Tumor-induced osteomalacia (TIO) is one of the paraneoplastic diseases characterized by hypophosphatemia caused by renal phosphate wasting. Because removal of responsible tumors normalizes phosphate metabolism, an unidentified humoral phosphaturic factor is believed to be responsible for this syndrome. To identify the causative factor of TIO, we obtained cDNA clones that were abundantly expressed only in a tumor causing TIO and constructed tumor-specific cDNA contigs. Based on the sequence of one major contig, we cloned 2,270-bp cDNA, which turned out to encode fibroblast growth factor 23 ( T umor-induced osteomalacia (TIO) is one of the hypophosphatemic diseases characterized by renal phosphate wasting. Because removal of responsible tumors normalizes phosphate metabolism, an unknown phosphaturic factor sometimes called phosphatonin is believed to be responsible for this paraneoplastic syndrome (1, 2). Although several groups have reported inhibitory activity of renal phosphate transport in conditioned media of tumor cells causing TIO (3-6), the responsible factor for TIO has not been identified. Similar biochemical findings to TIO also are observed in X-linked hypophosphatemic rickets͞ osteomalacia (XLH), its murine homologue, Hyp, and autosomal dominant hypophosphatemic rickets (ADHR) (7). In addition, several lines of evidence indicate that XLH and Hyp are caused by a humoral mechanism (7-10). Therefore, it is possible that TIO and XLH derive from a common or at least very similar humoral factor(s). Thus, identification of this phosphaturic factor causing TIO is indispensable for understanding normal phosphate metabolism and pathogenesis of several hypophosphatemic diseases. In this report, we describe the cloning of a humoral factor from a TIO tumor and show that this factor has the ability to rapidly induce hypophosphatemia and reproduce clinical, biochemical, and histological features of TIO in vivo. MethodsDifferential cDNA Screening of TIO and Adjacent Normal Bone Tissue.

With a view to initiating clinical trials, cell morphology and function for a newly developed artificial liver support system employing highly functional human liver cell line, FLC-7, cultured in a radial flow bioreactor were compared to cells grown in a conventional monolayer culture. The radial flow bioreactor consists of a vertically extended cylindrical matrix comprised of porous glass bead microcarriers through which liquid medium flows from the periphery in toward the central axis generating a beneficial concentration gradient of oxygen and nutrients, while preventing excessive shear stresses or buildup of waste products. The three-dimensional culture system supports high-density (1.1 x 10(8) cells/ml-matrix), large scale cultures (4.4 x 10(10) cells/400 ml-bioreactor) with long-term viability. Scanning and transmission electron microscopy (SEM and TEM) revealed that cells cultured in a monolayer system were flattened and extended with numerous cytoplasmic projections. Cells in the three-dimensional culture were spherical and covered with microvillilike processes resembling liver cells in vivo. The cells were solidly attached on the surfaces and within the pores of the microcarriers in highly dense colonies. The spherical cells remained in close contact with adjacent cells, while circulation of liquid medium flowed freely through spaces between cells. FLC-7 cells produced albumin at a rate of 6.41 micrograms/24 h/10(6) cells. Alpha-fetoprotein (AFP) production dropped nearly threefold in comparison to monolayer cultures. Results demonstrated that the new artificial liver support systems (ALSS) provides a superior three-dimensional culture environment that allows cells to perform at naturally functioning levels.

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Ginger, Zingiber officinale Roscoe, contains a fragrant oil mainly composed of sesquiterpenes and monoterpenes. We isolated a cDNA that codes for a sesquiterpene synthase from young rhizomes of ginger, Z. officinale Roscoe, Japanese cultivar "Kintoki". The cDNA, designated ZoTps1, potentially encoded a protein that comprised 550 amino acid residues and exhibited 49-53% identity with those of the sesquiterpene synthases already isolated from the genus Zingiber. Recombinant Escherichia coli cells, in which ZoTps1 was coexpressed along with genes for D-mevalonate utilization, resulted in the production of a sesquiterpene (S)-beta-bisabolene exclusively with a D-mevalonolactone supplement. This result indicated that ZoTps1 was the (S)-beta-bisabolene synthase gene in ginger. ZoTPS1 was suggested to catalyze (S)-beta-bisabolene formation with the conversion of farnesyl diphosphate to nerolidyl diphosphate followed by the cyclization between position 1 and 6 carbons. The ZoTps1 transcript was detected in young rhizomes, but not in leaves, roots and mature rhizomes of the ginger "Kintoki".

