bluuebunny/crossref_metadata_2025_split huggingface dataset:
Studies of human pituitary LH containing internally cleaved β subunit
Authors: Hartree A. Stockell , Shownkeen R. C. 
Published: December 2008

ABSTRACT
We have investigated the origin of the internal peptide bond cleavage found in the β subunit of a proportion of pituitary human LH (hLH) molecules, as well as the effects of this cleavage (or nick) on the interaction between α and β subunits and on binding of hLH to its receptor. The content of cleaved β subunit, assessed by the intensity of staining of an approximately 10 kDa component on sodium dodecyl sulphate-polyacrylamide gel electrophoresis of reduced hLH, was variable in batches of hLH prepared from pooled acetone-preserved human pituitary glands. There was evidence of a similar cleavage in purified hTSH, but not in the purified hFSH or human chorionic gonadotrophin examined. Although intact hLH was relatively resistant to cleavage in solution, urea dissociation of hormone followed by dialysis resulted in an increased content of nicked β subunit, which was largely prevented by incorporation of the proteolytic enzyme inhibitor phenylmethylsulphonyl fluoride (PMSF) and the metal-chelating agent EDTA. Hormone that was virtually free of nicked β subunit was obtained by urea dissociation of hLH subunits in the presence of PMSF and EDTA followed by dialysis to remove urea, reassociation of subunits at 37 °C (pH 7) and purification of reassociated hLH dimer by gel filtration on Sephadex G-100 in the presence of 1-anilinonaphthalene-8-sulphonic acid (ANS). When hLH was incubated at 37 °C at pH 55 or pH 95 in the absence of enzyme inhibitors, some cleaved β subunit was found in the hLH—ANS dimer form of the hormone, but most of the nicked subunit appeared in fractions of lower molecular weight and with low receptor-binding activity. Fractions isolated as hLH—ANS dimer had receptor-binding activities which were negatively correlated with their content of cleaved β subunit, the most active fraction (21 000 i.u./mg) being virtually free of this component. Our studies suggest (1) that the cleavage is caused by proteolytic enzyme activity present in human pituitary tissue and in purified preparations of hLH, (2) that free hLH-β subunit is cleaved far more readily than when it is combined with α subunit in the intact hormone, (3) that the presence of the cleavage in free hLH-β prevents refolding of this subunit to the correct conformation that permits its combination with α subunit to regenerate native hormone, and (4) that the site of cleavage is likely to be at a peptide bond located on the surface of the intact hLH molecule.
Inverse effects of activin and inhibin on the synthesis and secretion of FSH and LH by ovine pituitary cells in vitro
Authors: Muttukrishna S. , Knight P. G. 
Published: December 2008

ABSTRACT
Little information is available on the effects of activin and inhibin on the synthesis and secretion of pituitary gonadotrophins in species other than the rat. In this in-vitro study, ovine pituitary cell cultures derived from immature sheep were used to investigate the effects of recombinant human activin-A and native Mr 32 000 bovine inhibin on basal and gonadotrophin-releasing hormone (GnRH)-induced release of FSH and LH. Residual cellular contents of FSH and LH were also determined, allowing total content/well to be calculated.
Activin-A promoted a dose-dependent increase in basal (+72%; P<0·001) and GnRH-induced (+25%; P<0·001) release of FSH as well as in the residual cell content (+114%; P<0·001) and total FSH content/well (+67%; P<0·001). Conversely, inhibin significantly (P<0·001) suppressed each aspect of FSH production examined, confirming that in sheep, as in rats, activin and inhibin exert opposing effects on pituitary FSH production.
In contrast to the rat, however, in which activin is reported to have no effect on LH secretion, exposure of sheep pituitary cells to activin-A promoted a dose-dependent suppression (−42%; P<0·001) of GnRH-induced LH release. This was associated with a corresponding increase (P<0·001) in residual cellular content of LH. Consistent with a previous report from this laboratory, inhibin had the opposite effect and significantly enhanced (+47%; P<0·001) GnRH-induced LH release. This was associated with a corresponding fall (P<0·001) in residual cellular content of LH. Neither activin-A nor inhibin significantly affected total LH content/well, indicating a lack of effect of either agent on LH biosynthesis.
Comparison of the dose-dependent effects of inhibin on FSH and LH production by cells cultured in the presence and absence of a fixed dose level of activin-A (5 ng/ml) revealed a significant (P<0·01) interaction between the two agents which appeared to be of a non-competitive nature.
These findings support the concept that both activin and inhibin participate in the gonadal feedback control of pituitary FSH secretion in the sheep. They also indicate that both agents are capable of modulating GnRH-induced LH secretion in this species.
Identification of mRNA for epidermal growth factor and transforming growth factor-α present in low copy number in human endometrium and decidua using reverse transcriptase-polymerase chain reaction
Authors: Haining R. E. B. , Schofield J. P. , Jones D. S. C. , Rajput-Williams J. , Smith S. K. 
Published: December 2008

