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M J Low

Publications and source records attributed to M J Low.

At least 19 recordsLinked to original sources
PubMed1992

Identification of DNA elements cooperatively activating proopiomelanocortin gene expression in the pituitary glands of transgenic mice.

The proopiomelanocortin (POMC) gene is highly expressed in adult mouse pituitary anterior lobe corticotrophs and intermediate lobe melanotrophs. To identify the DNA elements important for this tissue-specific expression, we analyzed a series of POMC reporter genes in transgenic mice. A DNA fragment containing rat POMC 5'-flanking sequences from -323 to -34 recapitulated both basal pituitary cell-specific and hormonally stimulated expression in adult mice when fused to a heterologous thymidine kinase promoter. Developmental onset of the reporter gene expression lagged by 1 day but otherwise closely paralleled the normal ontogeny of murine POMC gene expression, including corticotroph activation at embryonic day 14.5 (E14.5) followed by melanotroph activation at E15.5 to E16.5. AtT20 corticotroph nuclear protein extracts interacted with three specific regions of the functional POMC promoter in DNase I protection assays. The positions of these protected sites were -107 to -160 (site 1), -182 to -218 (site 2), and -249 to -281 (site 3). Individual deletions of these footprinted sites did not alter transgene expression; however, the simultaneous deletion of sites 2 and 3 prevented transgene expression in both corticotrophs and melanotrophs. Electrophoretic mobility shift and Southwestern (DNA-protein) assays demonstrated that multiple AtT20 nuclear proteins bound to these footprinted sites. We conclude that the sequences between -323 and -34 of the rat POMC gene promoter are both necessary and sufficient for correct spatial, temporal, and hormonally regulated expression in the pituitary gland. Our data suggest that the three footprinted sites within the promoter are functionally interchangeable and act in combination with promoter elements between -114 and -34. The inability of any reporter gene construction to dissociate basal and hormonally stimulated expression suggests that these DNA elements are involved in both of these two characteristics of POMC gene expression in vivo.

Animals↗
PubMed1992

Ectopic corticotropin-releasing hormone produced by a transfected cell line chronically activates the pituitary-adrenal axis in transkaryotic rats.

Hypothalamic CRH is the primary positive regulatory factor of the pituitary-adrenal axis. The purpose of our study was to analyze the chronic effects of CRH on the production and secretion of POMC peptides from both the anterior lobe (AL) and neurointermediate lobe (NIL) of the pituitary by mimicking the syndrome of ectopic CRH secretion from neuroendocrine tumors. We first generated stably transfected W2 medullary thyroid carcinoma cell lines with a rat CRH expression vector under the transcriptional control of a cytomegalovirus gene promoter. These cell lines constitutively expressed the foreign gene, accurately processed the encoded prepro-CRH, and secreted biologically active CRH with an estimated potency equivalent to that of synthetic CRH-(1-41)NH2. The cell line designated W2CRH-7 was implanted sc in the syngeneic rat strain WAG/Rij and produced tumors that abundantly secreted CRH into the peripheral circulation. Four weeks postimplantation, W2CRH-7, but not wild-type W2, cells caused significant increases in the AL content of beta-endorphin-like immunoreactivity comparable to that caused by adrenalectomy (ADX). Plasma ACTH and serum beta-endorphin-like immunoreactivity were increased to a greater extent by ADX than by W2CRH-7 cell implantation. The NIL of both male and female rats showed either no change or a tendency to decreased beta-endorphin concentrations with no change in the acetylation or carboxy-shortening profiles judged by cation exchange chromatography in response to the ectopic CRH treatment. Rats of both sexes maintained a profound activation of the pituitary adrenal axis up to 16 weeks postimplantation, with normalized adrenal gland weights 5 times that of controls. The chronic secretion of CRH by W2CRH-7 cells resulted in a complete cessation of body growth in all rats up to the maximum time tested of 16 weeks. The lack of growth was partly ameliorated by concomitant ADX, suggesting an important role for adrenal glucocorticoids in these effects. We conclude that 1) the transplantable W2CRH-7 cell line provides a highly effective and reproducible means of sustained CRH treatment that mimics the syndrome of ectopic CRH expression by neuroendocrine tumors; 2) AL corticotrophs respond to chronic CRH by a sustained production and secretion of POMC peptides, leading to a marked adrenal cortical hyperplasia, with no evidence of biologically significant desensitization; 3) chronic CRH tends to decrease the NIL content of beta-endorphin,with remarkably little effect on posttranslational processing; and 4) the syndrome of chronic ectopic CRH in WAG/Rij rats includes a cessation of body growth at least partly due to products of the adrenal glands.

