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N Walther

Publications and source records attributed to N Walther.

31 records · Page 2Linked to original sources
PubMed1994

Two orphan receptors binding to a common site are involved in the regulation of the oxytocin gene in the bovine ovary.

The peptide hormone oxytocin is highly expressed in the hypothalamus within only a small number of magnocellular neurons. However, it is also expressed in a much larger number of cells in the bovine corpus luteum at high levels in an estrous cycle-dependent manner. By using nuclear extracts from this tissue for in vitro binding studies, two protein complexes have been shown to bind to a common site in the bovine oxytocin promoter. One of these proteins has been identified as the bovine homologue of the chicken ovalbumin upstream promoter transcription factor (COUP-TF). The second protein is here characterized as the bovine homologue of a tissue-specific transcription factor, steroidogenic factor 1 (SF-1). The relative expression of these two factors during luteal development correlates with the level of luteal oxytocin gene expression, with SF-1 being the factor binding to the promoter of the oxytocin gene when this promoter is activated. Cotransfection experiments using the murine testicular cell line TM4 show that SF-1 can stimulate the expression of a transfected oxytocin gene, suggesting that SF-1 may be involved in upregulation of the oxytocin gene in vivo, possibly by transducing a stimulatory signal to the RNA polymerase.

Animals↗
PubMed1994

Structure of the alpha-inhibin gene and its regulation in the ruminant gonad: inverse relationship to oxytocin gene expression.

The genes for the alpha subunit of inhibin and for the nonapeptide hormone oxytocin are both expressed in the granulosa cells of the ruminant follicle as well as in the Sertoli cells of the ruminant testis. Northern hybridization of mRNA from both ovary and testis indicate that in both gonads the expression of the two genes is inversely regulated. In the luteinizing granulosa cells, in vitro as in vivo, the alpha-inhibin gene is down-regulated when the oxytocin gene is up-regulated. In the Sertoli cells of the bull and sheep testis, the situation is similar, with the alpha-inhibin gene being up-regulated in the prepubertal gonad and down-regulated concomitantly with an up-regulation of the oxytocin gene in early puberty. The gene for the bovine alpha-inhibin subunit was cloned and characterized. Assessment of transcriptional initiation by primer extension and ribonuclease protection assays showed that several different sites were used in both granulosa cells and testis. Transient transfection of primary bovine granulosa cells with alpha-inhibin/luciferase gene constructs indicated that a major promoter element resided in the region -178 to -245 respective to the methionine start codon of translation, a region that contains a cAMP response element. The ability of forskolin to up-regulate the transcription of transfected gene constructs also depended on the integrity of this region. In contrast, transfection of TM4 cells led to transcriptional initiation from an unusual site in the alpha-inhibin gene and to a lack of forskolin regulation. Comparison of the alpha-inhibin and oxytocin genes indicates that although both can be up-regulated by FSH or by forskolin within the same cells, different mechanisms of signal transduction are involved to explain the temporal differences in expression. Together the results indicate that a differentiation step occurring in Sertoli cells at early puberty and in granulosa cells at luteinization involves comparable regulation of genes through the sequential action of different cAMP-linked transcription factors.

Animals↗
PubMed1994

The orphan receptor SF-1 binds to the COUP-like element in the promoter of the actively transcribed oxytocin gene.

The factors regulating oxytocin expression have not yet been characterized in detail. Although direct control by ligand-dependent binding of nuclear hormone receptors to the oxytocin promoter has been suggested, the presence of these receptors in the tissues expressing oxytocin has not been shown consistently. We have analyzed nuclear proteins from preovulatory bovine granulosa cells and corpus luteum, tissues actively expressing the oxytocin gene, and describe here the characterization of a tissue-specific factor binding to the conserved element in the oxytocin promoter that has been implicated in the control of this gene. This factor is the bovine homologue of SF-1, an orphan receptor expressed specifically in steroidogenic tissues. It is suggested that SF-1 binds to the oxytocin promoter in vivo and is involved in control of oxytocin gene expression possibly by interaction with other factors.

