bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural characteristics of growth hormone receptors on mouse 3T3 cells having different biological responses to growth hormone
Authors: Uchida E. , Hayakawa T. , Niimi S. , Tanaka A. , Morikawa M. 
Published: December 2008

ABSTRACT
Cultured 3T3-F442A preadipocytes are able to undergo GH-promoted differentiation into adipocytes. The relationship between the structure and function of GH receptors on 3T3 cells (3T3-F442A preadipocytes, differentiated adipocytes and 3T3-C2 cells, which vary in susceptibility to adipose conversion or with respect to carbohydrate and lipid metabolism) was studied by the covalent cross-linking of 125I-labelled human (h) GH to intact cells with the bifunctional reagent disuccinimidyl suberate. When preadipocytes were cross-linked and analysed using sodium dodecylsulphate-polyacrylamide gel electrophoresis, a prominent 125I-labelled hGH-receptor complex of Mr 130 000 was observed along with minor complexes (Mr 300 000, 230 000 and 60 000) on autoradiography. Non-reducing—reducing two-dimensional gel electrophoresis revealed that the higher molecular weight complexes also contained the Mr 130 000 complex. Neuraminidase and tunicamycin treatment demonstrated that the GH receptor on F442A preadipocytes is a sialo-glycoprotein with N-linked carbohydrate chains. When the differentiated 3T3-F442A adipocytes and 3T3-C2 cells (a sub-line with no susceptibility to adipose conversion with GH) were examined in the same way as 3T3-F442A preadipocytes, no differences were observed in the specificity of GH binding and in the molecular size of the 125I-labelled hGH-receptor complexes and their glycosylation characteristics. This suggests that the structural characteristics of the GH receptor are closely related in each cell type, but that the hormonal signals produced after GH binding to the receptor may cause different effects according to the cell type.
Inducible anti-sense RNA for angiotensinogen in stably transformed hepatoma cell lines
Authors: Clouston W. M. , Lloyd C. J. , Richards R. I. 
Published: December 2008

ABSTRACT
Angiotensinogen mRNA is found in many extrahepatic tissues, where it may participate in local angiotensin-generating systems. In this study we explore the feasibility of using anti-sense RNA to decrease angiotensinogen production in rat H4IIEC3 hepatoma cells. An amplifiable shuttle vector was modified to allow the production of high levels of stable anti-sense RNA from two regions of the mouse angiotensinogen gene under the control of the inducible sheep metallothionein promoter. Stably transformed, clonal cell lines expressing anti-sense RNA for angiotensinogen were isolated after selection with the aminoglycoside G418. Subsequently, the number of chromosomally integrated copies of the angiotensinogen anti-sense constructs was coamplified by methotrexate selection for dihydrofolate reductase activity carried on the shuttle vector. With a 20- to 30-fold induction of the anti-sense RNAs, the target angiotensinogen mRNA level was reduced to 25–30% of control values. The specificity of this effect was confirmed by showing no decrease in either β-tubulin or neomycin phosphotransferase mRNA levels. Using tissue-specific promoters, it should be possible to direct these effects to specific organs in transgenic mice. However, in agreement with results from other groups, our findings suggest that it will not be possible to eradicate completely the target gene product using the anti-sense RNA strategy.
Molecular cloning of cDNA for porcine prolactin precursor
Authors: Kato Y. , Hirai T. , Kato T. 
Published: December 2008

ABSTRACT
Porcine prolactin cDNA clones were screened using antiserum against ovine prolactin from a cDNA library of porcine anterior pituitary, and their nucleotide sequences were determined by the chain-termination method. The nucleotide sequence of the 5′ untranslated region and part of the signal peptide region were determined by direct RNA sequencing with reverse transcriptase. The composite sequence of 957 nucleotides showed a signal sequence of 30 amino acids and a further 199 amino acids corresponding to the mature prolactin molecule. The predicted sequence confirmed the amino acid sequence determined previously by direct protein analysis, except for one amide form at residue 122 (Gln instead of the reported Glu). Northern blot analysis showed that the length of the porcine prolactin mRNA was about 1·1 kb. The porcine prolactin amino acid sequence showed 81, 80, 64, 62, 80 and 31% homology with human, bovine, rat, mouse, chick and salmon forms respectively. The identical amino acid residues showed marked clustering in four domains, two of which are highly conserved throughout a wide range of species. The hydropathy and secondary structure of porcine prolactin were analysed and compared with those of porcine GH, which shares the same ancestral gene. The two highly conserved regions of both hormones showed similar hydrophilicity, and the predicted secondary structures indicated that these regions in each hormone form different structures with differences in extension of the hydrophilic residues outside the molecule.
Partial cDNA sequence for the donkey chorionic gonadotrophin-β subunit suggests evolution from an ancestral LH-β gene
Authors: Leigh S. E. A. , Stewart F. 
Published: December 2008

