Systematic isolation of peptide signal molecules regulating development in hydra: LWamide and PW families.

To isolate new peptide signal molecules involved in regulating developmental processes in hydra, a novel screening project was developed. Peptides extracted from the tissue of Hydra magnipapillata were systematically purified to homogeneity using HPLC. A fraction of each purified peptide was examined by differential display-PCR for its ability to affect gene expression in hydra. Another fraction was used to determine the tentative structure using an amino acid sequence analyzer and/or a mass spectrometer. Based on the results, peptides of potential interest were selected for chemical synthesis, followed by confirmation of the identity of the synthetic with the native peptides using HPLC. Using this approach, 286 peptides have been isolated, tentative amino acid sequences have been determined for 95 of them, and 19 synthetic peptides identical to native ones were produced. The 19 synthetic peptides were active in a variety of biological tests. For example, Hym-54 stimulated muscle contraction in adult polyps of hydra and sea anemone, Anthopleura fuscoviridis, and induced metamorphosis of planula, the larval stage, into polyps in a marine hydrozoan species, Hydractinia serrata. Another peptide, Hym-33H, inhibited nerve cell differentiation in hydra and induced tissue contraction in planula of Hydractinia serrata. The evidence obtained so far suggests that hydra contains a large number (>350) of peptide signal molecules involved in regulating developmental or other processes in cnidaria. These peptides can be isolated and their functions examined systematically with the new approach developed in this study.

Interactions of progesterone-dependent endometrial nuclear factors with the promoter of the rabbit uteroglobin gene.

We have studied the in vitro interactions of a promoter fragment of the rabbit uteroglobin (UG) gene with endometrial nuclear factors from either control rabbits or progesterone-treated animals, a treatment that induces the transcription of the gene in the endometrium. Nuclear factors from liver, which does not express UG, were also compared. Using DNase I footprinting, several protected zones were observed on the promoter; some of these were common to all three nuclear extracts, whereas others seemed to be specific to progesterone treated endometrium. One of these footprints was over the TATA box region. A 28-bp synthetic oligonucleotide encompassing the sequence of that region was used as a probe in electrophoretic mobility shift assays (EMSA) and UV-crosslinking assays. In EMSA experiments, endometrial nuclear extracts, but not liver extracts, generated a major retarded complex that was strongly increased following progesterone stimulation. Competition experiments with unlabeled mutated versions of the probe showed that sequences 3' downstream from the TATA box (but not this motif) were responsible for the formation of the complex. UV-crosslinking experiments indicated that the probe interacted with two progesterone-dependent nuclear proteins of 55 and 60 kDa, different from the TATA box-binding protein. The results suggest that progesterone induction of the UG gene in the endometrium might be mediated by both general and tissue-specific nuclear factors.

Binding of retinoids to uteroglobin.

Uteroglobin, a progesterone-binding secretory protein, was shown to bind retinoic acid and retinol in a non-saturable manner, at least up to concentrations of retinoids of 20 microM. Binding is increased about 10-fold by previous reduction of uteroglobin with 10 mM dithiothreitol and it is not affected by previous saturation of the progesterone binding site, suggesting different binding sites for the steroid and the retinoids. The results are discussed in relation to a possible physiological role for this protein.

Isolation and characterization of two isoforms of a retinoic acid-binding protein from rabbit epididymal secretions.

Two isoforms of a retinoic acid-binding protein have been purified from rabbit epididymal secretions using a combination of gel filtration and ion-exchange chromatography. The two polypeptides (EP21a and b) present similar molecular mass (21 kDa), under native or denaturing conditions and have very similar amino-acid composition and tryptic peptide maps but differ in net charge. Both isoforms are glycosilated though to a different extent (9.2% and 6.3% of carbohydrate content) and are major components of the epididymal fluid. Binding of retinoic acid to EP21s appears to be specific, since they do not bind retinol, but is non-saturable. EP21s seem to present some similarity to two retinoic acid-binding proteins from rat epididymal secretions (site of biosynthesis, androgen dependence, ligand specificity and association to the spermatozoa) but differ from the rat proteins in amino-acid composition and glycosilation.

Hydra tropomyosin TROP1 is expressed in head-specific epithelial cells and is a major component of the cytoskeletal structure that anchors nematocytes.