ABSTRACT
The presence of mRNA for epidermal growth factor (EGF) and transforming growth factor-α (TGFα) was demonstrated in small fragments of human endometrium and decidua by use of the technique of reverse transcriptase-polymerase chain reaction with nested oligonucleotide primers. The presence of mRNA encoding EGF and TGFα has not been shown in human endometrium previously. Other studies using conventional techniques, such as Northern blot or in-situ hybridization, showed the presence in low copy number of EGF but not TGFα in murine endometrium. Messenger RNA for EGF was not present in peripheral leukocytes or platelets, suggesting an endometrial source for the message. Messenger RNA for TGFα was found in these blood components, thus preventing confirmation of the source of TGFα mRNA.
The effect of anti-oestrogens on cell growth and progesterone receptor concentration in human endometrial cancer cells (Ishikawa)
Authors: Jamil A. , Croxtall J. D. , White J. O. 
Published: December 2008

ASTRACT
The effect of two anti-oestrogens, 4-OH tamoxifen and ICI 164,384, on growth and progesterone receptor (PR) concentration was investigated in the endometrial carcinoma cell line, Ishikawa. Growth stimulation in response to 4-OH tamoxifen was antagonized by ICI 164,384, the latter having no agonist effect when used as a single agent. Similarly, ICI 164,384 antagonized oestradiol-stimulated cell growth. PR was significantly increased following treatment with 4-OH tamoxifen, this response being antagonized in the presence of ICI 164,384. Oestradiol increased PR, although to a lesser extent than did 4-OH tamoxifen;the effect of oestradiol on PR was also antagonized by ICI 164,384. Used as a single agent, ICI 164,384 induced a moderate but statistically significant increase in PR, thus demonstrating partial agonist activity. This agonist property of ICI 164,384 may provide a mechanism of maintaining PR, which is down-regulated during conventional progestin therapy, without undesirable mitogenic activity.
Short-term effects of ACTH on the low-density lipoprotein receptor mRNA level in rat and hamster adrenals
Authors: Lehoux J.-G. , Lefebvre A. 
Published: December 2008

ABSTRACT
Low-density lipoprotein (LDL) receptor mRNA was found in both rat and hamster adrenals. Within 30 min after ACTH administration a significant increase in the levels of both LDL receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) mRNAs was observed in rat adrenals; these levels remained increased for up to 240 min. The increase in the levels of LDL receptor and HMG-CoA reductase mRNAs produced by ACTH was reduced by co-administration of aminoglutethimide while, at the same time, the adrenal cholesterol content of rats treated with both aminoglutethimide and ACTH was significantly increased compared with that in groups treated with ACTH alone. Cycloheximide also induced increased levels of rat adrenal mRNAs for LDL receptor and HMG-CoA reductase, but this effect was not additive with that of ACTH. These results suggest that, in the rat, the short-term effect of ACTH on the levels of mRNAs for the LDL receptor and HMG-CoA reductase is similarly controlled and might be mediated through changes in the adrenal cholesterol content. In the hamster adrenal, however, no significant fluctuations were found in the level of LDL receptor mRNA, although a marked increase was found in the level of HMG-CoA reductase mRNA, 2 h after ACTH administration. This indicates that an important effect of ACTH on cholesterol metabolism in the hamster adrenal is at the level of HMG-CoA reductase. In the hamster, therefore, where the main source of cholesterol for the adrenal gland is de-novo synthesis, it seems that a complex mechanism is involved in the control of LDL receptor gene expression.
Expression, purification and characterization of secreted recombinant human insulin-like growth factor-I (IGF-I) and the potent variant des(1–3)IGF-I in Chinese hamster ovary cells
Authors: McKinnon P. , Ross M. , Wells J. R. E. , Ballard F. J. , Francis G. L. 
Published: December 2008

ABSTRACT
Recombinant human insulin-like growth factor-I (hIGF-I) and a biologically potent variant lacking the N-terminal tripeptide (des(1–3)IGF-I) were produced from transfected Chinese hamster ovary cells. The constructs encoding the signal peptide, sequence of the mature peptide and a C-terminal extension peptide were expressed under the control of a Rous sarcoma virus promoter. Successfully transfected clones secreting correctly processed recombinant hIGF-I or des(1–3)IGF-I were selected by their secretion of IGF-I-like activity into the culture medium. The recombinant peptides were purified to homogeneity as assessed by high-performance liquid chromatography and N-terminal sequence analysis. The purified recombinant peptides exhibited biological potencies equivalent to authentic IGF-I and des(1–3)IGF-I respectively.
Differences in properties of the sheep testicular LH and FSH receptors
Authors: Yarney T. A. , Sairam M. R. 
Published: December 2008

