Establishment in culture of a multihormone-secreting cell strain derived from the MtT/F4 rat pituitary tumor.

A clonal cell strain F4C1 has been established from the transplantable rat pituitary tumor MtT/F4 and has been maintained in continuous culture for two years. The cells grow with a population doubling time of 48 hours; the karyotype with a modal number of 39 chromosomes includes a pair of large metacentric marker chromosomes. F4C1 cells in culture produce growth hormone and prolactin but not adrenocorticotropin in contrast to the MtT/F4 tumor which secretes all three hormones in the host rat. The cloned cells lack specific receptors for thyrotropin-releasing hormone and do not respond to this agent with increased prolactin or decreased growth hormone production. Treatment with hydrocortisone results in a small increase in growth hormone and a small decrease in prolactin production. Tumors generated in rats from injected F4C1 cells secrete prolactin and growth hormone but not adrenocorticotropin. The results suggest that growth hormone and prolactin are produced by a single cell type in the MtT/F4 tumor.     

Chemical regulators of plant hormones and their applications in basic research and agriculture*

Plant hormones are small molecules that play versatile roles in regulating plant growth, development, and responses to the environment. Classic methodologies, including genetics, analytic chemistry, biochemistry, and molecular biology, have contributed to the progress in plant hormone studies. In addition, chemical regulators of plant hormone functions have been important in such studies. Today, synthetic chemicals, including plant growth regulators, are used to study and manipulate biological systems, collectively referred to as chemical biology. Here, we summarize the available chemical regulators and their contributions to plant hormone studies. We also pose questions that remain to be addressed in plant hormone studies and that might be solved with the help of chemical regulators.     

Crystallization and X-ray data collection on human growth hormone

Single crystals of natural sequence human growth hormone have been grown from media containing ethanol, acetone or paraldehyde. Recombinant growth hormone in its native and desamidated form and pituitary hormone have been crystallized. A full native set of diffraction data extending to 3.5 A resolution has been obtained with synchrotron radiation for crystals of recombinant human growth hormone grown from ethanol. The identity of the material in these crystals has been established by anion-exchange chromatography.     

Nucleotide sequence of mouse prolactin and growth hormone mRNAs and expression of these mRNAs during pregnancy

The mRNAs for mouse prolactin and growth hormone have been isolated from anterior pituitary glands and cloned as cDNAs. The nucleotide sequences of these mRNAs have been determined, and these sequences, along with the predicted amino acid sequences, are compared to those of other mammalian prolactin and growth hormone mRNAs. Levels of prolactin and growth hormone mRNAs during pregnancy have been monitored by hybridization to the cloned cDNA probes. We find the levels of these mRNAs to remain nearly constant during mid-to-late gestation.     

Growth Hormone Secretion in Growth-retarded Asthmatic Children

The serum growth hormone response to Bovril was studied in 12 growth-retarded children with severe asthma, and was found to be normal. Eight children who were receiving corticosteroids had been small for their age before starting steroid treatment. It is concluded that there is no case for treating growth-retarded asthmatic children with growth hormone.     

Methionyl- and pituitary derived human growth hormone have identical effects on the expression of growth hormone responsive rat hepatic mRNA

Pituitary extracts of human growth hormone have been used extensively for therapy of growth hormone deficiency, although they are known to contain a variety of contaminating polypeptides. Biosynthetic human growth hormone is now available for this use and appears to be functionally identical in promoting growth. To establish additional criteria of identity we compared the effect of these two hormone preparations on a family of hepatic messenger RNA sequences in hypophysectomized adult male rats. Total hepatic RNA from these animals was translated in a cell-free rabbit reticulocyte lysate. Five translational products previously demonstrated to be responsive to ovine and methionyl-human growth hormone were found to be equally induced by pituitary derived human growth hormone, despite demonstrable heterogeneity in pituitary derived preparations. In addition, no significant alterations in approximately 200 non-growth hormone responsive translational products were identified. Methionyl and pituitary derived growth hormone have identical effects on the expression of hepatic mRNA.     

Quantitative determination of growth hormone by immunoblotting

Blotting techniques have been extensively used, not only analytically for protein identification, but also preparatively to isolate and purify specific proteins from a large variety of cellular extracts and biological fluids. The process involves the separation of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transfer of proteins to nitrocellulose membranes, and immunostaining to identify proteins which often are at very low concentrations. Because of the quantitative interactions of the proteins with specific antibodies, we have coupled the immunoblot procedure with photographic and densitometric methods for the quantitative determination of bovine growth hormone (bGH). In this way, the method is suitable for bGH detection and quantitation for a small number of samples by use of a single Western blot analysis. The sensitivity of this method permits determinations of bGH to 0.5 ng. The method uses a comparative procedure in which purified bovine growth hormone is used as a standard.     