A cDNA clone encoding a 253 amino acid tropomyosin was isolated from Hydra in a differential screen for head-specific genes. The Hydra tropomyosin gene, designated trop1, is a single copy gene, lacks introns and is strongly expressed in tentacle-specific epithelial cells. Analysis of protein synthesis in head and gastric tissue indicated a high rate of tropomyosin synthesis in head tissue. Immunolocalization of tropomyosin in tentacle tissue revealed a cushion-like tropomyosin-containing structure within battery cells at the base of nematocytes. The structure appears to form part of the cytoskeletal anchor for nematocytes. Tropomyosin cushions were also observed in epithelial cells along the body column, which contain mounted stenotele nematocytes.

Tissue-specific and hormonally regulated expression of the puromycin N-acetyltransferase-encoding gene under control of the rabbit uteroglobin promoter in transgenic mice.

Transgenic mice bearing two fragments of the rabbit uteroglobin 5'-flanking region fused to the new reporter gene (pac) encoding puromycin N-acetyltransferase (PAC) showed a different pattern of expression. Transgenic lines (C0.4) harboring a 404-bp fragment (-396/+8) had a uterus-specific expression slightly inducible by estrogen, lacking detectable expression in other tissues where the uteroglobin-encoding gene is naturally expressed in rabbit. Transgenic lines (C3.2) bearing a longer fragment of 3.2-kb (-3254/+8) showed hormonally regulated expression in the uterus and the male genital tract, and detectable expression in the lung. In addition, the nonstimulated uterine expression of the transgene was higher in C0.4 lines than in C3.2 lines. It could be concluded that all sequences required for uterus-specific expression should be present within the 404-bp fragment, and that other upstream (-396) sequences are responsible for expression in the lung and male genital tract, as well as for a possible down modulation of expression in the uterus.

Purified uteroglobin lacks anti-proteinase activity.

Rabbit uteroglobin was purified from both uterine fluids and lung lavages by a combination of gel filtration and ion-exchange column chromatography. Anti-trypsin and anti-papain activities were measured in the fractions of the eluates. Anti-proteinase activities were detected in minor contaminants eluting close to the uteroglobin peak but the protein itself was devoid of anti-proteinase activity. Ion-exchange-purified uteroglobin also lacked inhibitory activity of elastase, chymotrypsin or subtilisin. The presence of contaminants could explain the anti-proteinase activity reported occasionally for uteroglobin.

Localization of an estrogen receptor binding site near the promoter of the uteroglobin gene.

By means of a DNA-cellulose competitive binding assay, we have studied the interaction of the estrogen receptor with genomic fragments of the estrogen responsive rabbit uteroglobin gene. The fragments spanned from 3255 bp upstream to 1754 bp downstream of the initiation site. Only a fragment (-396/+8) showed strong affinity for the receptor. Within this fragment a unique palindromic sequence (GGTCAccaTGCCC) was found which is very similar to the canonical consensus sequence for the estrogen receptor. A synthetic oligonucleotide of that structure specifically competed for the binding of the receptor to DNA-cellulose.

In vivo estrogenic and antiestrogenic activity of phenolphthalein and derivative compounds.

The estrogenic activity of phenolphthalein and other related triphenylmethane dyes was evaluated in vivo in the immature rat uterus. Phenolphthalein behaved as a partial agonist of estradiol in stimulating the growth of rat uterus. Other specific estrogenic effects of the dye included an increase of the uterine DNA content, histological changes and induction of estrogen-modulated secretory proteins. The progressive introduction of side chains in the triphenylmethane skeleton concomitantly decreased the estrogenic activity. Triphenylmethanes competed with [3H]estradiol for the binding to the estrogen receptor in vitro, the relative binding affinity being correlated with the estrogenic potency observed in vivo. Phenolphthalein also showed antiestrogenic activity that could be overcome by increasing the dose of estradiol.

Evidence for the identity of anti-proteinase pulmonary protein CCSP and uteroglobin.

Purified Clara cell secretory protein (CCSP) from rabbit lung was analyzed by SDS gel electrophoresis, and immunoblotting with a specific anti-uteroglobin antibody as well as for its ability to bind [3H]progesterone. The results obtained indicate that proteins CCSP and uteroglobin are identical.

Testosterone induces the expression of the uteroglobin gene in rabbit epididymis.

