The effect of female hormones upon urate transport systems in the mouse kidney

Increased levels of serum urate in postmenopausal women are thought to be caused by a change in renal urate elimination associated with the loss of female hormones. In this study, we investigated the regulation of renal urate transporter expression by female hormones using ovariectomized mice with or without hormone replacement. Estradiol suppressed the protein levels of urate reabsorptive transporters urate transporter 1 and glucose transporter 9 (Urat1 and Glut9), and that of urate efflux transporter ATP-binding cassette sub-family G member 2 (Abcg2). Progesterone suppressed protein levels of sodium-coupled monocarboxylate transporter 1 (Smct1). However, neither estradiol nor progesterone influenced the respective levels of mRNA.     

Dickkopf (Dkk)-3 and β-catenin expressions increased in the transition from normal oral mucosal to oral squamous cell carcinoma

Dickkopf (Dkk)-3, an inhibitor of the Wnt/β-catenin pathway, is reported as a potential tumor suppressor gene in many cancers. To gain a better comprehension of the mechanisms involved in the carcinogenesis of oral squamous epithelium, protein expression and localization of Dkk-3 and β-catenin was investigated in normal epithelium, dysplasias and squamous cell carcinoma (SCC). An increase in β-catenin and Ki-67 expressions was observed from dysplasias to poorly differentiated SCC. Interestingly, an increase in Dkk-3 positive cells was also noted, which was correlated to the cancer progression step. A change in Dkk-3 localization during the transformation of normal oral epithelium to SCC was clearly observed. Dkk-3 was localized in the cell membrane in normal oral epithelium and in dysplasias, whereas that was localized in both cell membrane and cytoplasm in SCC. These results suggest that Dkk-3 is involved in the carcinogenesis of SCC with a distinct function from those in other cancers.     

Characterization of marine X-family DNA polymerases and comparative analysis of base excision repair proteins

While mammalian DNA polymerase β (Pol β), which is a member of the Pol X family, play important roles in base excision repair (BER) that efficiently removes DNA base lesions arising from both endogenous and exogenous agents, this protein has been found only a subset of animals. To understand natural evolution of this enzyme, we isolated and characterized Pol β from jellyfish Aurelia sp.1. (AsPol β). Despite of phylogenetic distance and environmental differences between jellyfish and mammals, in vitro assays showed biochemical characteristics of AsPol β were very similar to those of a mammalian counterpart. We also searched two other homologs of mammalian genes that were involved in short patch (sp) BER in the nucleotide sequence database, and found that both of these homologs were encoded in the genomes of a lineage from Cnidarians through mammals and Arthropods. This study suggests that a DNA repair mechanism resembling mammalian sp-BER may be largely limited to a subset of animals. On the basis of our findings and previous reports, we discuss possible evolutional model of Pol β and the other members of the Pol X family.     

Synthesis of L-Tyrosine or 3,4-Dihydroxyphenyl-L-alanine from DL-Serine and Phenol or Pyrocathechol

Reaction conditions for the synthesis of L-tyrosine or L-dopa from DL-serine and phenol or pyrocatechol were studied with intact cells of Erwinia herbicola (ATCC 21434) containing high tyrosine phenol lyase activity. The optimum pH for this reaction was around 8.0, and the optimum temperature range was between 37~40°C for the synthesis of L-tyrosine and between 15~25°C for that of L-dopa. Sodium sulfite and EDTA were added to protect the synthesized L-dopa from decomposition. As high concentrations of phenol or pyrocatechol denatured the enzyme, each substrate was fed to maintain the optimum concentration during incubation.

The reaction mixture (100 ml) containing 4.0 g of DL-serine, 1.0 g of phenol or 0.7 g of pyrocatechol, 0.5 g of ammonium acetate and the cells, was incubated. During incubation, phenol or pyrocatechol was fed at intervals to maintain the substrate at the initial concentration. 5.35 g of L-tyrosine or 5.10 g of L-dopa was synthesized in 100 ml of the reaction mixture.     

Structure of a New Antioxidative Phenolic Diterpene Isolated from Rosemary (Rosmarinus officinalis L.)

A new antioxidant, phenolic diterpene, named rosmanol, was isolated from the leaves of rosemary (Rosmarinus officinalis L.). Its structure has been determined as 7β,11,12-trihydroxy-6,10-(epoxymethano)abieta-8,11,13-trien-20-one on the basis of chemical and spectroscopic evidence. In addition, all the protons of rosmanol (II) as well as carnosol (I), including the methylene protons of the A ring, were analyzed by 400 MHz ¹-NMR.     

Developmental Changes in the Structure of Endosperm Starch of Rice (Oryza sativa L.)

The developmental changes in the structure and properties of endosperm starches were investigated using the near-isogenic lines for wx alleles of rice. The amylose content in nonwaxy starch was increased during the development of rice grains. Because the accumulation of amylose in endosperm stopped earlier than that of amylopectin during development, the percentages of amylose reached a maximum at the 17th day after flowering in nonwaxy endosperm. Since the distributions of the unit-chain length of amylopectin in waxy and nonwaxy starches were unchanged with the development of the grains, these amylopectins would be synthesized in a similar manner through development. The structure and properties of endosperm starches were reconfirmed to be conspicuously affected by the temperature at the early developmental stages of the grain-filling period, namely, they appeared to be characterized by the temperature at which the starch was accumulated in the endosperm.     