Animals↗
PubMed1994

Over-expression of oxytocin in the testes of a transgenic mouse model.

The bovine oxytocin gene has been expressed in the testes of two independent transgenic mouse lines. Hybridization and RNase protection analysis showed that the oxytocin transgene was transcribed from the normal functional promoter in the Sertoli cells of the seminiferous tubules in a developmentally regulated manner. Immunohistochemistry indicated that both oxytocin and neurophysin epitopes were expressed together in the Sertoli cells at stages I-V and X-XII of the cycle of the seminiferous epithelium. Furthermore, analysis with high-performance liquid chromatography showed that there was a tenfold increase in the amount of amidated oxytocin present in testicular extracts from the transgenic mice. However, there appeared to be no detectable effect of this overproduction of hormone on testicular morphology or fertility parameters. A significant decrease by 50% was detected only in the levels of intratesticular testosterone and dihydrotestosterone. The results point to a local paracrine role for oxytocin in the modulation of Leydig cell function.

Animals↗
PubMed1992

The COUP transcription factor (COUP-TF) is directly involved in the regulation of oxytocin gene expression in luteinizing bovine granulosa cells.

Competition with specific oligonucleotides in DNA-binding experiments, polyacrylamide gel electrophoresis, and recognition by specific antibodies have identified the ubiquitous transcription factor COUP as one of the nuclear proteins binding to the promoter region of the bovine oxytocin gene in endogenously expressing bovine granulosa cells. PCR cloning of partial cDNA sequences for bovine COUP-TF I and II and development of RNase protection assays demonstrated the up-regulation of COUP-TF in bovine granulosa cells and corpus luteum under conditions where the oxytocin gene is switched off. These experimental results from in vitro and in vivo studies point to the direct involvement of COUP-TF in oxytocin gene down-regulation during luteinization of bovine granulosa cells.

Animals↗
PubMed1991

Comparison of the estrogen responsiveness of the rat and bovine oxytocin gene promoters.

DNA sequences in the 5'-flanking region of rat and bovine oxytocin genes were examined for their capacity to confer estrogen responsiveness to their homologous promoters. In contrast to the 5'-flanking region of the rat oxytocin gene, upstream promoter sequences up to 3200 bp of the bovine gene linked to the chloramphenicol acetyltransferase (CAT) reporter gene which were transfected in estrogen receptor expressing MCF-7 cells did not respond to estrogen. Testing 5'-deletion mutants of the rat upstream region linked to the luciferase gene in P19 embryocarcinoma cells co-transfected with an estrogen receptor expression plasmid showed that two regions each associated with approximately 15-fold stimulation of promoter activity were located between nucleotides -172 and -149 and between -148 and +16 in the rat gene. The former region contains the imperfect palindrome GGTGACCTTGACC which differs in one nucleotide from the estrogen response element (ERE) consensus. It is concluded that the corresponding motive CATAACCTTGACC of the bovine gene is not a functional ERE. Thus, the estrogen responsiveness of oxytocin genes is species-dependent.

Animals↗
PubMed1986

Nucleotide sequence of the CMII v-myc allele.

The entire nucleotide sequence of the transduced v-myc allele in the genome of avian oncogenic retrovirus CMII was determined. The CMII v-myc and the chicken c-myc alleles differ in their shared coding sequences by a single nucleotide substitution causing a glutamic acid/alanine exchange in the predicted sequences of the corresponding protein products. This mutation has not been found in the transduced v-myc alleles of avian oncogenic retroviruses MC29, MH2, and OK10. We conclude that no specific, if any, missense mutation of the c-myc coding sequence is necessary for oncogenic activation upon transduction of the cellular gene.

Alleles↗
PubMed1985

Molecular and biological properties of MH2D12, a spontaneous mil deletion mutant of avian oncovirus MH2.