Adrenalectomy↗
PubMed1992

Pituitary corticotroph hyperplasia in rats implanted with a medullary thyroid carcinoma cell line transfected with a corticotropin-releasing hormone complementary deoxyribonucleic acid expression vector.

CRH stimulates both the synthesis and release of ACTH and other derivatives of POMC by the adenohypophysis. It is uncertain, however, whether it also causes proliferation of corticotrophs. Patients with CRH-producing tumors develop Cushing's syndrome, and some have been reported to have pituitary corticotroph hyperplasia. We now report an animal model that accurately reproduces the human disorder of ectopic production of CRH by a neoplasm. Prolonged CRH secretion by a transplanted medullary thyroid carcinoma cell line stably transfected with a CRH cDNA under transcriptional control of a cytomegalovirus promoter resulted in corticotroph hyperplasia and hypertrophy; the percentage of ACTH-containing cells in animals bearing W2CRH tumors was increased at 9.8 +/- 0.5% (controls, 6.2 +/- 0.3%; W2 implanted tumors, 7.7 +/- 0.4%). Occasional mitotic figures were identified, and the cells were larger, with abundant cytoplasm but generally less intense immunohistochemical staining for ACTH due to relative degranulation compared to controls. Melanotrophs of the intermediate lobe were also increased in number and were larger, with abundant cytoplasm. No corticotroph adenomas were found. Our experiment accurately reproduces the gradually increasing CRH levels in the general circulation produced by a growing tumor, as found in the human ectopic CRH syndrome, and confirms that long term exposure to CRH excess, as produced by a tumor, results in an increased number of corticotrophs in the adenohypophysis.

Adrenocorticotropic Hormone↗
PubMed1992

Gonadotrope-specific expression of the human follicle-stimulating hormone beta-subunit gene in pituitaries of transgenic mice.

The molecular basis for the cell-specific and hormonal regulation of the pituitary gonadotropin beta-subunit genes is unknown. To identify the essential DNA regulatory elements of the human FSH beta (hFSH beta) gene, we introduced a 10-kilobase cloned fragment encompassing the gene into the germline of transgenic mice. We showed by a combination of Northern blot hybridization, RIA, and immunofluorescence histochemistry that the hFSH beta gene was expressed exclusively in mouse pituitary gonadotropes. Human FSH beta was actively secreted, presumably as an interspecies heterodimer, with mouse alpha-subunit, and no free hFSH beta was detected in serum. Male transgenic mice consistently had higher basal pituitary expression and secretion of hFSH beta than the female transgenic mice. Castration combined with testosterone replacement caused a marked reduction in hFSH beta gene expression in transgenic males, but had no effect on the pituitary levels of FSH in normal male mice. These experiments demonstrate that the essential DNA regulatory elements and gonadotrope transcription factors required for cell-specific expression of the FSH beta gene must be conserved between human and mouse. There appear to be important differences in the hormonal regulation of the gene between species, however.

Animals↗
PubMed1992

Fine structure of adrenal cortex in rats harbouring a medullary thyroid carcinoma transfected with a corticotrophin-releasing hormone cDNA expression vector.

We report the light microscopic, transmission and scanning electron microscopic features of the adrenal cortices in rats bearing a medullary thyroid carcinoma cell line transfected with a corticotrophin-releasing hormone (CRH) cDNA expression vector. The animals had elevated CRH, ACTH and corticosterone blood levels, involuted thymuses and markedly enlarged adrenal glands with prominent lipid-depleted cortices and dilated congested capillaries, similar to those of animals treated with ACTH. Using electron microscopy it was found that the enlarged fasciculata and reticularis zones were composed of large, compact cells with abundant smooth endoplasmic reticulum, prominent Golgi complexes, increased number of large mitochondria with focal loss of cristae and cavitation of the internal compartments, numerous lysosomes and prominent elongated microvilli. In addition, small cytoplasmic fragments were seen within the capillary lumina; these structures resembled microvilli that were apparently detached from adrenocortical cells and entered the blood stream via discontinuous endothelium of dilated capillaries. By scanning electron microscopy it was found that the cells had bulging surfaces with scattered pits and numerous long microvilli pointing in different directions. This animal model allows analysis of the effects of protracted CRH excess resembling tumoural CRH-dependent Cushing's syndrome in human patients. Our findings call attention to the role of microvilli in adrenocortical secretion. The increased number and size of microvilli has been thought to lead to an increase in the surface area of adrenocortical cells, thereby facilitating hormone discharge. The detachment of microvilli from adrenocortical cells may represent a form of apocrine secretion and may contribute to hypercorticosteronaemia in CRH excess.