Small molecule approaches in plants

Small molecules offer exciting opportunities for plant science. So far, bioactive small molecules have been identified as plant hormones, herbicides, growth regulators, or taken from animal research. Recently, plant scientists have started to explore further the chemical space for novel modulators of plant hormone signalling, and have followed up this work with exciting discoveries illustrating the potential of small molecules such as brassinazole and sirtinol. New chemical genetic screens have been designed to generate chemical tools for the investigation of membrane trafficking, gravitropism and plant immunity. Further novel 'chemetic' tools to identify targets and modes of action are currently generated through an intimate interdisciplinary collaboration between biologists and small molecule chemists.     

Transgenic mice containing growth hormone fusion genes

New genes composed of the mouse metallothionein I promoter-regulator and the rat or human growth hormone structural gene have been introduced into mice. A large proportion of mice containing these genes express them, and some animals grow to be up to twice as large as controls. The technique has been used in part to correct the genetic defect that results in the small stature of the mutant little mouse.     

Altered release of growth hormone from dispersed adenohypophysial cells of streptozotocin diabetic rats. I. Effects of growth hormone releasing factor and somatostatin

As growth hormone has been implicated in the "dawn phenomenon," an early morning rise in serum glucose, we have studied the control of growth hormone release in diabetes using an acutely dispersed system of adenohypophysial cells from normal or diabetic rats (65 mg/kg streptozotocin, 8 days before sacrifice; serum glucose, 490 +/- 17 mg/dL). Growth hormone release is normally controlled by the two hypothalamic hormones, growth hormone releasing factor and somatostatin. We have found cells of the diabetic rats exhibit changes in sensitivity that result in increased growth hormone release in static incubation. In normal cells, rat growth hormone releasing factor increases growth hormone release three- to four-fold with an EC50 of 151 +/- 27 pM (n = 7). In contrast, in cells from diabetic rats, there was a significant (twofold) increase in sensitivity to growth hormone releasing factor (EC50 = 75 +/- 15 pM, n = 7) which resulted in increased growth hormone release with lower but not maximal (10 nM) growth hormone releasing factor. Basal nonstimulated release was unchanged. Somatostatin inhibition of stimulated growth hormone release was reduced (n = 7); half-maximal inhibition occurred with 0.21 +/- 0.03 nM (normal) and 0.76 +/- 0.17 nM somatostatin (diabetic). In perifusion the peak secretion rate was significantly lower for diabetic cells stimulated by a maximal dose of growth hormone releasing factor. These studies suggest somatotrophs of diabetic rats have altered sensitivity in vitro to the controlling hormones growth hormone releasing factor and somatostatin.     

Hormones and body size evolution in papionin primates

This study examines the evolution of size differences among papionin primates by measuring hormones that regulate size growth during ontogeny and influence ultimate adult size (insulin‐like growth factor‐I (IGF‐I), insulin‐like growth factor binding protein‐3 (IGFBP‐3), growth hormone binding protein (GHBP), dehydroepiandrosterone sulfate (DHEAS), testosterone, estradiol). The analyses assess longstanding ideas about circulating hormone levels and body size. Importantly, because the consensus papionin molecular phylogeny implies at least two episodes of size increase, this study offers opportunities to determine whether or not similar hormone profiles regulate this apparent evolutionary convergence (i.e., do larger‐bodied papionins have higher levels of growth‐related hormones than smaller‐bodied papionins?). Five hundred and sixty serum samples (from 161 individuals) from 11 papionin species were analyzed using a two‐level approach to address this issue. One used mixed longitudinal samples from two papionin species to test whether, during growth, large‐ and small‐bodied species have higher and lower hormone levels, respectively. The second compared multiple papionin species to assess whether or not hormone levels covary with size in adult animals. Result show that size and hormone levels do not covary consistently across papionins, either during growth or in adulthood. Specifically, some smaller‐bodied papionin species have higher absolute hormone levels than larger‐bodied species. Differences in some hormone levels appear to track phylogeny more closely than body size. In contrast to studies based on single species, we demonstrate that, while the hormones analyzed affect growth, absolute circulating hormone levels either during growth or adulthood may be decoupled from interspecific differences in body size. Am J Phys Anthropol, 2007. © 2006 Wiley‐Liss, Inc.     