Avian oncogenic retrovirus MH2 carries two cell-derived oncogenes, v-mil and v-myc. From an infectious stock of MH2 a spontaneous deletion mutant, MH2D12, that has lost most of the v-mil gene but has retained a complete and functional v-myc gene, has been isolated. Nonproducer quail embryo cells transformed by MH2D12 in the absence of helper virus contain two virus-specific proteins: a gag-related protein of 53,000 Da (p53gag), and a v-myc gene product of 59,000/61,000 Da (p59/61v-myc) indistinguishable from the v-myc protein encoded by MH2. MH2D12 viral RNA contains all T1-oligonucleotides specific for the MH2 v-myc gene but none of those characteristic for the v-mil gene. The genetic structure of molecularly cloned proviral DNA of MH2D12 was revealed by restriction mapping, blot hybridization, heteroduplex analysis, and nucleotide sequencing. The MH2D12 provirus is homologous to the MH2 genome but has suffered a deletion of 1271 nucleotides from the central region encompassing the 3' end of delta gag and all of v-mil except the very 3' 31 nucleotides directly adjacent to the v-myc gene. A nine-nucleotide overlap of homology to gag or mil at the delta gag/delta mil junction suggests that recombination between homologous sequence elements of the delta gag and v-mil domains of MH2 was involved in the genesis of MH2D12. The nucleotide sequence analysis predicts that the carboxyterminal 17 amino acids of p53gag are encoded by the residual v-mil sequences and by intron-derived v-myc sequences. Transformation of quail embryo cells by MH2D12 can be assayed by focus and colony formation of transformed cells. This indicates that the v-mil gene is not essential for these activities. However, size and morphology of foci and colonies, and cellular morphology of cultured MH2D12-transformed cell lines can easily be distinguished from those observed in cell transformation by MH2 and resemble more those seen in cell transformation by viruses containing the myc oncogene only.

Amino Acid Sequence↗
PubMed1985

Molecular cloning of proviral DNA and structural analysis of the transduced myc oncogene of avian oncovirus CMII.

Molecularly cloned proviral DNA of avian oncogenic retrovirus CMII was isolated by screening a genomic library of a CMII-transformed quail cell line with a myc-specific probe. On a 10.4-kilobase EcoRI fragment, the cloned DNA contained 4.4 kilobases of CMII proviral sequences extending from the 5' long terminal repeat to the EcoRI site within the partial (delta) complement of the env gene. The gene order of CMII proviral DNA is 5'-delta gag-v-myc-delta pol-delta env-3'. All three structural genes are partially deleted: the gag gene at the 3' end, the env gene at the 5' end, and the pol gene at both ends. The delta gag (0.83 kilobases)-v-myc (1.50 kilobases) sequences encode the p90gag-myc transforming protein of CMII. In comparison with the p110gag-myc protein of acute leukemia virus MC29, p90gag-myc lacks amino acids corresponding to additional 516 bases of gag sequences and 12 bases of 5' v-myc sequences present in the MC29 genome. Nucleotide sequence analysis of CMII proviral DNA at the delta gag-v-myc and the v-myc-delta pol junctions revealed significant homologies between avian retroviral structural genes and the cellular oncogene c-myc precisely at the positions corresponding to the gene junctions in CMII. Furthermore, the delta gag-v-myc junction in CMII corresponds to sequence elements in gag and C-myc that are possible splicing signals. The data suggest that transduction of cellular oncogenes may involve RNA splicing and recombination with homologous sequences on retroviral vectors. Different sequence elements of both the retroviral vectors and the c-myc gene recombined during genesis of highly oncogenic retroviruses CMII, MC29, or MH2.

Animals↗
PubMedDate not supplied

Ultrastructural changes of venous autologous bypass grafts in rabbits: correlation of patency and development.

Electron microscopic techniques were used to correlate the patency of venous autografts in rabbits with ultrastructural changes within a time range from 5 days to 6 months p.o. Early degenerative changes of the grafts include endothelial desquamation followed by fibrin deposition or platelet adhesion, mural edema and extensive medial and adventitial degeneration. The regeneration of the grafts is due to an early adventitial vascularization (10 days p.o.), which enables the surviving smooth muscle cells and fibrocytes to proliferate and to develop an arterial-like vessel. The organization of the 6-month-old graft is comparable to the carotid artery of the animal, but shows, in contrast, many features of a muscular artery.

Animals↗