bluuebunny/crossref_metadata_2025_split huggingface dataset:
LH receptor RNA and protein levels after hormonal treatment of porcine granulosa cells in primary culture
Authors: Goxe B. , Salesse R. , Remy J. J. , Genty N. , Garnier J. 
Published: December 2008

ABSTRACT
Granulosa cells were prepared from small follicles (<3 mm) from the ovaries of 5-month-old gilts. They were cultured in plastic dishes coated with a synthetic adhesion peptide in a chemically defined medium supplemented with 2% serum substitute. After 3 days of culture, the cells reached confluence and expression of the LH receptor could be stimulated in a hormonally defined medium. LH receptor RNAs were estimated by autoradiography using Northern blots and dot blots of total cell RNA. LH receptor RNAs were hybridized with a homologous 32P-labelled random-primed DNA probe. The LH receptor was measured using 125I-labelled human chorionic gonadotrophin (hCG) as tracer.
Northern blots of LH receptor RNAs revealed a predominant signal of 4.4kb and two less-intense hybridization bands of 7.5 and 1.9kb. The 4.4kb band was used for quantification of LH receptor RNAs because it was the most intense and may be attributed to the full-length messenger RNA. In these conditions, after 72h stimulation, FSH (0.6 nm), insulin (5 μg/ml), oestradiol (30 nm) and deoxycorticosterone (0.3 nm) yielded high LH receptor RNA levels (eight times unstimulated cell level), while dibutyryl cyclic AMP (1 mm), cortisol (5.4 nm), thyroxine (100 nm) and epidermal growth factor (16pm) gave low LH receptor RNA levels (one to five times). However, the respective amounts of the receptor RNA did not give yield to the same proportion of LH receptor for every factor, indicating some post-transcriptional regulations.
The kinetic study of the production of the LH receptor obtained in a defined medium supplemented with FSH, oestradiol and insulin showed that the receptor appeared after 48 h of stimulation and reached a maximum of about 7000 receptors per cell at 72 h. The three hybridization bands on Northern blots evolved in parallel and appeared as early as 24 h. They were at maximal level from 24 to 48 h of stimulation. When the granulosa cells were pulse-treated for 2 h with cycloheximide (10 μg/ml), they exhibited a transient rise in LH receptor RNA content which was followed by a delayed receptor increase especially at 72 h of stimulation.
Taken together, these results indicate that the LH receptor in primary culture of granulosa cells seems to be regulated by different physiological factors both at the transcriptional and the translational levels.
Interleukin-1β stimulates growth of adrenocortical cells in primary culture
Authors: Hanley N. , Williams B. C. , Nicol M. , Bird I. M. , Walker S. W. 
Published: December 2008

ABSTRACT
Using tritiated-thymidine incorporation as a measure of cell growth, interleukin-1β stimulated the growth of bovine zona fasciculata/reticularis adrenocortical cells after 72h in primary culture. Within the range of 10–1000pg/ml, interleukin-1β produced over 40% of angiotensin II-stimulated [3H]thymidine incorporation (P<0.005 compared with basal for 10pg/ml and 1000pg/ml; P<0.05 for 100pg/ml; two-tailed unpaired Student's t-test). Interleukin-1β did not directly stimulate cortisol secretion.
By stimulating adrenocortical growth, the increase in interleukin-1 during fever provides a potential mechanism for chronically raising glucocorticoid output. This study is the first demonstration of a long-term effect involving interleukin-1β on the adrenal cortex.
Expression of a human thyrotrophin receptor fragment in Escherichia coli and its interaction with the hormone and autoantibodies from patients with Graves' disease
Authors: Huang G. C. , Collison K. S. , McGregor A. M. , Banga J. P. 
Published: December 2008

ABSTRACT
Graves' disease is an autoimmune thyroid disease characterized by the presence of pathogenic autoantibodies to the TSH receptor (TSH-R). By using polymerase chain reaction, the extracellular region of the human TSH-R cDNA has been amplified and used to prepare recombinant TSH-R (extracellular) protein fused with glutathione-S-transferase (GST). Purification of the recombinant TSH-R (extracellular)-GST fusion protein was achieved by preparative gel electrophoresis in SDS or by preparative isoelectric focusing in urea. Following removal of SDS by detergent exchange or urea by dialysis, the purified recombinant receptor preparations were assessed for binding to the hormone or to autoantibodies from Graves' disease patients. The purified recombinant receptor preparations fail to show any binding to the hormone or autoantibodies either by inhibition of binding assays or by immunoblotting. The results imply that the correct folding and/or post-translational modifications of the polypeptide chain which are not achieved in recombinant proteins produced in Escherichia coli may be important for the binding of the hormone or Graves' disease autoantibodies to the TSH-R. The recombinant receptor prepared in this manner will be useful for immunological and cellular investigations in patients with Graves' disease.
Differential expression of the insulin-like growth factor binding proteins in spontaneously diabetic rats
Authors: Luo J. , Murphy L. J. 
Published: December 2008