ABSTRACT
A 246 bp cDNA clone representing the C-terminal region of the donkey (Equus asinus) chorionic gonadotrophin (CG)-β subunit was isolated from a placental library. The transcript contained the 3′ untranslated region and 42% of the CG-β subunit coding region (amino acid residues 85–146 of the mature peptide). Comparison of the deduced donkey amino acid sequence with the published horse CG-β subunit protein sequence (where they overlapped) revealed an overall homology of 61%. However, most of the differences were in the C-terminal extension, which is thought not to be important for gonadotrophic activity, and appeared to be due to two nucleotide insertions in the donkey sequence (compared with a deduced horse nucleotide sequence) leading to a reading-frame shift. Amino acid homology in the disulphide 'core' region was 81%. Some of the differences in this region were in the 'determinant loop' (residues 93–100) and these are interpreted in relation to the observed biological activities of horse and donkey CG.
The deduced amino acid sequence of the donkey cDNA indicated that it was larger than the majority of gonadotrophin-β subunits due to a C-terminal extension. Primate and horse CG (and horse LH) β subunits have analogous C-terminal extensions. The extension in the donkey subunit is 25 amino acid residues in length, compared with 28 in the horse and 24 in man. Comparisons with other available subunit DNA sequences indicated that, like the human CG-β gene, the donkey gene probably evolved from an ancestral LH-like β gene, following nucleotide deletions that allowed readthrough into previously untranslated DNA. Furthermore, both the human and donkey CG-β genes make use of the original LH polyadenylation sequence AAUAAA for translational termination and polyadenylation. We conclude that the C-terminal extension arose independently in equids and primates but through similar mechanisms.
Poly(A) tail length of oxytocin- and lysine vasopressin-encoding mRNAs increases during development in the porcine hypothalamus
Authors: Rehbein M. , Richter D. 
Published: December 2008

ABSTRACT
Lysine vasopressin- and oxytocin-encoding mRNAs have been analysed in the developing hypothalamus of the pig. The two hormone-encoding mRNAs were first detectable on fetal day 49 by Northern blot analysis. Whereas RNase mapping revealed identical transcripts throughout the developmental stages studied, Northern blots showed that the early transcripts appeared to be shorter (by 100–200 nucleotides) and more heterogeneous in size than those of later stages. This developmentally related length polymorphism was shown to be due to different poly(A) lengths and was abolished by removal of the poly(A) tails with RNase H. These results indicate that maturation of neurones in the developing porcine hypothalamus is accompanied by an increase in length of the poly(A) tail of vasopressin and oxytocin mRNAs.
Molecular cloning, sequence analysis and expression of putative chicken insulin-like growth factor-I cDNAs
Authors: Fawcett D. H. , Bulfield G. 
Published: December 2008

ABSTRACT
A cDNA library was constructed from mRNA isolated from the liver of a 5-week-old female broiler chicken; at this age the level of insulin-like growth factor-I (IGF-I) mRNA was expected to be high. Three clones, of sizes 23, 0·86 and 0·2 kb, were isolated by using a homologous human (IGF-I) probe. The DNA sequence of these clones has been determined and the potential amino acid sequence deduced. The sequence of the mature chicken IGF-I peptide shows a high degree of homology with IGF-I from other species, providing evidence for the identity of these clones. Alternative splicing of the chicken IGF-I mRNA has been found in the region potentially encoding the leader peptide. This may give rise to two forms of prepeptide, differing in the length and nature of their leader peptide. The 086 kb cDNA has been used as a probe to Northern blots of chicken mRNA. A major band of approximately 0·65–0·85 kb was seen, plus several minor bands of larger molecular weights. Analysis of genomic Southern blots shows that there is one copy of the chicken IGF-I gene.
Alteration in the expression of genes for cholesterol side-chain cleavage enzyme and 21-hydroxylase by hypophysectomy and ACTH administration in the rat adrenal
Authors: Imai T. , Seo H. , Murata Y. , Ohno M. , Satoh Y. , Funahashi H. , Takagi H. , Matsui N. 
Published: December 2008

