Selective binding of steroid hormone receptors to octamer transcription factors determines transcriptional synergism at the mouse mammary tumor virus promoter.

Transcriptional synergism between glucocorticoid receptor (GR) and octamer transcription factors 1 and 2 (Oct-1 and Oct-2) in the induction of mouse mammary tumor virus (MMTV) transcription has been proposed to be mediated through directed recruitment of the octamer factors to their binding sites in the viral long terminal repeat. This recruitment correlates with direct binding between the GR DNA binding domain and the POU domain of the octamer factors. In present study, in vitro experiments identified several nuclear hormone receptors to have the potential to bind to the POU domains of Oct-1 and Oct-2 through their DNA binding domains, suggesting that POU domain binding may be a property shared by many nuclear hormone receptors.

Involvement of the sonic hedgehog, patched 1 and bmp2 genes in patterning of the zebrafish dermal fin rays.

The signaling molecule encoded by Sonic hedgehog (shh) participates in the patterning of several embryonic structures including limbs. During early fin development in zebrafish, a subset of cells in the posterior margin of pectoral fin buds express shh. We have shown that regulation of shh in pectoral fin buds is consistent with a role in mediating the activity of a structure analogous to the zone of polarizing activity (ZPA) (Akimenko and Ekker (1995) Dev.

Sequence-specific binding of Ku autoantigen to single-stranded DNA.

Glucocorticoid-induced transcription of mouse mammary tumor virus is repressed by Ku antigen/DNA-dependent protein kinase (DNA-PK) through a DNA sequence element (NRE1) in the viral long terminal repeat. Nuclear factors binding to the separated single strands of NRE1 have been identified that may also be important for transcriptional regulation through this element. We report the separation of the upper-stranded NRE1 binding activity in Jurkat T cell nuclear extracts into two components.

Stable transfer of zebrafish chromosome segments into mouse cells.

Whole-cell fusion between zebrafish fibroblast-like ZF4 cells and mouse B78 melanoma cells resulted in hybrids containing one or a few zebrafish chromosome segments in a murine chromosomal background. Fluorescence in situ hybridization to hybrid cell metaphases with a zebrafish genomic DNA probe revealed that many hybrids contained zebrafish chromosome segments that were either inserted or translocated to a mouse chromosome, whereas other hybrids contained zebrafish chromosomes with no evidence of insertion or translocation. We have assigned hybrids to 17 linkage groups of the genetic map of the zebrafish genome.

Sequence-specific DNA binding by Ku autoantigen and its effects on transcription.

DNA-dependent protein kinase (DNA-PK) has been implicated in several nuclear processes including transcription, DNA replication, double-stranded DNA break repair, and V(D)J recombination. Linkage of kinase and substrate on DNA in cis is required for efficient phosphorylation. Recruitment of DNA-PK to DNA is by Ku autoantigen, a DNA-end-binding protein required for DNA-PK catalytic activity.

Nuclear factor binding to a DNA sequence element that represses MMTV transcription induces a structural transition and leads to the contact of single-stranded binding proteins with DNA.

NRE1 is a DNA sequence element in the long terminal repeat of mouse mammary tumor virus through which viral transcription is repressed. In addition to double-stranded DNA binding, both upper- and lower-stranded NRE1 binding activities occur in nuclear extracts. All three binding activities appear to be important for transcriptional effects.

Anterior duplication of the Sonic hedgehog expression pattern in the pectoral fin buds of zebrafish treated with retinoic acid.

The Sonic hedgehog gene has been identified as a candidate for the signal mediating the function of the zone of polarizing activity (ZPA) during limb development in tetrapods. To better understand the early steps of development of paired fin buds in fish, we have analyzed the regulation of the zebrafish Sonic hedgehog gene (shh/vhh-1) in response to retinoic acid. Systemic administration of retinoic acid (RA) to zebrafish embryos during the initial stages of pectoral fin bud development resulted in the induction of ectopic expression of shh/vhh-1 on the anterior margin of the bud under the apical ectodermal ridge and in abnormal pectoral fin bud morphology.

5 alpha-Reductase type 1 is localized to the outer nuclear membrane.