Human growth hormone association and binding study in vitro: high performance size exclusion chromatography and radioimmunoassay-measured levels in plasma of normal and acromegalic subjects

Human growth hormone (hGH), like other protein hormones, consists of several molecular forms both in the pituitary and in plasma. In recent years carrier proteins have been detected and studied for growth hormone, using different experimental approaches. In the present study we have attempted to investigate whether hGH could be separated and identified in molecular or aggregated forms using high performance size exclusion chromatography and a small plasma sample, without particular treatment, in order to investigate specific hGH-binding proteins in normal as well as in acromegalic subjects. Results showed that it was possible to observe binding or/and the formation of a complex between free hormone and other molecules that could be specific binding proteins. Equilibrium was reached in a few minutes (7-10 min) and was reversible, as observed with labelled hormone using low and high concentrations of hGH in the incubation medium. At 37 degrees C the associated form at equilibrium was 30% of the total (measured as percent of total radioactivity with labelled hormone) in a plasma medium in which the original growth hormone was absent. Acromegalic plasma demonstrated that the percent of the associated form (namely the 80-kDa form) was less than that in normal humans. This may be due to the fact that capacity binding and competition between labelled and non-labelled growth hormone were favorable to the non-labelled form, if only for the high concentrations in this type of pathology. Therefore our results seem to be in agreement with the hypothesis that acromegalic subjects do not lack this aggregation capacity.     

A bacterial biosensor of endocrine modulators

The nuclear hormone receptors comprise one of the largest classes of protein targets for drug discovery, as their function has been linked to a variety of serious diseases, including several forms of cancer. Identifying novel compounds with the ability to modulate the function of these targets could lead to the development of effective therapeutics. In vivo sensors of ligand binding have emerged as tools that can greatly accelerate the lead identification process, allowing new drugs to be discovered more rapidly and cheaply. In this work, a novel sensor of nuclear hormone binding has been developed in Escherichia coli by constructing a fusion of the ligand-binding domain of the human estrogen receptor with a thymidylate synthase enzyme (TS). Expression of this fusion protein in TS-deficient bacterial cells resulted in growth phenotypes that were dependent on the presence of estrogen. Subsequent replacement of the estrogen receptor with the ligand-binding domain of the human thyroid hormone receptor led to specific thyroid hormone-enhanced growth that was insensitive to estrogen. This biosensor was then challenged with a small library of estrogen and thyroid hormone analogues, and it was observed that levels of cell growth correlate well with ligand-binding affinity. Remarkably, this simple biosensor was able to discriminate between agonistic and antagonistic activities, as combinations of estrogen agonists had an additive impact on cell growth, whereas known estrogen antagonists were found to neutralize agonist effects. This system constitutes a technique for facile selection of lead compounds with potential medical applications.     

Study of growth hormone gene in non-familial complete somatotropin deficiency

Familial growth hormone deficiency has been often associated to homozygous gene deletions. In this work we have looked for the possible absence of this gene in patients with isolated GH deficiency. The patient genomic DNAs have been digested with two restriction enzymes and hybridized with a 32P labelled growth hormone cDNA. The presence of the growth hormone gene has been proved in the patients. This situation, in which the gene is present but not expressed, might be due to changes in gene regulation or to punctual gene deletions or mutations.     

A systematic immunohistochemical survey of the distribution patterns of GH, prolactin, somatolactin, β–TSH, β–FSH, β–LH, ACTH, and α–MSH in the adenohypophysis of Oreochromis niloticus, the Nile tilapia

Fish pituitary plays a central role in the control of growth, development, reproduction and adaptation to the environment. Several types of hormone-secreting adenohypophyseal cells have been characterised and localised in diverse teleost species. The results suggest a similar distribution pattern among the species investigated. However, most studies deal with a single hormone or hormone family. Thus, we studied adjacent sections of the pituitary of Oreochromis niloticus, the tilapia, by conventional staining and immunohistochemistry with specific antisera directed against growth hormone (GH), prolactin (PRL), somatolactin (SL), thyrotropin (beta-TSH), follicle-stimulating hormone (beta-FSH), luteinising hormone (beta-LH), adrenocorticotropic hormone (ACTH) and melanocyte-stimulating hormone (alpha-MSH). The pituitary was characterised by a close interdigitating neighbourhood of neurohypophysis (PN) and adenohypophysis. PRL-immunoreactive and ACTH-immunoreactive cells were detected in the rostral pars distalis. GH-immunoreactive cells were present in the proximal pars distalis (PPD). A small region of the PPD contained beta-TSH-immunoreactive cells, and beta-LH-immunoreactive cells covered approximately the remaining parts. Centrally, beta-FSH-immunoreactive cells were detected in the vicinity of the GH-containing cells. Some of these cells also displayed beta-LH immunoreactivity. The pars intermedia was characterised by branches of the PN surrounded by SL-containing and alpha-MSH-immunoreactive cells. The ACTH and alpha-MSH antisera were observed to cross-react with the respective antigens. This cross-reactivity was abolished by pre-absorption. We present a complete map of the distinct localisation sites for the classical pituitary hormones, thereby providing a solid basis for future research on teleost pituitary.     