ABSTRACT
The changes in steady-state levels of mRNA for cholesterol side-chain cleavage cytochrome P-450 (P-450scc) and steroid 21-hydroxylase cytochrome P-450 (P-450c21) caused by hypophysectomy and ACTH treatment were determined in rat adrenals. Hypophysectomy caused marked decreases in adrenal weight and total RNA per gland. Administration of ACTH resulted in increases in adrenal weight and total RNA. A significant correlation between the amount of RNA and adrenal weight was observed. Both P-450scc and P-450c21 mRNAs were decreased by hypophysectomy and increased by ACTH treatment. P-450scc mRNA decreased to 20% and P-450c21 mRNA to 76% of control values 1 day after hypophysectomy. ACTH caused a significant increase in P-450scc mRNA after 3 h. However, a significant increase in P-450c21 mRNA was observed 12 h after administration of ACTH. These results are concordant with previous studies in vitro utilizing cultured adrenocortical cells. Moreover, the induction of steady-state levels of P-450scc mRNA was faster than that observed by other investigators in studies in vitro. These results may indicate that integrity of the adrenal gland in vivo is important for the action of ACTH.
Glucagon-like peptide-1(7–36)amide: characterization of the domain responsible for binding to its receptor on rat insulinoma RINm5F cells
Authors: Gallwitz B. , Schmidt W. E. , Conlon J. M. , Creutzfeldt W. 
Published: December 2008

ABSTRACT
Glucagon-like peptide-1(7–36)amide (GLP-1(7–36)amide) is a potent stimulator of insulin secretion. Receptors for this hormone have been found on different insulinoma-derived cell lines, e.g. the RINm5F cell line which is derived from a radiation-induced rat insulinoma. To characterize the part of the GLP-1(7–36)amide molecule that is responsible for binding to its receptor on RINm5F cells, binding studies with synthetic C-terminal (GLP-1(21–36)amide) and synthetic N-terminal (GLP-1(7–25)) GLP-1 fragments were carried out. GLP-1(21–36)amide showed dose-dependent binding to the GLP-1(7–36)amide receptor but was approximately 1500 times less potent in inhibiting binding of 125I-labelled GLP-1(7–36)amide than the intact hormone. GLP-1(7–25) at concentrations up to 10 μmol/l did not inhibit binding of label. Neither fragment changed intracellular cyclic AMP concentrations, in contrast to GLP-1(7–36)amide which increased intracellular cyclic AMP. GLP-1(21–36)amide, however, acted as a weak partial antagonist of GLP-1(7–36)amide with respect to GLP-1(7–36)amide-dependent stimulation of cyclic AMP production.
Differential effects of β-adrenergic agonists on insulin secretion from pancreatic islets isolated from rat and man
Authors: Lacey R. J. , Berrow N. S. , London N. J. M. , Lake S. P. , James R. F. , Scarpello J. H. B. , Morgan N. G. 
Published: December 2008

ABSTRACT
The selective β2-adrenergic agonist clenbuterol was ineffective as a stimulus for insulin secretion when isolated rat pancreatic islets were incubated with glucose at concentrations between 4 and 20 mM. Inclusion of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine led to potentiation of glucose-induced insulin secretion, but did not facilitate stimulation by clenbuterol. Furthermore, maintenance of isolated rat islets for up to 3 days in tissue culture also failed to result in the appearance of a secretory response to β-agonists. By contrast, clenbuterol induced a dose-dependent increase in insulin release from isolated human islets incubated with 20 mm glucose. Clenbuterol did not increase the basal rate of insulin secretion (4 mm glucose) in human islets. Under perifusion conditions, the secretory response of human islets to clenbuterol was rapid, of similar magnitude to that seen under static incubation conditions and could be sustained for at least 30 min. The increase in insulin secretion induced by clenbuterol was inhibited by propranolol, indicating that the response was mediated by activation of β-receptors. In support of this, a similar enhancement of glucose-induced insulin secretion was elicited by a different β2-agonist, salbutamol, in human islets. The results indicate that the B cells of isolated rat islets are unresponsive to β-agonists, whereas those of human islets are equipped with functional β-receptors which can directly influence the rate of insulin secretion.
Secretory patterns of 1α-hydroxycorticosterone in the isolated perifused interrenal gland of the dogfish, Scyliorhinus canicula
Authors: O'Toole L. B. , Armour K.J. , Decourt C. , Hazon N. , Lahlou B. , Henderson I. W. 
Published: December 2008