ABSTRACT
Diabetes-induced growth retardation in the rodent is associated with both reduced circulating insulin-like growth factor-I (IGF-I) and enhanced levels of inhibitors of somatomedin activity. IGF-binding proteins (IGFBPs) are present in the circulation and tissue fluids and are believed to modulate the actions of IGF-I. Since elevated concentrations of the IGFBPs may contribute to the enhanced somatomedin-inhibitor activity observed in serum from diabetic animals, we have examined the amounts of hepatic IGFBP-1, -2, -3 and -4 mRNA in the spontaneously diabetic BioBreeding/Worcester rat.
The study used two types of diabetic animal: mildly diabetic animals, which received suboptimal insulin treatment (0.5–1 U/day) and diabetic animals, which received intensive insulin treatment (3–6 U/day). A significant increase in the amount of IGFBP-1 and IGFBP-2 mRNA was seen 1 month and 3 months after the onset of diabetes. Intensive insulin treatment for 3 weeks normalized the amount of IGFBP-1 mRNA in diabetic rats and resulted in a decrease in IGFBP-2 mRNA. In contrast to the increase in IGFBP-1 and IGFBP-2 mRNA, a significant decrease in IGFBP-3 mRNA was seen in diabetic rats (54.6% of control, P < 0.0005 and 64.6% of control, P < 0.005 for 1 and 3 months respectively) and intensive insulin treatment for 3 weeks did not restore the IGFBP-3 mRNA level in diabetic rats. No significant difference in IGFBP-4 mRNA levels was seen in diabetic compared with non-diabetic rats. When serum was analysed by ligand blotting the major finding was a reduction in the 39–42kDa binding protein. No increase in 29–30kDa IGFBP in the serum was detected in the diabetic rats. From these studies we conclude that the major change in IGFBPs in mildly hyperglycaemic spontaneously diabetic rats is a decrease in IGFBP-3. The changes in hepatic IGFBP-1 and -2 mRNA do not appear to be of sufficient magnitude to result in an increase in serum concentrations of these binding proteins.
Recombinant mouse prolactin: expression in Escherichia coli, purification and biological activity
Authors: Yamamoto M. , Harigaya T. , Ichikawa T. , Hoshino K. , Nakashima K. 
Published: December 2008

ABSTRACT
Transformation of Escherichia coli cells with a recombinant plasmid containing modified mouse prolactin (mPRL) cDNA and a pKK223-3 vector resulted in efficient expression of mPRL protein. Cloned mPRL cDNA was modified by removing the 5′ non-translating sequence as well as the sequence which encoded the signal peptide of preprolactin for recombination. In addition, approximately 100 nucleotides of the 5′-terminal region of the cDNA, which include the ATG initiation codon and the following 31 codons of mature mPRL, were replaced by a chemically synthesized oligonucleotide duplex. The sequence of this duplex was chosen to be rich in AT without changing the amino acid sequence of the protein. The modified cDNA was finally inserted into the multicopy plasmid, pUC19, before high-level expression of mPRL in E. coli cells was obtained. Western blotting analysis of total protein from transformed E. coli cells showed that both 23 and 16kDa peptides were recognized by specific mPRL antisera. The purified and refolded 23 kDa protein exhibited a growth-stimulating effect on rat Nb 2 Node lymphoma cells, and was very similar to that of natural pituitary PRL.
Phosphorylation of prolactin and growth hormone
Authors: Arámburo C. , Montiel J. L. , Proudman J. A. , Berghman L. R. , Scanes C. G. 
Published: December 2008

