An androgen-dependent mouse mammary carcinoma cell line (SC-3) requires androgen for growth stimulation. We have shown previously that androgen acts on SC-3

CD13, a receptor for human coronavirus 229E (HCoV-229E), was identified as a major component of the Triton X-100-resistant membrane microdomain in human fibroblasts. The incubation of living fibroblasts with an anti-CD13 antibody on ice gave punctate labeling that was evenly distributed on the cell surface, but raising the temperature to 37°C before fixation caused aggregation of the labeling. The aggregated labeling of CD13 colocalized with caveolin-1 in most cells. The HCoV-229E virus particle showed a binding and redistribution pattern that was similar to that caused by the anti-CD13 antibody: the virus bound to the cell evenly when incubated on ice but became colocalized with caveolin-1 at 37°C; importantly, the virus also caused sequestration of CD13 to the caveolin-1-positive area. Electron microscopy confirmed that HCoV-229E was localized near or at the orifice of caveolae after incubation at 37°C. The depletion of plasmalemmal cholesterol with methyl ␤-cyclodextrin significantly reduced the HCoV-229E redistribution and subsequent infection. A caveolin-1 knockdown by RNA interference also reduced the HCoV-229E infection considerably. The results indicate that HCoV-229E first binds to CD13 in the Triton X-100-resistant microdomain, then clusters CD13 by cross-linking, and thereby reaches the caveolar region before entering cells.Recent studies have revealed that the plasma membranes of cells contain microdomains with discrete molecular compositions. Rafts are sphingolipid-and cholesterol-rich membrane microdomains that are thought of as platforms for signal transduction (39, 40). Although there are still many controversies regarding how rafts exist in living cells, it is generally agreed that cholesterol is indispensable for their integrity and that the detergent-resistant membrane (DRM) fraction is the in vitro correlate of the raft. Because acyl chains of sphingolipids and glycosylphosphatidylinositol (GPI)-anchored proteins enriched in the DRM fraction are more highly saturated than those of glycerolipids in the bulk membrane, the raft domain is thought to show less fluidity than nonraft areas of the plasma membrane. However, it is difficult to capture rafts morphologically because their shape and size are likely to change dynamically (40).On the other hand, caveolae were first defined morphologically as invaginations of the plasma membrane (49). They are also susceptible to cholesterol depletion (31). Moreover, caveolin-1, -2, and -3, which were identified as major components of caveolae (31,35,44,47), are highly enriched in the DRM fraction (2,12,14,36). Several results suggest that many molecules are shared by rafts and caveolae but that at least several molecules that are enriched in the DRM fraction are not concentrated in caveolae (11). Thus, caveolae are not simply a stabilized form of rafts, but there should be a regulatory mechanism (as yet unknown) to control the molecular distribution between caveolae and rafts.It has been shown that cross-linked raft molecules, such as GPI-anchored proteins,...

Identification of the second gonadotropin-releasing hormone in chicken hypothalamus: Evidence that gonadotropin secretion is probably controlled by two distinct gonadotropin-releasing hormones in avian species (reproduction/neuropeptide) KAORU MIYAMOTO*, YOSHIHISA HASEGAWA*, MITSuo NOMURA*, MASAO Since the structural determination of porcine hypothalamic luteinizing hormone (LH)-releasing hormone (LHRH) in 1971 (1), the concept has widely been acepted that gonadotropin secretion is controlled by a sole gonadotropin-releasing hormone (GnRH), such as mammalian LHRH. On the other hand, we have demonstrated that gonadotropin secretion is not controlled by LHRH under certain physiological and experimental conditions (2). However, there has been no structural evidence that two or more than two GnRHs coexist and function in any vertebrate.The present paper represents the structural demonstration of the existence of two different GnRHs at least in avian species.Recently, we (3, 4) and King and Millar (5-7) have independently isolated chicken LHRH and determined its structure as [Gln8]LHRH. We have purified chicken LHRH by monitoring its biopotency towards rat anterior pituitary cells. During the course of the purification, another gonadotropin-releasing activity was found in the chicken hypothalamic extract, which was well separated from that due to chicken LHRH on ion-exchange chromatography.The present paper describes the isolation and structural determination of the second GnRH in chicken hypothalamic extract (designated chicken GnRH-II).Chicken GnRH-II has been purified by successive gel filtration, ion-exchange HPLC and reverse-phase HPLC. A pure peptide has been isolated in a yield of 7 pg (6 nmol), starting from 10,000 chicken hypothalamic fragments.The amino acid composition of the peptide indicated a decapeptide structure consisting of SerGlxPro1Gly2Tyrl-His2Trp2. By amino acid analyses and terminal analyses of enzymatic fragments derived from the purified decapeptide, the complete structure of chicken GnRH-II has been determined to be: pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly- NH2.The present paper also describes the gonadotropin-releasing activity of chicken GnRH-II compared with that of mammalian LHRH. MATERIALS AND METHODSAssay of Gonadotropin-Releasing Activity. Gonadotropinreleasing activity was estimated on the rat anterior pituitary cell culture system as reported (3).Anterior pituitaries were obtained from female Holzman rats (180-to 220-g body weight), agitated in trypsin solution (0.25%), and finally dispersed with 1% Viokase (GIBCO) solution. Cells were collected and washed three times with Dulbecco's modified Eagle's medium (DME medium) containing gentamycin (100 ,ug/ml), fungizone (25 ttg/ml), 5% horse serum, 5% human cord serum, 2.5% bovine calf serum, 0.1% glutamine, and 1% nonessential amino acids (GIBCO). Cells were resuspended in an appropriate volume of the medium described above, and 0.2-ml portions (containing 5 x 104 cells) were put into 96 multi-well tissue culture plates (Nunc,. Th...

