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X-linked hypophosphatemic rickets (XLH) is an inheritable type of rickets caused by inactivating variants in the phosphate regulating endopeptidase homolog X-linked (PHEX) gene, which results in the overproduction of fibroblast growth factor 23 (FGF23). The mechanism by which PHEX impairment leads to FGF23 overproduction is unknown. Because little is known regarding the genotype-phenotype correlation in Japanese XLH, we summarized the available clinical and genetic data and analyzed the genotype-phenotype relationships using 3-dimensional (3D) structure modeling to clarify the XLH pathophysiology. We retrospectively reviewed the clinical features and performed genetic analysis of 39 Japanese patients with XLH from 28 unrelated pedigrees carrying any known or novel PHEX variant. To predict changes in the 3D structure of mutant PHEX, we constructed a putative 3D model of each mutant and evaluated the effect of structural alteration by genotype-phenotype correlation analysis. Genetic analysis revealed 23 PHEX variants, including eight novel variants. They were associated with high i-FGF23 levels, hypophosphatemia, phosphaturia, high alkaline phosphatase levels, and short stature. No gene dosage effect or genotype-phenotype correlation was observed when truncating and non-truncating variants were compared. However, the conservation of the zinc-binding site and cavity in PHEX had an impact on the elevation of i-FGF23 levels. Via genotype-phenotype relationship analysis using 3D modeling, we showed that the zinc-binding site and cavity in PHEX can play a critical role in its function. These findings provide new genetic clues for investigating the function of PHEX and the pathogenesis of XLH.

Enterovirus and human parechovirus (HPeV) are RNA viruses belonging to the family Picornaviridae that frequently infect infants. These infections show a wide variety of clinical manifestations, from mild to severe. However, there are no known early clinical markers for diagnosis and prediction of disease severity. The aim of this study was to examine the clinical utility of urinary beta 2-microglobulin (β2MG) for the early detection and prognosis of infantile enterovirus and HPeV infections. This retrospective study included 108 full-term infants younger than 60 days of age, including 15 with enterovirus or HPeV-3 (enterovirus/HPeV-3), 22 with respiratory syncytial virus (RSV), and 24 with bacterial infections. Laboratory data and clinical characteristics were compared among these 3 groups. Of the 15 patients with enterovirus/HPeV-3, 6 were treated with intravenous immunoglobulin (IVIG subgroup) because of severe clinical conditions. Urinary β2MG to creatinine ratio (β2MG/Cr) was significantly higher in the enterovirus/HPeV-3 group compared to bacterial and RSV infection groups (both P

Ni complex was prepared from bipyridine dicarboxylic acid and Ni salt. Then, the Ni complex was reacted with Al salt in dimethyl formamide in an autoclave to prepare metal-organic framework (MOF) compounds containing Ni-complex. IR analysis indicated disappearance of absorption peaks related to carboxylic acid, and nitrogen-adsorption isotherm analysis revealed the presence of micropores. MOF compounds containing Ni complex were used as catalysts for oligomerization of ethylene. Diethyl aluminum chloride or MOF containing Ni complex was inactive for the reaction. However, MOF compounds containing Ni complex used with diethyl aluminum chloride had high potential as a catalyst for the oligomerization of ethylene with high selectivity for linear butenes.

The purpose of the current study was to examine the binding of pulmonary surfactant protein A (SP-A) to TLR4 and MD-2, which are critical signaling receptors for lipopolysaccharides (LPSs). The direct binding of SP-A to the recombinant soluble form of extracellular TLR4 domain (sTLR4) and MD-2 was detected using solid-phase binding, immunoprecipitation, and BIAcore. SP-A bound to sTLR4 and MD-2 in a Ca2+-dependent manner, and an anti-SP-A monoclonal antibody whose epitope lies in the region Thr184–Gly194 blocked the SP-A binding to sTLR4 and MD-2, indicating the involvement of the carbohydrate recognition domain (CRD) in the binding. SP-A avidly bound to the deglycosylated forms of sTLR4 and MD-2, suggesting a protein/protein interaction. In addition, SP-A attenuated cell surface binding of smooth LPS and smooth LPS-induced NF-κB activation in TLR4/MD-2-expressing cells. To know the role of oligomerization in the interaction of SP-A with TLR4 and MD-2, the collagenase-resistant fragment (CRF), which consisted of CRD plus neck domain of SP-A, was isolated. CRF assembled as a trimer, whereas SP-A assembled as a higher order oligomer. Although CRD was suggested to be involved in the binding, CRF exhibited approximately 600- and 155-fold higher KD for the binding to TLR4 and MD-2, respectively, when compared with SP-A. Consistently significantly higher molar concentrations of CRF were required to inhibit smooth LPS-induced NF-κB activation and tumor necrosis factor-α secretion. These results demonstrate for the first time the direct interaction between SP-A and TLR4/MD-2 and suggest the importance of supratrimeric oligomerization in the immunomodulatory function of SP-A.