ABSTRACT
To determine whether GH and prolactin could be phosphorylated, turkey GH, chicken GH, chicken prolactin and turkey prolactin were incubated in vitro with the catalytic subunit of protein kinase A and [γ-32P]ATP. Phosphorylation was assessed after sodium dodecyl sulphate-polyacrylamide gel electrophoresis, Western blotting and autoradiography. Polyacrylamide electrophoresis showed that both purified native chicken GH and turkey GH were phosphorylated under the conditions employed. However, the glycosylated variant of chicken GH did not appear to be labelled. Chicken prolactin, turkey prolactin and the glycosylated variant of turkey prolactin were all intensely phosphorylated by protein kinase A. Ovine and rat prolactins could also be phosphorylated by protein kinase A. The phosphate content of different native prolactin (turkey, ovine and rat) and GH (ovine and chicken) preparations was also determined and found to be significant. Chicken pituitary cells in primary culture incorporated P in GH- and prolactin-like bands isolated by non-denaturing polyacrylamide gel electrophoresis, and this was stimulated by phorbol myristate acetate. Phosphorylation of GH and prolactin may thus explain some of the charge heterogeneity of these hormones.
Effects of cisplatin on human thyrocytes in monolayer or follicle culture
Authors: Massart C. , Le Tellier C. , Lucas C. , Gibassier J. , Leclech G. , Nicol M. 
Published: December 2008

ABSTRACT
Cis-diamminedichloroplatinum (II) (cisplatin) is a widely used anticancer drug which induces many sideeffects, but its action on the thyroid gland is still unknown. We have investigated the effects of this drug on human thyrocytes cultured in monolayers or in follicles and stimulated with 200 μU TSH/ml.
After 72h in culture, different concentrations of cisplatin (15, 30 and 75 μm) caused partial or total inhibition of cyclic AMP (cAMP), thyroglobulin (Tg) and tri-iodothyronine (T3) production, whereas thyroxine levels increased in the medium of thyrocytes cultured as follicles. Small doses of the drug did not affect thyrocyte production. Decreases in neutral-red uptake by thyroid cells and in intracellular lactate dehydrogenase, α-hydroxybutyryldehydrogenase and creatine phosphokinase activities were induced by 30 and 75 μm cisplatin.
These data show that high concentrations of cisplatin had a cytotoxic effect on thyrocytes. Cisplatin also induced inhibition of the production of cAMP, Tg and T3.
Characterization of a rat adrenocortical inner zone-specific antigen and identification of its putative precursor
Authors: Barker S. , Laird S. M. , Ho M. M. , Vinson G. P. , Hinson J. P. 
Published: December 2008

ABSTRACT
We have previously reported the production of a monoclonal antibody (IZAb) which interacts with an antigen, found predominantly in rat adrenal inner zone tissue, which may have a role in steroidogenesis. Here we describe initial studies on its characterization.
Immunoblot analysis of rat adrenocortical proteins obtained from fresh tissue and separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, showed that the IZAb interacted with a protein with a molecular mass of approximately 30 000 Da (IZAg1). This protein was found predominantly in rat adrenal inner zone tissue. Small amounts were seen in the zona glomerulosa, while no corresponding protein was seen in rat ovary, heart, liver, testis or kidney tissue. Subcellular fractionation of rat adrenocortical inner zone tissue and immunoblot analysis showed that the IZAg1 was present in the microsomal and mitochondrial fractions of the cell, but was absent from the cytosol. Invivo treatment with ACTH (100 μg/day) for more than 5 days also increased the expression of this protein by rat adrenal inner zone tissue, and this was coincident with increased corticosterone and 18-hydroxydeoxycorticosterone (18-OH-DOC) production in incubations of inner zone tissue in vitro.
In experiments involving the short-term culture of rat adrenal inner zone cells, IZAb interacted with two protein bands. IZAg1 was detected as a minor band in untreated control cells, while another protein with a molecular mass of approximately 60 000 Da, designated IZAg2, was present in greater amounts. Treatment of cells for 48 h with either ACTH (1 μmol/l) or dibutyryl-cAMP (100 μmol/l) resulted in apparent increased expression of IZAg1 and diminished levels of IZAg2. As in the in-vivo treatments, the increase in IZAg1 was associated with a corresponding increase in corticosterone and 18-OH-DOC production.
These findings suggest that the IZAb recognizes a protein (IZAg2) which occurs in unstimulated adrenal cells. On stimulation by steroidogenic agents, this protein becomes processed to yield a smaller protein (IZAg1) which is associated with enhanced adrenal steroidogenesis.
The size of the mature membrane receptor for follicle-stimulating hormone is larger than that predicted from its cDNA
Authors: Dattatreyamurty B. , Smith R. A. , Zhang S.-B. , Santa-Coloma T. A. , Reichert L. E. 
Published: December 2008

