Perinuclear localization of cytosolic phospholipase A(2)alpha is important but not obligatory for coupling with cyclooxygenases

In response to Ca(2+) signaling, cytosolic phospholipase A(2)alpha (cPLA(2)alpha) translocates from the cytosol to the perinuclear membrane, where downstream eicosanoid-synthetic enzymes, such as cyclooxygenase (COX), are localized. Although the spatiotemporal perinuclear colocalization of cPLA(2)alpha and COXs has been proposed to be critical for their functional coupling leading to prostanoid production, definitive evidence for this paradigm has remained elusive. To circumstantiate this issue, we took advantage of a chimeric cPLA(2)alpha mutant harboring the C2 domain of protein kinase Calpha, which translocates to the plasma membrane following cell activation. Transfection analyses of the native or chimeric cPLA(2)alpha in combination with COX-1 or COX-2 revealed that, even though the arachidonate-releasing capacities of native and mutant cPLA(2)alpha were comparable, prostaglandin production by mutant cPLA(2)alpha was markedly impaired as compared with that by native cPLA(2)alpha. We thus conclude that the perinuclear localization of cPLA(2)alpha is preferential, even if not obligatory, for efficient coupling with COXs.     

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.     

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.     

Single nucleotide polymorphisms of thrifty genes for energy metabolism: evolutionary origins and prospects for intervention to prevent obesity-related diseases

The "thrifty" genotype and phenotype that save energy are detrimental to the health of people living in affluent societies. Individual differences in energy metabolism are caused primarily by single nucleotide polymorphisms (SNPs), some of which promote the development of obesity/type 2 diabetes mellitus. In this review, four major questions are addressed: (1) Why did regional differences in energy metabolism develop during evolution? (2) How do genes respond to starvation and affluence? (3) Which SNPs correspond to the hypothetical "thrifty genes"? (4) How can we cope with disease susceptibility caused by the "thrifty" SNPs? We examined mtDNA and genes for energy metabolism in people who live in several parts of Asia and the Pacific islands. We included 14 genes, and the SNP frequencies of PPAR gamma 2, LEPR, and UCP3-p and some other genes differ significantly between Mongoloids and Caucasoids. These differences in SNPs may have been caused by natural selection depending on the types of agriculture practiced in different regions. Interventions to counteract the adverse effects of "thrifty" SNPs have been partially effective.