The mammalian MCM protein family, presently with six members, exists in the nuclei in two forms, chromatin-bound and unbound. The former dissociates from chromatin with progression through the S phase. Recently, we have established a procedure to isolate chromatin-bound and unbound complexes containing all six human MCM (hMCM) proteins by immunoprecipitation. In the present study, we applied this procedure to HeLa cells synchronized in each of the G1, S, and G2/M phases and could detect hMCM heterohexameric complexes in all three. In addition, depending on the cell cycle and the state of chromatin association, hMCM2 and 4 in the complexes were found to variously change their phosphorylation states. Concentrating attention on G2/M phase hyperphosphorylation, we found hMCM2 and 4 in the complexes to be good substrates for cdc2/cyclin B in vitro. Furthermore, when cdc2 kinase was inactivated in temperature-sensitive mutant murine FT210 cells, the G2/M hyperphosphorylation of the murine MCM2 and MCM4 and release of the MCMs from chromatin in the G2 phase were severely impaired. Taken together, the data suggest that the six mammalian MCM proteins function and undergo cell cycle-dependent regulation as heterohexameric complexes and that phosphorylation of the complexes by cdc2 kinase may be one of mechanisms negatively regulating the MCM complex-chromatin association.

In vitro shoot propagation from shoot tip explants of zoysiagrass (Zoysia matrella Merr.) was achieved in agar and liquid media based on Murashige and Skoog (1962) media supplemented with various combinations of α-naphthaleneacetic acid (NAA) and 6-benzyl-aminopurine (BA) concentrations. Maximum shoot propagation was obtained with 0.5μM NAA and 5.0μM BA in agar media, and 0.05μM NAA and 0.5μM BA in liquid media. Stolons were formed in liquid media but not in agar media. Liquid media was superior for propagation of shoots to agar media as the formations of multiple shoots, stolons and roots were higher in rates.

細胞遺伝学的知識の集積に伴って染色体分析の必要性と需要が増し, 簡便で迅速な検査法の確立が望まれている.しかし, 分裂した細胞のみを対象とする従来の染色体分析法では熟練と時間が要求され, また培養に伴う細胞群の選択がおこる.蛍光インサイツーハイブリダイゼーション (FISH法) は間期細胞核においても染色体分析を可能にした (間期細胞遺伝学).ハプテン化染色体特異的プローブを用いたFISH法は, 染色体数の計算と数的異常の検出に汎用されてきたが, 約48時間を要し, ハプテン検出に伴う煩雑な免疫細胞化学的処理過程が必要である.この煩雑さを解消するため, 蛍光色素直接標識セントロメアプローブを用いて, 18トリソミー, 21トリソミーおよびXXYの染色体異常患者の末梢リンパ球にFISH法を適用したところ, おのおのの症例に対応するコピー数が得られた.しかも所要時間を2時間まで短縮できたうえ, 操作段階を半減することが可能であった.このように, 蛍光色素直接標識プローブによるFISH法は, 染色体の数的異常の, 迅速かつ手技的間違いのない簡便な検査法として, 染色体数異常疾患の診断に有用であることを明らかにした.

BUF/Mna strain rats spontaneously develop renal glomerular sclerotic lesions (RSL) at a nearly 100% incidence, diagnosed by hyperalbuminuria (greater than 500 mg/dl) and glomerular lesions morphologically resembling one type of human focal glomerular sclerosis (FGS). Genetic segregation of RSL development was studied by crossing the BUF/Mna strain with two other rat strains, WKY/NCrj and ACI/NMs, which were free of RSL. Two autosomal recessive genes in the BUF/Mna rats were found to determine the susceptibility to RSL in both combinations of crosses.