The subcellular distribution of the two isozymes of 5 alpha-reductase has been controversial. To resolve this issue which could provide clues about the respective functions of the two isozymes, two antisera were generated, one which was specific for the Type 1 5 alpha-reductase and one which recognized both isozymes. In COS cells transfected separately with the Type 1 or Type 2 cDNA, both isozymes were detected on Western blots at an M(r) of 26,000.

Multi-strand binding of nuclear factors to a repressor of mouse mammary tumor virus transcription can be distinguished kinetically.

NRE1 is a DNA sequence element in the long terminal repeat of mouse mammary tumor virus that represses viral transcription in mature T cells. In addition to double-stranded binding activity, factors in Jurkat T cell nuclear extracts bind specifically to each of the two single-strands of NRE1. Here we show that binding to the three forms of NRE1 can be distinguished kinetically.

Differential induction of four msx homeobox genes during fin development and regeneration in zebrafish.

To study the genetic regulation of growth control and pattern formation during fin development and regeneration, we have analysed the expression of four homeobox genes, msxA, msxB, msxC and msxD in zebrafish fins. The median fin fold, which gives rise to the unpaired fins, expresses these four msx genes during development. Transcripts of the genes are also present in cells of the presumptive pectoral fin buds.

Extracellular matrix components secreted by microvascular endothelial cells stimulate preadipocyte differentiation in vitro.

Paracrine interaction between preadipocytes and microvascular endothelial cells may play a role in the regulation of adipose tissue growth. We report here a study of the effect of extracellular matrix factors secreted by microvascular endothelial cells, derived from adipose tissue, on preadipocyte differentiation in primary culture. Extracellular matrix components (EC) were prepared by differential centrifugation of medium conditioned by microvascular endothelial cells (CM).

Combinatorial expression of three zebrafish genes related to distal-less: part of a homeobox gene code for the head.

We describe analysis of zebrafish distal-less-related homeobox genes that may serve as specifiers of positional information in anterior regions of the CNS and in peripheral structures. We isolated three zebrafish genes, dlx2, dlx3, and dlx4, by screening embryonic cDNA libraries. Comparisons of the predicted sequences of the Dlx2, Dlx3, and Dlx4 proteins with distal-less proteins from other species suggest that vertebrate distal-less genes can be divided into four orthologous groups.

Repression of mouse mammary tumor virus transcription by a transcription factor complex. Binding of individual components to separated DNA strands.

Expression of mouse mammary tumor virus in T lymphocytes appears to be required for accession of horizontally transmitted virus to the mammary gland. Further, deletions in the long terminal repeat which relax constraints on viral transcription promote T cell lymphoma. We have identified a polypurine transcriptional repressor element (NRE1) that is deleted from viruses that induce T cell lymphoma.

Extraction of DNA-binding proteins from cryogenically preserved cells.

We demonstrate that DNA-binding protein extracts can be effectively prepared directly from tissue culture cells preserved under liquid nitrogen without returning the cells to culture. We prepared DNA-binding protein extracts directly upon thawing of T47D, Jurkat and CAC-L153S cell lines after storage in liquid nitrogen for periods of up to one year. Our results show that DNA binding of a repressor of mouse mammary tumor virus transcription in these extracts is indistinguishable from binding activity in similar extracts prepared from cells maintained in culture.

Identification of binding proteins for nuclear localization signals of the glucocorticoid and thyroid hormone receptors.

Nuclear entry of proteins the size of the glucocorticoid and thyroid hormone receptors appears to be mediated by an interaction of nuclear localization signals (NLSs) within the proteins and specific NLS-binding proteins. NLSs have been identified in the hinge region of both receptors. We have identified the cellular binding proteins of the glucocorticoid receptor NLS and the thyroid hormone receptor NLS after cross-linking of radiolabeled signal peptides to subcellular fractions.

Extracellular matrix composition and resilience: two parameters that influence the in vitro migration and morphology of rat inner cell mass-derived cells.

Parietal endodermal (PE) migration along rat trophectodermal (TE) cells coincides with the deposition of Reichert's membrane between these two cell layers. In this study, we compared the influences of fibronectin and laminin, two components of Reichert's membrane, on the migration and replication of PE-like cells from cultured rat inner cell masses (ICMs). We also explored the role of substrate nondeformability by comparing cell translocation on gels versus coatings of Matrigel (a tumor cell-derived basement membrane preparation) or of collagen.