Uteroglobin was characterized in the rabbit epididymis by radioimmunoassay and electrophoretic determinations, as well as by analysis of its mRNA by means of 'Northern blot' and nuclease-S1 mapping. Treatment of sexually immature rabbits with testosterone during 5 days increased up to 8-fold the concentrations of both uteroglobin and its mRNA in the epididymis. The amounts of beta-tubulin mRNA, measured as reference, remained unchanged after the hormonal treatment. The synthesis of uteroglobin occurred mainly in the middle region of the epididymis, progressively decreasing toward the distal part of the organ. Uteroglobin was not detected in the testis by radioimmunoassay. The results are discussed in relation to a possible role of uteroglobin in the reproductive functions.

Ultrastructural and kinetic studies on uteroglobin secretion in the uterus and oviduct of the pseudopregnant rabbit.

The intracellular localization of uteroglobin, a progesterone-induced protein, was studied in uterus and oviduct by means of immunoelectron microscopy with the protein A-gold technique. In the uterus, uteroglobin was synthesized in the columnar epithelium of the endometrium where most of the cells were immunoreactive. The protein was localized mainly in small secretory granules which were seen in the process of release into the uterine lumen. The luminal microvilli were also heavily stained. In the oviduct, the secretory cells contained large immunoreactive granules at the apical zone, some of which were observed while discharging into the lumen. Within these secretory granules, uteroglobin accumulated particularly in lens-shaped patches at the periphery of the granules. In vitro kinetic studies on the secretion of newly synthesized uteroglobin indicated that the ability to store uteroglobin is greater in the oviduct than in the uterus; however, the rate of uteroglobin secretion is greater in the uterus than in the oviduct. Thus, there appears to be a good correlation between the microscopic and the functional observations.

Nucleotide and derived amino acid sequences of a cDNA coding for pre-uteroglobin from the lung of the hare (Lepus capensis).

An almost full-length cDNA coding for pre-uteroglobin from hare lung was cloned and sequenced. The derived amino acid sequence indicated that hare pre-uteroglobin contained 91 amino acids, including a signal peptide of 21 residues. Comparison of the nucleotide sequence of hare pre-uteroglobin cDNA with that previously reported for the rabbit gene indicated five silent point substitutions and six others leading to amino acid changes in the coding region. The untranslated regions of both pre-uteroglobin mRNAs were very similar. The amino acid changes observed are discussed in relation to the different progesterone-binding abilities of both homologous proteins.

Primary structure of rabbit lung uteroglobin as deduced from the nucleotide sequence of a cDNA.

Double-stranded cDNA was synthesized from partially purified uteroglobin mRNA from rabbit lung. A cDNA coding for lung uteroglobin was then cloned in the plasmid pUC18 and both the nucleotide sequence and the derived amino acid sequence were determined. This allowed us to demonstrate unequivocally that uteroglobins from lung and uterus are identical proteins.

Glucocorticoids induce the expression of the uteroglobin gene in rabbit foetal lung explants cultured in vitro.

In 27-day-old rabbit foetal lung explants cultured in vitro, the synthesis of the protein uteroglobin decreased progressively during several days of culture. Addition of glucocorticoids to the medium progressively induced the synthesis of uteroglobin in a dose-dependent manner without affecting the synthesis of total proteins. The glucocorticoid-mediated induction of uteroglobin appears mainly due to increased amounts of uteroglobin mRNA and seems to be independent of simultaneous cell proliferation, suggesting a glucocorticoid-triggered differentiation of pre-existing cells. The results suggest a major role of glucocorticoids in the developmental regulation of the uteroglobin gene in the lung.

Uteroglobin mRNA from the lung of the hare (Lepus capensis): cell-free translation and properties.

Poly(A)-containing RNA was obtained from the lung of hares (Lepus capensis) and uteroglobin mRNA was characterized by cell-free translation and molecular hybridization to a rabbit uteroglobin cDNA probe. In the cell-free system, hare uteroglobin mRNA was preferentially translated as compared to the whole lung mRNA and it directed the synthesis of a precursor, preuteroglobin, containing about twenty additional amino acids. Hare uteroglobin mRNA was about 40 nucleotides larger than the homologous rabbit one, as analyzed by electrophoresis on agarose gel. Thermal stability of the hybrids formed between rabbit or hare uteroglobin mRNAs and the rabbit cDNA probe indicated differences in the nucleotide sequence of both mRNAs. The levels of uteroglobin mRNA and uteroglobin synthesis in lung are about two-fold higher in hare than in rabbit lung.