Adrenal Cortex↗
PubMed1990

Somatostatin gene regulation: an overview.

The somatostatinergic system has proven to be one of the best models of neuropeptide biology. Originally characterized as a hypothalamic regulator of growth hormone secretion, somatostatin also regulates the secretion of several other pituitary, pancreatic, and gastrointestinal (GI) hormones including thyrotropin-stimulating hormone, insulin, glucagon, and gastrin. Disorders in somatostatin metabolism have been proposed to contribute to the pathogenesis of Alzheimer's disease, epilepsy, GI motility disorders, and diabetes. On a more basic level, studies of somatostatin action have integrated divergent concepts of intracellular signal transduction. Advances in the understanding of somatostatin biosynthesis have had an impact on areas outside the field of endocrinology by providing new concepts of eukaryotic gene regulation. This report focuses on the transcriptional regulation of somatostatin gene expression. Two aspects of somatostatin gene transcription will be considered--regulated expression by second messengers and tissue-specific basal expression.

Amino Acid Sequence↗
PubMed1990

Pituitary-specific and hormonally regulated gene expression directed by the rat proopiomelanocortin promoter in transgenic mice.

All aspects of POMC biosynthesis exhibit tissue-specific regulation. The single copy gene is highly expressed in anterior lobe (AL) corticotrophs and intermediate lobe (IL) melanotrophs of the pituitary gland and in the arcuate nucleus of the hypothalamus. POMC gene transcription in corticotrophs is induced by hypothalamic CRH and vasopressin and inhibited by adrenal glucocorticoids, while in melanotrophs it is predominantly regulated by beta-adrenergic neural input and dopamine. To identify the rat POMC (rPOMC) gene sequences necessary and sufficient to target expression and hormonal regulation in corticotrophs and melanotrophs, we generated 13 transgenic mice carrying rPOMC fusion genes. The genes consisted of 706 or 480 basepairs of rPOMC 5' flanking sequences ligated to either the E. coli LacZ gene encoding beta-galactosidase or the K1 mutant of the SV40 large T-antigen gene. Overall, half of the transgenic lines had reporter gene expression in their AL and IL in a pattern indistinguishable from ACTH immunohistochemistry. In three of these lines, beta-galactosidase or K1 T-antigen was localized by double immunofluorescence exclusively to ACTH-positive corticotrophs and melanotrophs. Transcriptional regulation of the rPOMC-LacZ fusion gene in response to hormonal manipulation was quantified by a fluorescence assay for beta-galactosidase enzyme activity in pituitary extracts. There was a 15-fold increase in AL enzyme activity after adrenalectomy and a 3-fold increase in IL activity after haloperidol treatment. X-gal histochemistry of pituitaries from hormonally treated mice confirmed the cellular specificity of these effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗
PubMed1989

Cryptic human growth hormone gene sequences direct gonadotroph-specific expression in transgenic mice.

Our laboratory reported previously that chimeric genes encoding either rat somatostatin (SS) or human GH (hGH), but containing the identical mouse metallothionein-I (MT) promoter/enhancer sequences and hGH 3'-flanking sequences, were selectively expressed in the gonadotrophs of transgenic mice. The experiments reported here were designed to identify the DNA sequences responsible for this unexpected cell-specific expression within the anterior pituitary. We produced new transgenic mice expressing fusion genes that tested separately the requirement of the MT or 3'-hGH sequences for gonadotroph expression. A fusion gene that retained the original MT and SS sequences, with a simian virus 40 polyadenylation signal exchanged for the 3'-hGH sequences, no longer directed strong pituitary expression, but was active in the liver. In contrast, a cytomegalovirus promoter/enhancer-SS-hGH fusion gene was expressed at the same high level in the anterior pituitaries of transgenic mice as the originally studied MT-SS-hGH gene. Immunohistochemical analysis indicated that pituitary expression of the cytomegalovirus promoter/enhancer-SS-hGH fusion gene was also restricted to gonadotroph cells in adult mice. These studies indicate that sequences within the 3'-flanking region of the hGH gene can direct expression of chimeric genes to pituitary cells that do not normally produce growth hormone.

Animals↗
PubMed1988

A Moloney MLV-rat somatotropin fusion gene produces biologically active somatotropin in a transgenic pig.