ABSTRACT
A 240 kDa protein isolated from bovine calf testis has been shown to have properties characteristic of an FSH receptor. However, rat testis FSH receptor has, on the basis of cloning experiments, been found to have a much lower molecular mass of 75 kDa (peptide only). To examine this point, the size of the FSH receptor in membranes obtained from cultured Sertoli cells of immature rats was determined after polyacrylamide gel electrophoresis under non-reducing conditions, followed by transfer to polyvinylidene difluoride membranes and direct identification of the FSH receptor by ligand blot analysis utilizing radioiodinated human FSH. In this system, the rat Sertoli cell membrane FSH receptor also showed a molecular mass of 240 kDa. Bovine testis contains LH and FSH receptors. We compared the sizes of FSH and LH receptors present in the same bovine testis membrane preparation by ligand blot analysis. The FSH receptor again showed a molecular mass of 240 kDa, whereas the LH receptor showed a molecular mass of 90 kDa. The latter value is similar to that deduced by cloning techniques (75 kDa, peptide only). The evidence seems to suggest that, whereas the molecular mass deduced for the LH receptor on the basis of its cDNA is similar to that of the mature membrane receptor, the size of the FSH membrane receptor is considerably different from that deduced on the basis of its cDNA, presumably as a result of post-translational processing. The marked difference in size between mature FSH (240 kDa) and LH (90 kDa) receptors may reflect significant structural differences of importance with regard to mechanisms of signal transduction.
Microheterogeneity of pituitary follicle-stimulating hormone in male rats: differential effects of the chronic androgen deprivation induced by castration or androgen blockade
Authors: Simoni M. , Weinbauer G. F. , Chandolia R. K. , Nieschlag E. 
Published: December 2008

ABSTRACT
Testicular androgens are known to influence not only the secretion but also the bioactivity and molecular composition of pituitary FSH. In the present study, we investigated the effects of chronic androgen blockade and castration on the molecular heterogeneity of the gonadotrophin. Groups of male adult rats (five animals per group) received one of the following treatments: vehicle, the non-steroidal anti-androgens casodex (20 mg/kg per day) or flutamide (20 mg/kg per day), or castration. After 8 weeks, the animals were killed and individual pituitary homogenates fractionated by isoelectric focusing (IEF) on sucrose density gradients in the pH range 2·5–8. FSH was measured by radioimmunoassay (RIA) in the individual fractions and by in-vitro bioassay (Sertoli cell aromatase bioassay) in pools of fractions which were combined according to pH intervals of 0·5 units. Bioactive and immunoreactive FSH were also measured in sera and unfractionated pituitary extracts. Testosterone and inhibin were assayed in sera by RIA.
A significant increase in serum immunoreactive and bioactive FSH was demonstrated in flutamide-treated and castrated animals, whereas the pituitary content of bioactive FSH remained unchanged in the four groups. Serum testosterone and inhibin were undetectable in castrated animals and significantly increased in those treated with flutamide. By RIA, the IEF profiles of the flutamide-treated and castrated rats showed a significant reduction of the FSH isoforms with 3·5<pI<4, with a significant increase in the isoforms with pI>4 only in the castrated group. By bioassay, there was a significant decrease in the isoforms with 3·5<pI<4 in both casodex- and flutamide-treated animals, with no significant differences between the two groups. Castration caused a further significant shift in the relative distribution of FSH isoforms towards the less acidic components, with a significant increase in the isoforms with 5<pI<5·5 not attained by androgen blockade alone.
These results suggest that the effects of long-lasting castration on pituitary FSH heterogeneity cannot be entirely reproduced by the androgen blockade. Since inhibin, eliminated by castration but not by androgen blockade, is a major regulator of FSH in the male rat, we speculate that it might not only influence FSH secretion but also modulate its qualitative properties.
The first disulphide loop of the rabbit growth hormone receptor is required for binding to the hormone
Authors: Gobius K. S. , Rowlinson S. W. , Barnard R. , Mattick J. S. , Waters M. J. 
Published: December 2008