ABSTRACT
An isolated in-vitro perifused interrenal gland preparation from the dogfish Scyliorhinus canicula was used to study production of quantitatively the major corticosteroid 1α-hydroxycorticosterone (1α-OH-B), measured by radioimmunoassay. Basal secretory rates were 877·1 ± 145 (s.e.m.) fmol/mg per 15 min (n=14) and the preparation remained viable for up to 22 h, as reflected in a brisk response to 10 μm cyclic AMP (cAMP) after this time. Steroid production responded in a dose-dependent manner to porcine ACTH, with 10 μm producing a maximum stimulation of 225% above the basal secretory rate. cAMP (10 μm) produced an increase of 278% above basal, while 1 μm forskolin increased basal secretory rates by 127%. [Val5]- and [Ile5]-angiotensin II (0·1 μm) increased 1α-OH-B production by 120 and 372% respectively over basal secretory rates. Increasing the concentration of K+ in the perfusate from 8 mm to 12, 18, 28 and 40 mm produced a significant rise only at 28 mm. Alterations in the concentration of Na+ and osmolarity of the perifusion medium had inconsistent effects on steroid production. Increased concentrations of urea (from 360 to 720 mm) increased the basal secretory rate by 121%, whilst reducing the concentration of urea (from 360 to 90 mm) had no effect.
Production and utilization of monoclonal antibodies to human/rat corticotrophin-releasing factor-41
Authors: Milton N. G. N. , Hillhouse E. W. , Nicholson S. A. , Self C. H. , McGregor A. M. 
Published: December 2008

ABSTRACT
Murine monoclonal antibodies against human/rat corticotrophin-releasing factor-41 (CRF-41) were produced and characterized for use in the immunological and biological characterization of CRF-41. Spleen cells from BALB/c mice immunized with CRF-41 conjugated to bovine γ-globulin were fused with a BALB/c-derived non-secretor X-63 myeloma line. Hybridomas were selected for CRF antibody production by enzyme-linked immunosorbent assay, and positive hybridomas cloned twice. Three monoclonal antibodies were obtained (KCHMB001, KCHMB002 and KCHMB003) and characterized as IgG1, IgG1 and IgG2a isotypes respectively, with affinity constants for rat CRF-41 of 30, 53 and 34 nmol/l respectively. All three monoclonal antibodies recognize an epitope contained between residues 34 and 41 of the human/rat sequence. The antibodies were able to neutralize the ACTH-releasing activity of rat CRF-41, applied to rat pituitary fragments in vitro, in a dose-dependent manner. Isoelectric focusing showed that KCHMB 003 detected bands of synthetic rat CRF-41 and rat [Met(O)21,38]-CRF-41 at pH 7·1 and 6·8 respectively. Use of KCHMB003 in a two-site enzyme-amplified immunoassay showed that this antibody recognizes both synthetic rat CRF-41 and immunoreactive CRF-41 in rat hypothalamic tissue extracts.
Relaxin gene expression in the porcine follicle during preovulatory development induced by gonadotrophins
Authors: Bagnell C. A. , Tsark W. , Tashima L. , Downey B. R. , Tsang B. K. , Ainsworth L. 
Published: December 2008

ABSTRACT
Northern analysis was used to identify relaxin gene expression in ovaries of prepubertal pigs primed with pregnant mare's serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG). The cellular distribution of relaxin transcript in the developing follicle was localized by in-situ hybridization histochemistry. Three probes complementary to non-overlapping regions of the porcine prorelaxin molecule were used to identify relaxin gene expression in ovarian follicular tissue collected 0, 48, 60, 72 and 84 h after treatment with PMSG/hCG. A 1 kb transcript was detected in ovarian extracts of prepubertal gilts from 48 to 84 h after PMSG stimulation. This corresponds to the molecular size of the relaxin transcript reported in the pregnant sow ovary. Relaxin mRNA levels increased in ovaries from animals 48 through 84 h after PMSG. In-situ hybridization showed that the site of relaxin synthesis was the theca interna layer of the developing follicle. Relaxin mRNA was not observed in other follicular cell types, in small or atretic follicles or in follicles from unstimulated animals. The distribution and relative concentration of relaxin mRNA showed a good correlation with in-vitro production and immunohistochemical localization of relaxin previously reported in the developing pig follicle. The presence of both protein and mRNA for relaxin in the growing follicle supports a role for relaxin as a local regulator of ovarian function.
Growth hormone receptors in hypothalamic and extra-hypothalamic tissues
Authors: Fraser R. A. , Attardo D. , Harvey S. 
Published: December 2008