Sulfite plays an important role in beer flavor stability. Although breeding of bottom-fermenting Saccharomyces strains that produce high levels of SO 2 is desirable, it is complicated by the fact that undesirable H 2 S is produced as an intermediate in the same pathway. Here, we report the development of a high-level SO 2 -producing bottom-fermenting yeast strain by integrated metabolome and transcriptome analysis. This analysis revealed that O-acetylhomoserine (OAH) is the rate-limiting factor for the production of SO 2 and H 2 S. Appropriate genetic modifications were then introduced into a prototype strain to increase metabolic fluxes from aspartate to OAH and from sulfate to SO 2 , resulting in high SO 2 and low H 2 S production. Spontaneous mutants of an industrial strain that were resistant to both methionine and threonine analogs were then analyzed for similar metabolic fluxes. One promising mutant produced much higher levels of SO 2 than the parent but produced parental levels of H 2 S.The bottom-fermenting yeast Saccharomyces pastorianus is used to produce beer and has been proposed to be a natural hybrid between Saccharomyces cerevisiae and Saccharomyces bayanus (30). Bottom-fermenting yeasts have two types of genes, one set highly homologous (more than 90% identity) to those of S. cerevisiae and the other less so but highly homologous to S. bayanus (i.e., non-S. cerevisiae [Lg type]) (8,14,27,33). One way in which S. pastorianus differs from baker's yeast (S. cerevisiae) is its tendency to produce higher levels of both sulfite (SO 2 ) and hydrogen sulfide (H 2 S).It is well known that sulfur compounds in beer make significant contributions to flavor and aroma. SO 2 , for example, acts as an antioxidant, which slows the development of oxidation haze and staling of flavors in beer. In contrast, H 2 S has an aroma of rotten eggs and is also a precursor of other compounds with undesirable sensory characteristics. SO 2 and H 2 S are produced by yeast during reductive sulfate assimilation (Fig. 1). Inorganic sulfate is taken up through a sulfate permease and reduced to SO 2 by enzymes encoded by MET3, MET14, and MET16. SO 2 is then reduced to H 2 S by SO 2 reductase encoded by MET5 and MET10 (29). The next intermediate, homocysteine, which is synthesized from H 2 S and O-acetylhomoserine (OAH) by OAH sulfhydrylase encoded by MET17, leads to the formation of cysteine, methionine, and S-adenosylmethionine (SAM). SAM transcriptionally represses all of the genes involved in sulfate assimilation. Park and Bakalinsky previously reported that SSU1 encodes an SO 2 efflux pump that exports intracellular SO 2 through the plasma membrane (18).In the postgenomic era, systematic and high-throughput analyses of mRNA and proteins have become central to recent functional genomics initiatives. Metabolomics entails the analysis of all cellular metabolites and has become a powerful new tool for gaining insight into functional biology. Measurement of numerous metabolites within a cell and tracking concentration changes as a fu...

Liver endothelial cells are important components of the tissue along the hepatic sinusoid. They are responsible for microcirculation in the liver and scavenger functions. It would therefore be important to include these cells in any hybrid type of artificial liver in addition to hepatocytes. However, it is difficult to culture these cells in vitro. The development of a liver endothelial cell line, which maintains the characteristics of the primary culture, would thus be of great benefit in the development of an artificial liver. In the present study we established immortalized liver endothelial cells from the liver of an H-2Kb-tsA58 transgenic mouse, which harbors the SV40 TAg gene. Hepatic sinusoidal cells isolated from H-2Kb-tsA58 mouse proliferated in the presence of gamma-interferon at 33 degrees C. Four clones were established, out of which clone M1 had the highest amounts of PGI2 production, as well as plasminogen activator activity and internalized acetylated low density lipoprotein. On culture dishes the M1 cells grew individually and spread. Sieve plates on the cell surface were not readily visible, but small pores were detected under electron microscopic observation. These results suggest that M1 clone cells originated from liver endothelial cells. Moreover it was possible to culture the immortalized liver endothelial cells in a radial-flow bioreactor for 5 days, with a maximum 6-keto prostaglandin F1alpha production of 25 microg per day. This suggests that immortalized liver endothelial cells and a radial-flow bioreactor can prove useful tools in the development an artificial liver.

It has been proposed that bottom-fermenting yeast strains of

Serial electrophoretic experiments concerning serum and placental oxytocinases were carried out with a direct staining method for cystine aminopeptidase activity. The pregnancy serum oxytocinase and the lysosomal oxytocinase in human placenta shared the same enzymatic characteristics, with regard to heat resistence, sensitivity to L-methionine inhibition and electrophoretic pattern. These similarities suggest that the possible site of production of pregnancy serum oxytocinase is the lysosomes of the placenta, from which the enzyme is released into maternal circulation during pregnancy. Absence of oxytocinase activity in fetal serum indicates that this enzyme does not leak into fetal circulation. The physiological significance of oxytocinase was discussed with special reference to the oxytocin-oxytocinase equilibrium during pregnancy and labor.