Pulmonary surfactant protein D (SP-D), a member of the collectin group of innate immune proteins, plays important roles in lipopolysaccharide (LPS) recognition. We have previously shown that surfactant protein A (SP-A), a homologous collectin, interacts with Toll-like receptor (TLR) 2, resulting in alteration of TLR2-mediated signaling. In this study, we found that natural and recombinant SP-Ds exhibited specific binding to the extracellular domains of soluble forms of recombinant TLR2 (sTLR2) and TLR4 (sTLR4). Binding was concentration- and Ca2+-dependent, and SP-D bound to N-glycosidase F-treated sTLRs on ligand blots. Anti-SP-D monoclonal antibody 7A10 blocked binding of SP-D to sTLR2 and sTLR4, but there was no inhibitory effect of monoclonal 7C6. Epitope mapping with recombinant proteins consisting of the carbohydrate recognition domain (CRD) and the neck domain plus CRD (NCRD) localized binding sites for 7A10 and 7C6 to sequential epitopes associated with the CRD and the neck domain, respectively. Interactions with 7A10 but not 7C6 were blocked by prior binding of the NCRD to sTLRs. Although antibody 7A10 significantly inhibited the binding of SP-D to its major surfactant-associated ligand, phosphatidylinositol (PI), and Escherichia coli Rc LPS, 7C6 enhanced binding to both molecules. An SP-D(E321Q, N323D) mutant with altered carbohydrate specificity exhibited attenuated PI binding but showed an increased level of binding to sTLRs. Thus, human SP-D binds the extracellular domains of TLR2 and TLR4 through its CRD by a mechanism different from its binding to PI and LPS.

The purpose of the current study was to examine the binding of pulmonary surfactant protein A (SP-A) to TLR4 and MD-2, which are critical signaling receptors for lipopolysaccharides (LPSs). The direct binding of SP-A to the recombinant soluble form of extracellular TLR4 domain (sTLR4) and MD-2 was detected using solid-phase binding, immunoprecipitation, and BIAcore. SP-A bound to sTLR4 and MD-2 in a Ca2+-dependent manner, and an anti-SP-A monoclonal antibody whose epitope lies in the region Thr184-Gly194 blocked the SP-A binding to sTLR4 and MD-2, indicating the involvement of the carbohydrate recognition domain (CRD) in the binding. SP-A avidly bound to the deglycosylated forms of sTLR4 and MD-2, suggesting a protein/protein interaction. In addition, SP-A attenuated cell surface binding of smooth LPS and smooth LPS-induced NF-kappaB activation in TLR4/MD-2-expressing cells. To know the role of oligomerization in the interaction of SP-A with TLR4 and MD-2, the collagenase-resistant fragment (CRF), which consisted of CRD plus neck domain of SP-A, was isolated. CRF assembled as a trimer, whereas SP-A assembled as a higher order oligomer. Although CRD was suggested to be involved in the binding, CRF exhibited approximately 600- and 155-fold higher KD for the binding to TLR4 and MD-2, respectively, when compared with SP-A. Consistently significantly higher molar concentrations of CRF were required to inhibit smooth LPS-induced NF-kappaB activation and tumor necrosis factor-alpha secretion. These results demonstrate for the first time the direct interaction between SP-A and TLR4/MD-2 and suggest the importance of supratrimeric oligomerization in the immunomodulatory function of SP-A.

Four clonal cell lines of two types were established from a heterotransplantable mixed mesodermal tumor of the human ovary. Biologic properties of these cell lines (designated CS-C1, CS-S1, CS-S2, and CS-S3) were examined. Cells of one line (CS-C1) had an epithelioid shape and grew in monolayers (C-type). The cells showed alkaline phosphatase activity, stained positively with antikeratin antiserum, and had an ultrastructure with carcinomatous characteristics. Cells of the other three cell lines (CS-S1, CS-S2, and CS-S3) had an irregular shape and grew in multilayers (S-type). Most of the cells did not show alkaline phosphatase activity. They stained, not with antikeratin antiserum, but in fibrillar array with antifibronectin antiserum. Their ultrastructure had sarcomatous characteristics. By low cell density cultures, S-type sublines arose from CS-C1 cell line, but no C-type sublines arose from CS-S1 cell line. These findings may support the theory of the combination tumor as the cytogenesis of mixed mesodermal tumor of the ovary; they also suggest the conversion of carcinomatous cells to sarcomatous cells.

A case of clear cell carcinoma of the ovary with colony-stimulating-factor (CSF) production is presented. Granulocytosis (54,200/mm3) of unknown cause was observed in a patient at the terminal stage of the clinical course. This tumor was successfully heterotransplanted into athymic nude mice, and transplantable tumor line was designated as OCL-1. OCL-1 tumor reproduced granulocytosis up to 640,000/mm3 in nude mice, and CSF production was demonstrated by colony-forming assay using tumor extract and mouse bone marrow cells in vitro. This is the first report that describes the case of CSF-producing clear cell carcinoma of the ovary and its heterotransplantation in nude mice.