鲤鱼（Cyprinus carpio）生长激素基因的亚克隆及其序列分析

本文对ＰＣＲ扩增的６６８ｂｐ的ＤＮＡ片段进行了亚克隆,然后以Ｓａｎｇｅｒ双脱氧中止法为原理,利用美国ＡＢＩ公司３７０Ａ自动核酸序列分析仪,确定了６６８ｂｐ的核苷酸序列。序列分析表明鲤鱼生长激素基因的开放读框含有６３０ｂｐ,并推测其中包括２２个氨基酸的信号肽和１８８个氨基酸的成熟多肽。鲤鱼生长激素基因的酶切图谱和序列分析的结果都证明我们已获得了全长的鲤鱼生长激素基因。     

Human insulin-like growth-factor-binding protein. Low-molecular-mass form: protein sequence and cDNA cloning

Two different insulin-like growth-factor (IGF)-binding proteins have been found in human blood, one of high molecular mass and dependent on growth hormone for synthesis, the other of low molecular mass and independent of growth hormone. The small IGF-binding protein is abundant in human amniotic fluid. Its amino acid sequence has now been determined by direct analysis of the protein and its proteolytic fragments. Also, by immunoscreening a partial cDNA clone was isolated from a human hepatoma cell line. The mature protein consists of 234 amino acids and is coded for by an mRNA of approximately 1700 nucleotides in length. The primary structure of the protein reveals 18 Cys residues in N-terminal and C-terminal clusters and an Arg-Gly-Asp peptide sequence, common to extracellular proteins binding to receptors of the integrin family. A protein-sequence polymorphism was detected at position Ile/Met-228, indicating possible allelic variation. The 3'-untranslated mRNA sequence has a high A + T content and shows five copies of an ATTTA sequence, which has been shown to be involved in the regulation of the stability of certain mRNAs coding for growth-regulating proteins.     

Homogeneity studies on human growth hormone

1. Human growth hormone preparations (Raben) have been found to contain 10-40% of denatured growth hormone as shown by radioimmunoassay, reaction of radioiodinated subfractions with antiserum to whole growth hormone, and amino acid analysis, and confirmed by bioassay using the tibia test. 2. The altered fraction was more electronegative than the intact hormone on starch-grain electrophoresis in barbitone buffer, pH8.6. Some heterogeneity of the active material was detectable in simple buffer extracts of a single acetone-stored pituitary gland. 3. The inert fraction was more completely separated from the active hormone as an unretarded fraction from Sephadex G-200 columns with a borate buffer. This separation was due to aggregation of the denatured growth hormone in borate buffer. The active fraction from the Sephadex column still contained some inert material and the amount of this remaining varied considerably between different batches of growth hormone. 4. The radioimmunoassay procedure detects only the immunologically and biologically intact fraction.     

Purification of antibodies to protein hormones by affinity chromatography on divinylsulfonyl sepharose

Antibodies to human growth hormone and ovine interstitial cell stimulating hormone have been purified from rabbit antisera by affinity chromatography using a newly developed divinylsulfonyl activated agarose. Elution of the antibodies was accomplished by neutral solutions containing chaotropic ions.     

Activation of adenylate cyclase in renal medulla by bovine growth hormone. An artifact attributable to vasopressin.

The effect of bovine growth hormone on adenylate cyclase activity was studied in bovine and rat renal medulla. Highly purified growth hormone (lot B1003A) increased adenylate cyclase activity in plasma membranes from bovine renal medulla from 132+/-6 pmol cyclic AMP formed/mg protein per 10 min to 364+/-10 pmol cyclic AMP formed/mg protein per 10 min. Similar results were seen with homogenates of rat renal medulla. The minimum effective concentration of bovine growth hormone required to activate adenylate cyclase was 0.5 mug/ml and maximum activation was detected at 500 mug/ml. The amount of vasopressin determined by radioimmunoassay to contaminate the growth hormone caused an increase in adenylate cyclase activity comparable to that of the corresponding concentration of growth hormone that was tested. Dialysis of growth hormone and vasopressin resulted in parallel reductions in the effect of each hormone on adenylate cyclase activity. Similarly, both growth hormone and vasopressin produced increases in short circuit current in isolated toad bladders but these effects were not detectable after dialysis of the hormones. In contrast, the effect of growth hormone on the uptake of 35SO2-4 by cartilage from hypophysectomized rats was not decreased after dialysis. These results indicate that available preparations of growth hormone are contaminated by small but physiologically significant amounts of vasopressin and that the activation of adenylate cyclase activity in renal medulla in response to growth hormone can be explained by this contamination rather than by an effect of growth hormone per se.