ABSTRACT
Central GH receptors (GHR) have been identified in hypothalamic and extra-hypothalamic tissues of rabbit and chicken brains. Plasma membranes of the rabbit brain demonstrated specific saturable high-affinity, low-capacity binding sites for 125I-labelled GH. RNA extracted from hypothalamic and extra-hypothalamic tissues of rabbit and chicken brains contained mRNA that hybridized with a cDNA probe for the rabbit liver GHR. This transcript was of a similar size to the major GHR mRNA moiety in rabbit liver. The expression of these moieties was age related, and higher in adult than in neonatal animals.
Molecular cloning and nucleotide sequence analysis of complementary DNA for bullfrog prolactin
Authors: Takahashi N. , Yoshihama K. , Kikuyama S. , Yamamoto K. , Wakabayashi K. , Kato Y. 
Published: December 2008

ABSTRACT
A prolactin cDNA was cloned from a cDNA expression library constructed from total RNA of bullfrog (Rana catesbeiana) adenohypophyses by immunoscreening with antiserum against bullfrog prolactin. The cDNA clone thus obtained contained a 249 bp insert. Using this clone as a probe, plaque hybridizations were performed and two additional clones obtained. These clones had a polyadenylation site different from that of the first obtained clone, suggesting that the 3′-untranslated sequence was heterogeneous in length. The longest clone contained 830 bp, which encoded part of the signal peptide and the entire sequence of mature prolactin. The deduced amino acid sequence was in good accord with that determined by direct protein sequencing of purified bullfrog prolactin. The length of the bullfrog prolactin mRNA was estimated by Northern blot analysis to be about 1·0 kb. Homologies of prolactin nucleotide and amino acid sequences between bullfrog and other vertebrates were 64 and 65% for man, 66 and 68% for pig, 61 and 52% for rat, 69 and 74% for chicken, and 50 and 35% for salmon respectively. Highly conserved regions reported for mammalian prolactins also existed in bullfrog prolactin. Homologies of nucleotide and amino acid sequences between prolactin and GH of bullfrog origin were 49 and 25% respectively. Using the cDNA, the content of prolactin mRNA in the pituitary glands of metamorphosing tadpoles was measured. Prolactin mRNA levels rose at the mid-climax stage, suggesting that the increase in plasma and pituitary prolactin levels known to occur at the climax stage accompanies the increase in prolactin synthesis.
A marsupial β-lactoglobulin gene: characterization and prolactin-dependent expression
Authors: Collet C. , Joseph R. , Nicholas K. 
Published: December 2008

ABSTRACT
Analysis of the tammar wallaby β-lactoglobulin cDNA and inferred amino acid sequences reveal extensive sequence divergence from the eutherian β-lactoglobulins. Conserved residues include the cysteines and a number of individual amino acids involved in structure and ligand-binding. The only region of extended similarity is a heptapeptide sequence which may impart specificity to its interaction with a receptor protein. Northern analysis of total mammary RNA revealed two transcripts which result from differential utilization of polyadenylation signals. The concentration of β-lactoglobulin mRNA increased in late lactation and correlates with increases in milk production and levels of milk fat. Quantification of β-lactoglobulin mRNA levels in hormone-stimulated mammary gland explants from tammars in late pregnancy suggests that maximal induction of the gene is dependent on prolactin alone and that expression is not modulated by other hormones known to play a role in the initiation of lactation in eutherians.
The ovine insulin-like growth factor-I gene: characterization, expression and identification of a putative promoter
Authors: Dickson M. C. , Saunders J. C. , Gilmour R. S. 
Published: December 2008

ABSTRACT
Genomic DNA encoding the ovine insulin-like growth factor-I (IGF-I) gene was cloned and sequenced. The predicted amino acid sequence of the mature form of ovine IGF-I was highly homologous to that of human, rat and mouse. Analysis of the DNA sequence between exons 1 and 2 suggested the existence of an alternative 5′ exon (exon 1A) and this was confirmed by polymerase chain reaction (PCR) analysis of sheep liver mRNA. Primer extension of mRNA from exon 1A indicated a class of transcripts which initiated at a point 32 nucleotides 5′ to the Met codon of exon 1A to give a mRNA comprising exons 1A, 2, 3 and 5. In liver these transcripts co-existed with the alternative exon 1, 2, 3 and 5 mRNA form. Analysis by PCR of the 3′ terminus of liver RNA indicated heterogeneity arising from multiple polyadenylation sites; however, of the two possible alternatively spliced 3′ exons, only exon 5 could be detected.
Expression of IGF-I mRNA, as measured by a solution hybridization/RNase protection assay, predominated in the liver of the neonate and the late-gestation fetus; however, lower levels of expression were seen in multiple tissues throughout fetal and neonatal development.