an important role in the first line of defense against LPS challenge, and the proinflammatory cytokines transiently produced by various host cells may be involved in the initiation of inflammation and subsequent periodontal tissue destruction.     

Carbohydrate carriers affect adhesion of H. pylori to immobilized Leb-oligosaccharide

The present study involved comparison of adhesion of Helicobacter pylori KH202 to immobilized Le(b)-oligosaccharide carried on different carriers, i.e. Leb-oligosaccharide conjugated with polyacrylamide, bovine serum albumin, and dipalmitoylphosphatidylethanolamine (Le(b)-PAA, Le(b)-BSA, and Le(b)-DPPE). All of the Le(b)-oligosaccharide-carrying neoglycoconjugates served as ligands for H. pylori. However, H. pylori required 10-fold and 100-fold quantities of Le(b)-antigen to adhere to Le(b)-PAA and to Le(b)-DPPE in comparison to the quantity of Le(b)-antigen needed to adhere to Le(b)-BSA, respectively. H. pylori adhesion to Le(b)-PAA and Le(b)-DPPE was clearly inhibited by Le(b)-oligosaccharide, but adhesion to Le(b)-BSA was hardly inhibited by the oligosaccharide. Therefore, the carbohydrate carrier affects the affinity of H. pylori KH202 toward Le(b)-antigen, although the bacteria recognize Le(b)-antigen regardless of the carbohydrate carrier.     

Molecular cloning and immunohistochemical localization of ubiquitin C-Terminal hydrolase expressed in testis of a teleost,the Nile Tilapia,Oreochromis niloticus

We previously produced four monoclonal antibodies to testicular proteins of a teleost, the Nile tilapia. One of the monoclonal antibodies, TAT(Testicular Antigen of Tilapia)-10, recognizes a Mr=27,000 protein (27 kD protein), which is present in A and early B type spermatogonia, spermatids, and spermatozoa in testis. In order to clarify the function of this protein, molecular cloning was conducted. The cDNA for the 27 kD protein contains a complete open reading frame encoding 220 amino acid residues. The predicted amino acid sequence of the 27 kD protein was homologous to those of the ubiquitin carboxy-terminal hydrolases (UCH) reported in mammals. The measurement of the ubiquitin-releasing activity of the recombinant 27 kD protein revealed that the protein is the active form of UCH. Northern blot analysis showed that the UCH mRNA was expressed in ovary and brain in addition to the testis. Immunohistochemical study showed that, in brain, UCH was localized especially on the olfactory organ including the olfactory bulb and olfactory epithelium in olfactory rosetta, suggesting the involvement of the protein in chemoreceptive function. In the Tilapia ovary, UCH localized especially in pre-vitellogenic oocytes, suggesting that the enzyme activity could be important in oocyte growth. This is the first report for the cDNA cloning and cellular localization of UCH in fish. J. Exp. Zool. 293:368-383, 2002.     

Molecular requirements for CD8-mediated rejection of a MUC1-expressing pancreatic carcinoma: implications for tumor vaccines

Previous studies have indicated that different effector cells are required to eliminate MUC1-expressing tumors derived from different organ sites and that different vaccine strategies may be necessary to generate these two different MUC1-specific immune responses. In this study, we characterized molecular components that are required to produce immune responses that eliminate Panc02.MUC1 tumors in vivo by utilizing mice genetically deficient in molecules related to immunity. A parallel study has been reported for a B16.MUC1 tumor model. We confirmed that a CD8(+) effector cell was required to eliminate MUC1-expressing Panc02 tumors, and demonstrated that T cells expressing TCR-alpha/beta and co-stimulation through CD28 and CD40:CD40L interactions played critical roles during the initiation of the anti-Panc02.MUC1 immune response. TCR-alpha/beta(+) cells were required to eliminate Panc02.MUC1 tumors, while TCR-gamma/delta(+) cells played a suppressive non-MUC1-specific role in anti-Panc02 tumor immunity. Type 1 cytokine interferon-gamma (IFN-gamma), but not interleukin-12 (IL-12), was essential for eliminating MUC1-expressing tumors, while neither IL-4 nor IL-10 (type 2 cytokines) were required for tumor rejection. In vitro studies demonstrated that IFN-gamma upregulated MHC class I, but not MHC class II, on Panc02.MUC1 tumor cells. Surprisingly, both perforin and FasL played unique roles during the effector phase of immunity to Panc02.MUC1, while lymphotoxin-alpha, but not TNFR-1, was required for immunity against Panc02.MUC1 tumors. The findings presented here and in parallel studies of B16.MUC1 immunity clearly demonstrate that different effector cells and cytolytic mechanisms are required to eliminate MUC1-expressing tumors derived from different organ sites, and provide insight into the immune components required to eliminate tumors expressing the same antigen but derived from different tissues.