Expression of a Moloney murine leukemia virus (MLV) rat somatotropin fusion gene was examined in a transgenic pig. The fusion gene was integrated in a single site within the genome in a tandem array with approximately eight copies per cell. The integrated in a single site within the genome in a tandem array with approximately eight copies per cell. The integrated MLV-rat somatotropin fusion gene produced high levels of circulating rat somatotropin and resulted in an elevation in the circulating levels of insulin-like growth factor I. Although there was no increase in the rate of growth of the transgenic animal during the rapid growth phase, several phenotypic changes were evident. Skeletal growth was markedly increased and fat deposition was reduced throughout the animal. Blood glucose levels were elevated without ketosis. Northern blot analyses of rat somatotropin RNA revealed that expression of the fusion gene was highest in the spleen, lung, intestine, lymph nodes, and bone marrow. These results show that the MLV promoter can be used to express high levels of biologically active rat somatotropin in transgenic swine.

Animals↗
PubMed1987

Cell-specific processing of preprosomatostatin in cultured neuroendocrine cells.

We have previously found that preprosomatostatin is processed accurately to both somatostatin-14 and somatostatin-28 in pituitary gonadotrophs of transgenic mice. The foreign somatostatin peptides have been shown to enter the regulated secretory pathway of these cells. To determine whether accurate preprosomatostatin processing can occur in any neuroendocrine cell, we introduced preprosomatostatin cDNA expression vectors into several different neuroendocrine cell lines. We found that prosomatostatin was cleaved efficiently to somatostatin-14 and somatostatin-28 in RIN 5F and AtT20 cells, but not in GH4 or PC12 cells. The ability of a particular cell type to process prosomatostatin did not correlate with cellular storage capacity and was independent of the level of biosynthesis of the precursor. These data suggest that prosomatostatin processing requires specific pathways which are present in some neuroendocrine cells, but not in others.

Adrenal Gland Neoplasms↗
PubMed1986

Somatostatin is targeted to the regulated secretory pathway of gonadotrophs in transgenic mice expressing a metallothionein-somatostatin gene.

The pituitaries of transgenic mice that express a metallothionein-somatostatin fusion gene contain high concentrations of somatostatin-14 exclusively in the gonadotrophic cells. The purpose of this study was to determine whether somatostatin expressed from the foreign fusion gene enters the normal secretory pathway within these cells. Immuno-gold labeling of serial thin sections localized somatostatin to the secretory granules of gonadotropin-producing cells. The gonadotroph-specific hypophysiotropic factor, luteinizing hormone-releasing hormone caused a dose-dependent secretion of somatostatin when applied to primary pituitary cultures from these mice. Growth hormone-releasing hormone, thyrotropin-releasing hormone, corticotropin releasing factor, and dopamine did not affect somatostatin secretion. These experiments demonstrate that a neurosecretory peptide encoded by a foreign gene can enter the regulated secretory pathway of pituitary cells from transgenic mice.

Animals↗
PubMed1986

Gonadotroph-specific expression of metallothionein fusion genes in pituitaries of transgenic mice.

Transgenic mice expressing a metallothionein-somatostatin fusion gene contain high concentrations of somatostatin in the anterior pituitary gland, a tissue that does not normally produce somatostatin. Immunoreactive somatostatin within the anterior pituitaries was found exclusively within gonadotrophs. Similarly, a metallothionein-human growth-hormone fusion gene was also expressed selectively in gonadotrophs. It is proposed that sequences common to the two fusion genes are responsible for the gonadotroph-specific expression.

Animals↗
PubMed1986

Cyclic AMP regulates somatostatin mRNA accumulation in primary diencephalic cultures and in transfected fibroblast cells.

Although the factors controlling the secretion of the neuropeptide somatostatin have been extensively studied, little is known about the mechanisms that control somatostatin biosynthesis. Somatostatin secretion is regulated by numerous agents that increase intracellular levels of cAMP. We sought to determine whether cAMP also regulates somatostatin mRNA accumulation. We found that forskolin elicited an increase in somatostatin secretion and mRNA levels in primary cultures of rat diencephalic cells. Another secretagogue, KCl, was as effective as forskolin in causing somatostatin secretion but had no effect on mRNA accumulation. Somatostatin expression in fibroblast cells transfected with the somatostatin gene was also regulated by forskolin. These results demonstrate that somatostatin mRNA accumulation can be regulated through a cAMP-dependent pathway, that this pathway is operative in heterologous cells transfected with the somatostatin gene, and that stimulation of somatostatin secretion and mRNA accumulation can be uncoupled from one another.

Animals↗
PubMed1985

Biosynthesis of rat preprosomatostatin.