Construction of human Fab (gamma1/kappa) library and identification of human monoclonal Fab possessing neutralizing potency against Japanese encephalitis virus

A combinatorial human Fab library was constructed using RNAs from peripheral blood lymphocytes obtained from Japanese encephalitis virus hyper-immune volunteers on pComb3H phagemid vector. The size of the constructed Fab library was 3.3x10(8) Escherichia coli transformants. The library was panned 3 times on the purified Japanese encephalitis virus (JEV) virion, and phage clones displaying JEV antigen-specific Fab were enriched. The enriched phage pool was then screened for clones producing Fab molecule with JEV neutralizing activity by the focus reduction-neutralizing test. Among 188 randomly selected clones, 9 Fab preparations revealed neutralizing activities against JEV strain Nakayama. An E. coli transformed with TJE12B02 clone, which produced human monoclonal Fab with the highest neutralizing activity was cultured in a large scale, and the Fab molecule was purified using affinity chromatography. The purified FabTJE12B02 showed the 50% focus reduction endpoint at the concentration of 50.2 microg/ml (ca. 1,000 nM) when JEV strain Nakayama was used. The FabTJE12B02 recognized E protein of JEV strain Nakayama, and the dissociation equilibrium constant (Kd) of the FabTJE12B02 against purified JEV antigen was calculated as 1.21x10(-8) M. Sequence analysis demonstrated that TJE12B02 used a VH sequence homologous to the VH3 family showing 88.8% homology to germline VH3-23, and used a Vkappa sequence homologous to the VkappaII subgroup showing 92.8% homology to germline A17.     

Mechanism of bone induction by KUSA/A1 cells using atelocollagen honeycomb scaffold

In order to induce new bone formation, mesenchymal stem cells were seeded onto atelocollagen honeycomb scaffold. We evaluated the mechanism of bone induction by KUSA/A1 cells combined with honeycomb atelocollagen scaffold. Scaffold alone, KUSA/A1 cells alone and with scaffold were implanted in the subcutaneous pockets of 4-week-old male SCID mice. The transplants were subjected to radiographical, histological and immunohistochemical examinations after 2 and 4 weeks of implantation. Radiographically, both KUSA/A1 cells alone and KUSA/A1-Scaffold showed some radiopaque areas formation but the latter disclosed a larger amount. Scaffold alone did not show any radiopacity. Histologically, Scaffold alone demonstrated only fibrous connective tissues in the periphery of the scaffold. KUSA/A1 cells alone showed few small islands of new bone formation surrounded by a thin layer of cellular proliferation. On the other hand, KUSA/A1-Scaffold revealed abundant new bone formation as well as cellular proliferation. We also determined the immunolocalization of type I collagen, CD34, Osteocalcin and PCNA in this newly formed bone. Our results indicated that less amount of stem cells are capable to induce the more amount of new bone in tissue engineering. This study support that atelocollagen honeycomb scaffold plays an important role in cellular anchorage and in vessel invasion, giving the precise shape and size for the new bone formation.     

Activation of Pyk2 by CaM kinase II in cultured hypothalamic neurons and gonadotroph cells

Gonadotropin-releasing hormone (GnRH) is secreted from hypothalamic GnRH neurons and stimulates a GnRH receptor in gonadotroph cells and GnRH neurons. The GnRH receptor belongs to the G-protein-coupled receptors, and stimulation of the GnRH receptor activates extracellular signal-regulated protein kinase (ERK). We reported previously that the δ2 isoform of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase IIδ2) was involved in GnRH-induced ERK activation in cultured GnRH neurons (GT1–7 cells). Recently, we found that GnRH treatment of GT1–7 cells activated proline-rich tyrosine kinase 2 (Pyk2), and Pyk2 was involved in ERK activation. In the current study, we examined the possibility that CaM kinase IIδ2 might activate Pyk2. Knockdown of CaM kinase IIδ2 and KN93, an inhibitor of CaM kinases, inhibited the GnRH-induced activation of Pyk2. In the case of cultured gonadotroph cells (αT3-1 cells), knockdown of CaM kinase IIβ’e inhibited GnRH-induced Pyk2 activation. In addition, our inhibitor studies indicated that Pyk2 and CaM kinase II were involved in the GnRH-induced shedding of proHB-EGF in GT1–7 cells. These results suggested that CaM kinase II activated the ERK pathway through Pyk2 activation and HB-EGF production in response to GnRH.     

Eumelanin levels in rufous feathers explain plasma testosterone levels and survival in swallows

Pigment‐based plumage coloration and its physiological properties have attracted many researchers to explain the evolution of such ornamental traits. These studies, however, assume the functional importance of the predominant pigment while ignoring that of other minor pigments, and few studies have focused on the composition of these pigments. Using the pheomelanin‐based plumage in two swallow species, we studied the allocation of two pigments (the predominant pigment, pheomelanin, and the minor pigment, eumelanin) in relation to physiological properties and viability in populations under a natural and sexual selection. This is indispensable for studying the evolution of pheomelanin‐based plumage coloration. Pheomelanin and eumelanin share the same pathway only during their initial stages of development, which can be a key to unravel the functional importance of pigment allocation and thus of plumage coloration. Using the barn swallow, Hirundo rustica, a migratory species, we found that plasma testosterone levels increased with increasing the proportion of eumelanin pigments compared with pheomelanin pigments, but not with the amount of pheomelanin pigments, during the mating period. In the Pacific swallow Hirundo tahitica, a nonmigratory congener, we found that, during severe winter weathers, survivors had a proportionally smaller amount of eumelanin pigments compared with pheomelanin pigments than that in nonsurvivors, but no detectable difference was found in the pheomelanin pigmentation itself. These results indicated that a minor pigment, eumelanin, matters at least in some physiological measures and viability. Because the major pigment, pheomelanin, has its own physiological properties, a combination of major and minor pigments provides multiple information to the signal receivers, potentially enhancing the signaling function of pheomelanic coloration and its diversification across habitats.