Thyroid hormones precociously increase nerve growth factor gene expression in the submandibular gland of neonatal mice.

The developmental regulation of the expression of nerve growth factor (NGF) was studied in the mouse submandibular gland (SMG). Having demonstrated that, in the neonatal mouse, maturation of the SMG can be accelerated by treatment with thyroid hormones, with the resulting induction in SMG content of NGF, studies were undertaken to further examine the locus of thyroid hormone action. Because of the sexual dimorphism of the SMG, both male and female neonatal mice were used.

Thyroid hormone and androgen regulation of nerve growth factor gene expression in the mouse submandibular gland.

The nerve growth factor (NGF) content of the mouse submandibular gland (SMG) is under hormonal control and is modulated by both thyroid hormones (TH) and androgens. The sexual dimorphism of the gland is well documented. In the adult male mouse, the SMG contains 10 times more NGF compared to the female.

Levels of cholesterol and phospholipids in freshly ejaculated sperm and Percoll-gradient-pelletted sperm from fertile and unexplained infertile men.

Cholesterol and phospholipid levels were determined in individual sperm samples obtained from 20 fertile and 20 unexplained infertile men. The determination was performed on both washed freshly ejaculated sperm and Percoll-gradient-pelletted sperm. Although sperm cholesterol levels in unexplained infertile patients were significantly lower, i.

Immunolocalization of fibronectin and laminin within rat blastocysts cultured under serum-free conditions.

Blastocysts (approximately 50 per female) were collected on Day 5 of gestation from immature Sprague-Dawley rats superovulated using FSH/hCG-loaded mini-osmotic pumps and a single injection of the LHRH analogue, des-gly10 (D-ala6)-LHRH-ethylamide. The cytoplasmic distribution of fibronectin and laminin was determined by immunofluorescence within these blastocysts, either immediately following their isolation or after they had been cultured in serum-free medium for 48-96 h (to allow trophectodermal cell attachment and outgrowth). In addition, inner cell masses (ICMs; isolated by immunosurgery) were cultured under serum-free conditions and immunofluorescently stained for the presence of the two adhesive glycoproteins.

Secretion of fibronectin by rat granulosa cells occurs primarily during early follicular development.

Rats were pretreated with oestradiol-17 beta or PMSG, treatments producing mainly preantral and antral follicles respectively. The granulosa cells from these two treatments, E2- and PMSG-cells respectively, were cultured for 2 successive 24-h periods. Basal progesterone secretion, stimulated 3- to 5-fold by FSH, was almost 8-fold higher by PMSG-cells than by E2-cells at 24 and 48 h.

Characterization of high affinity and low affinity dexamethasone binding sites on male rat liver nuclear envelopes.

Steroids must traverse the nuclear envelope before exerting their action at the chromatin. However, few studies have been done to elucidate the mechanism by which steroids traverse this membrane barrier. As first steps towards investigating the mechanism, we have characterized the binding sites for dexamethasone on male rat liver nuclear envelopes.

Inhibitory effects of gossypol analogs on human sperm motility.

Aqueous-soluble gossypol Schiff's bases, SP562: bis-8,8'-[(N-(2-(dimethylamino)ethyl]-iminomethylene]- [1,1',6,6',7,7'-hexahydroxy-5,5'-diisopropyl-3,3'-dimethyl-2,2- binaphthalene dihydrochloride; SP563: bis-8,8'-[(N-(2-(diethylamino)ethyl]-iminomethylene]-1,1',6,6',7,7 '- hexahydroxy-5,5'-diisopropyl-3,3'-dimethyl-2,2'-binaphthalene++ + dihydrochloride; and SP564: bis-8,8'-[(N-(2-(diethylamino)propyl]-iminomethylene]- 1,1',6,6',7,7'-hexahydroxy-5,5'-diisopropyl-3,3'-dimethyl-2,2'- binaphthalene dihydrochloride, were investigated for their effects on human sperm motility. SP564, which has the longest alkyl substituent of the Schiff's base, appeared to exert the greatest inhibitory effects on human sperm motility. These inhibitory effects were even greater than those caused by (+/-)gossypol acetic acid at the same concentration.