The biologically active forms of somatostatin, somatostatin-14 (SS-14) and somatostatin-28 (SS-28) arise by post-translational cleavage of prosomatostatin. Prosomatostatin in turn is derived from a larger precursor, preprosomatostatin. We have previously reported the structure of a complementary DNA molecule encoding rat preprosomatostatin. The nucleotide sequence of this cDNA indicated that SS-14 and SS-28 are located at the carboxy-terminus of a 116 amino acid precursor. At the amino-terminus of the precursor is a hydrophobic region characteristic of a leader or pre-sequence. Sequential Edman degradations of cell-free translation products synthesized in the presence of microsomal membranes indicate that preprosomatostatin is cleaved within the endoplasmic reticulum to form prosomatostatin, a precursor of 92 amino acids. To begin to elucidate the factors which regulate the expression of the rat somatostatin gene, we have determined the sequence of the gene isolated from recombinant bacteriophage libraries. The gene spans 1.2 kilobases in length and is interrupted within the coding sequence of prosomatostatin by a single intron of 630 bases. A variant of the Goldberg-Hogness promotor, TTTAAA, is located 31 bases upstream from the transcriptional start point. A repetitive sequence was identified in the 5' region of the gene within 650 bases of the promoter. The nucleotide sequence of this region reveals an alternating GT sequence 42 bases in length characteristic of DNA with Z-forming potential. Such sequences are thought to influence the expression of other eukaryotic genes.

Amino Acid Sequence↗
PubMed1985

Tissue-specific posttranslational processing of pre-prosomatostatin encoded by a metallothionein-somatostatin fusion gene in transgenic mice.

The somatostatins are neuropeptides of 14 and 28 amino acids that inhibit the release of growth hormone and other hypophyseal and gastrointestinal peptides. These neuropeptides are cleaved posttranslationally from a common precursor, pre-prosomatostatin. We report here the production and processing of pre-prosomatostatin by transgenic mice carrying a metallothionein-somatostatin fusion gene. The most active site of somatostatin production, as determined by hormone concentrations in the tissues, is the anterior pituitary, a tissue that does not normally synthesize somatostatin-like peptides. Anterior pituitary processed pre-prosomatostatin almost exclusively to the two biologically active peptides, somatostatin-14 and somatostatin-28, whereas the liver and kidney synthesized much smaller quantities of predominantly a 6000 dalton somatostatin-like peptide. The growth of the transgenic mice was normal despite high plasma levels of the somatostatin-like peptides. These studies indicate that proteases which cleave prosomatostatin to somatostatin-28 and somatostatin-14 are not specific to tissues that normally express somatostatin.

Animals↗
PubMed1984

High plasma levels of immunoreactive somatostatin in transgenic mice expressing a metallothionein-somatostatin fusion gene.

To test the hypothesis that processing of pre-prosomatostatin (pre-proSS) can be accomplished by cells that do not normally synthesize the precursor, we have introduced the rat pre-proSS gene under control of the mouse metallothionein promoter into the germ line of mice. Four of the 11 resultant transgenic mice had markedly elevated plasma levels of somatostatin-like immunoreactivity (SLI); however, their growth was identical to control littermates. Liver contained 263 +/- 89 pg of SLI/mg of protein and kidney had 152 +/- 19 pg/mg. Gel filtration chromatography of tissue extracts resolved one major 6000-dalton peak of SLI and three minor peaks of 8500, 3000, and 1600 daltons. The latter two corresponded in elution position to synthetic somatostatin-28 (S-28) and somatostatin-14 (S-14). Almost all of the plasma SLI corresponded in size to the 6000-dalton peptide. These findings indicate that a metallothionein-somatostatin fusion gene was successfully integrated into the mouse genome and was expressed in tissues that do not normally synthesize pre-proSS. Pre-proSS was processed to S-28 and S-14 but atypical processing to a 6000-dalton peptide also occurred.

Animals↗
PubMed1980

Effects of immunizing rabbits against oestradiol-6-oxime-BSA on ovarian follicular development.

Female rabbits were actively immunized either against bovine serum albumin (BSA) or oestradiol-6-oxime BSA. When the antibody titres were elevated they were sacrificed and various parameters measured. Uterine weights were significantly lower, and a large number of haemorrhagic and some cystic follicles were present in the ovaries of the oestradiol immunized group. Increased serum gonadotrophins, testosterone, oestradiol, and progesterone levels as well as greater serum binding of oestradiol were also noted. Examination of the response of isolated follicles to gonadotrophin treatment in vitro indicated that oestradiol immunization decreased the ability of isolated follicles to form androgen and oestrogen following gonadotrophin treatment; however, progesterone levels were high regardless of the presence or absence of gonadotrophins. These data suggest that oestradiol plays an important role in follicular development.

Animals↗