Zinc-fingers and homeoboxes (ZHX) 1 is a transcription factor that interacts with the activation domain of the A subunit of nuclear factor-Y (NF-YA). Using a yeast two-hybrid system, a novel ubiquitous transcription factor ZHX2 as a ZHX1-interacting protein was cloned. ZHX2 consists of 837 amino acid residues and contains two zinc-finger motifs and five homeodomains (HDs) as well as ZHX1. The mRNA is expressed among various tissues. ZHX2 not only forms a heterodimer with ZHX1, but also forms a homodimer. Moreover, ZHX2 interacts with the activation domain of NF-YA. Further analysis revealed that ZHX2 is a transcriptional repressor that is localized in the nuclei. Since ZHX2 shares a number of properties in common with ZHX1, we conclude that all these come under the ZHX family. The minimal functional domains of ZHX2 were then characterized. The dimerization domain with both ZHX1 and ZHX2 is the region containing HD1, the domain that interacts with NF-YA is the HD1 to HD2 region, the repressor domain is the HD1 to a proline-rich region. Lastly, using an immunoprecipitation assay, we showed that ZHX2 intrinsically interacts with NF-YA in HEK-293 cells and that ZHX2 represses the promoter activity of the cdc25C gene stimulated by NF-Y in Drosophila Schneider line 2 cells. Thus the ZHX family of proteins may participate in the expression of a number of NF-Y-regulated genes via a more organized transcription network.

Adult stem cells from bone marrow, referred to as mesenchymal stem cells or marrow stromal cells (MSCs), are defined as pluripotent cells and have the ability to differentiate into multiple mesodermal cells. In this study, we investigated whether MSCs from rat, mouse, and human are able to differentiate into steroidogenic cells. When transplanted into immature rat testes, adherent marrow-derived cells (including MSCs) were found to be engrafted and differentiate into steroidogenic cells that were indistinguishable from Leydig cells. Isolated murine MSCs transfected with green fluorescence protein driven by the promoter of P450 side-chain cleaving enzyme gene (CYP11A), a steroidogenic cell-specific gene, were used to detect steroidogenic cell production in vitro. During in vitro differentiation, green fluorescence protein-positive cells, which had characteristics similar to those of Leydig cells, were found. Stable transfection of murine MSCs with a transcription factor, steroidogenic factor-1, followed by treatment with cAMP almost recapitulated the properties of Leydig cells, including the production of testosterone. Transfection of human MSCs with steroidogenic factor-1 also led to their conversion to steroidogenic cells, but they appeared to be glucocorticoid- rather than testosterone-producing cells. These results indicate that MSCs represent a useful source of stem cells for producing steroidogenic cells that may provide basis for their use in cell and gene therapy.

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Human zinc-fingers and homeoboxes (ZHX) 1, a transcriptional repressor, was originally cloned as an interacting protein with the activation domain of the A subunit of nuclear factor-Y (NF-YA). As the first step in investigating the mechanism by which ZHX1 acts as a transcriptional repressor, we conducted a search of ZHX1-interacting proteins using a yeast two-hybrid system. Nuclear proteins such as ZHX1, transcriptional co-factors and DNA-binding proteins, zyxin, androgen-induced aldose reductase and eleven-nineteen lysine-rich leukaemia gene, as well as some unknown proteins, were cloned. Molecular cloning and determination of the nucleotide sequence of the full-length cDNA encoding a novel protein revealed that it consists of 956 amino acid residues and contains two zinc-finger (Znf) motifs and five homeodomains (HDs) as well as ZHX1. We concluded that the protein forms the ZHX family with ZHX1 and denoted it ZHX3. ZHX3 not only dimerizes with both ZHX1 and ZHX3, but also interacts with the activation domain of the NF-YA. Further analysis revealed that ZHX3 is a ubiquitous transcriptional repressor that is localized in nuclei and functions as a dimer. Lastly, the dimerization domain, the interaction domain with NF-YA, and the repressor domain are mapped to a region including the HD1 region, and two nuclear localization signals are mapped to the N-terminal through Znf1 and the HD2 region, respectively.