ABSTRACT
Residues within the first disulphide loop of the GH receptor are highly conserved, and the two cysteines forming this motif are conserved across many cytokine receptors. We have used site-directed mutagenesis and the polymerase chain reaction with splicing by overlap extension to show that these residues are essential for binding of bovine (b)GH and human (h)GH to the rabbit GH receptor. When all residues within this loop were replaced with an equivalent polyalanine sequence, hormone binding was abolished. Conversion of Arg 39 within the loop to aspartate (R39D) decreased affinity for bGH by up to 20-fold. Conversion of Glu 42 to lysine (E42K) also resulted in a fivefold loss of affinity for bGH. However, charge reversals at Lys 37, Glu 44 and the conversion of Leu 43 to an arginine (as in the human receptor) were without a major effect on bGH binding. The lack of effect of the L43R mutation on bGH affinity, despite a significant (threefold) decrease in hGH affinity, argues against a role for Arg 43 as a residue conferring primate GH-binding specificity on the human receptor.
Examination of the affinities of poly Ala, R39D and E42K mutants for a variety of hormone-binding-site directed and other monoclonal antibodies (MAbs) to the GH receptor revealed that these mutations were without a major effect on tertiary structure. It is of interest that the epitopes for the hormone-binding inhibitory MAbs 263 and 7 are located within this first loop, since the poly Ala mutation abolished the binding of both MAbs, and the R39D mutation abolished binding of MAb 7.
In conclusion, residues within the first disulphide loop of the GH receptor are critical for hormone binding, and are epitopes for the binding of inhibitory MAbs. These findings may be applicable to other cytokine receptors.
Analysis of DNA structure in the human insulin gene-linked polymorphic region in vivo
Authors: Hammond-Kosack M. C. U. , Kilpatrick M. W. , Docherty K. 
Published: December 2008

ABSTRACT
An altered DNA structure exists within the hypervariable region located 360 bp upstream of the human insulin gene. The aim of the present study was to determine whether this structure exists in the insulin gene in vivo, and whether its presence is related to the expression of the insulin gene. However, since there were no clonal human β-cell lines available for such studies, the human insulin gene was transfected into a rat insulinoma-derived β-cell line and several human insulin-expressing clones were selected. One such cell line was treated in vivo with the DNA structural probe bromoacetaldehyde and the chromosomal DNA was extracted. Following digestion with TaqI and subsequent digestion with S1-nuclease to cleave at the bromoacetaldehydereactive sites, the DNA was subjected to agarose gel electrophoresis, and insulin gene fragments were detected by Southern blot analysis. Bromoacetaldehyde generated subfragments of 2500, 1700 and 800 bp in the human insulin gene isolated from the rat β-cell line, while the human insulin gene in the non-expressing HeLa cell line was unreactive to bromoacetaldehyde. These results suggest that an altered structure might exist in the insulin gene-linked polymorphic region of the human insulin gene in vivo, and that this structure may play a role in the expression of the insulin gene.
Cell-specific expression of aromatase and LH receptor mRNAs in rat ovary
Authors: Whitelaw P.F. , Smyth C.D. , Howles C.M. , Hillier S.G. 
Published: December 2008

ABSTRACT
Current understanding of the endocrine and paracrine regulation of follicular oestrogen synthesis predicts that aromatase cytochrome P450 (P450arom) mRNA is inducible by FSH in granulosa cells. LH receptor mRNA is constitutively expressed in thecal/interstital cells, and is also thought to be induced in granulosa cells in response to joint stimulation by FSH and oestrogen. This study provides direct evidence that FSH induces the ovarian P450arom gene selectively, perhaps exclusively, in the granulosa cells of Graafian follicles. FSH-induction of LH receptor mRNA occurs simultaneously but is independent of oestrogen synthesis per se.
Tri-iodothyronine and cycloheximide enhance insulin-like growth factor-binding protein-1 gene expression in human hepatoma cells
Authors: Angervo M , Leinonen P , Koistinen R , Julkunen M , Seppälä M 
Published: December 2008

ABSTRACT
The growth-regulating actions of IGFs are modulated by their binding proteins (IGFBPs). The serum concentration of IGFBP-1 is down-regulated by insulin, and in-vitro studies have demonstrated that IGFBP-1 secretion from various tissues and cells can be stimulated by theophylline, forskolin, oestrogen and progesterone. We have studied the effects and mechanisms of thyroid hormone action on IGFBP-1 gene expression and secretion by human hepatoma cells in vitro.
Tri-iodothyronine dose-dependently enhanced IGFBP-1 secretion in serum-free HepG2 cell cultures after 24–48 h of exposure, as measured by a specific immunofluorometric assay. This was accompanied by an increase (+ 50%) in the amount of IGFBP-1 mRNA, which could be prevented by cycloheximide, a protein synthesis inhibitor. Cycloheximide transiently enhanced (+ 200%) the accumulation of IGFBP-1 mRNA at 3–12 h of incubation, when no effect of tri-iodothyronine was observed. It is concluded that thyroid hormone stimulates IGFBP-1 secretion slowly by enhancing IGFBP-1 gene expression by a protein mediator. The acute stimulation of IGFBP-1 gene transcription by cycloheximide associates this gene with a number of growth-related genes encoding growth- and tumour-associated peptides.