ABSTRACT
Differences in binding and structural properties of ovine testicular FSH and LH receptors were investigated. The ovine FSH receptor did not discriminate between FSH of different species, although equine FSH was more reactive. In the same tissue, however, the LH receptor showed marked preference for ovine and bovine LH, reacting very weakly with other preparations of pituitary LH. Human chorionic gonadotrophin also reacted partly with the ovine LH receptor at 25 °C. However, at 4 °C. the optimum temperature for binding of the LH receptor to its homologous hormone, the receptor displayed no recognition for chorionic gonadotrophin preparations. Affinity cross-linking studies with ovine testicular membrane suggested that the ovine FSH receptor has an Mr of 70 000, which is very similar to that observed in the porcine ovary. The Mr of the ovine LH receptor was estimated to be 150 000, which is different from those of other mammalian species, including those that have been cloned. The data suggest that the binding and structural properties of the ovine FSH receptor are similar to those of other mammalian FSH receptors, whereas the ovine LH receptor appears to differ from other mammalian LH receptors in having a different Mr and in being more stringent in its requirement for pituitary LH.
Studies on the activation of the oestrogen receptor bound to the anti-oestrogens 4-hydroxytamoxifen and ICI 164,384 by using three monoclonal antibodies
Authors: Giambiagi N. , Pasqualini J. R. 
Published: December 2008

ABSTRACT
Three monoclonal antibodies, H222, H226 and D547, which provided evidence of the structural transformation and change in exposure of the functional domains of the oestrogen receptor from fetal guineapig uterus upon activation, were used to study the receptor bound to the anti-oestrogens 4-hydroxytamoxifen and ICI 164,384. No differences in the structure of non-activated 4-hydroxytamoxifen— and ICI 164,384—receptor complexes, as compared with the oestradiol—receptor complex, were detected by the three monoclonal antibodies. When heated at 28°C, both anti-oestrogen—receptor complexes became capable of binding the D547 antibody, which reacts selectively with the activated receptor; however, this binding was lower than that of the oestradiol-receptor complex. The interaction with the H226 antibody showed that anti-oestrogens can induce receptor dimerization, but to a lesser extent than oestradiol. In addition, both anti-oestrogen—receptor complexes can bind to DNA—cellulose and are retained in nuclei from intact cells at 28°C, but less efficiently than the oestradiol—receptor complex. On the other hand, the nuclear receptor seems to have a similar dimeric structure when bound to either anti-oestrogens or oestradiol, as detected by the three monoclonal antibodies. The data suggest that 4-hydroxytamoxifen and ICI 164,384 induce an impaired activation of the oestrogen receptor; this difference, although quantitative rather than qualitative, might be related to the partial agonistic action of these anti-oestrogens in the fetal guinea-pig uterus.
Purification of human c-erb A β protein
Authors: Ichikawa K. , Hashizume K. , Nishii Y. , Takeda T. , Kobayashi M. , Suzuki S. , Yamada T. 
Published: December 2008

ABSTRACT
Human thyroid hormone receptor (c-erb A protein) produced by Escherichia coli expression vector plasmid was purified sequentially using polyethylenimine precipitation of DNA, hydroxylapatite column chromatography, ammonium sulphate precipitation, Sephacryl S-300 gel filtration and mono Q-Sepharose column chromatography. These column procedures resulted in 41.3-fold purification of 3,5,3′-tri-iodo-l-thyronine (T3) binding activity over the initial E. coli extract. Purified protein as well as crude preparation showed high-affinity binding to T3. The c-erb A protein enriched by column purification was further purified by electroelution after electrophoresis. Rabbit antibody against the c-erb A protein was prepared and used for the Western blotting analysis. The antibody recognized c-erb A protein but not the bacterial proteins in crude E. coli extract. When partially purified rat hepatic nuclear thyroid hormone receptor was analysed, a 56kDa receptor was specifically recognized by the antibody.
Expression of a growth hormone-responsive exon of the ovine insulin-like growth factor-I gene
Authors: Saunders J. C. , Dickson M. C. , Pell J. M. , Gilmour R. S. 
Published: December 2008

ABSTRACT
The sheep insulin-like growth factor-I (IGF-I) gene encodes mRNAs containing three different 5′-untranslated sequences as a consequence of alternate splicing of leader exons. Using a combination of RNase protection and primer extension assays, we have mapped the transcriptional start sites of one of the leader exons, exon 1A. Transcription from exon 1A appeared to initiate from multiple points within a 20 bp region situated about 60 bp upstream of the exon 1A splice site. The presence of this transcript in the liver of animals treated with GH was enhanced five- to tenfold and contributed to about 95% of the total hepatic increase in IGF-I mRNA. This exon is generally expressed in a number of tissues immediately after birth; by about 4 weeks postpartum, however, expression is confined to liver. The regulation of hepatic and non-hepatic IGF-I synthesis by GH may involve different mechanisms.
Nucleotide sequence of the cDNA encoding the common α subunit of the chicken pituitary glycoprotein hormones
Authors: Foster D. N. , Galehouse D. , Giordano T. , Min B. , Lamb I. C. , Porter D. A. , Intehar K. J. , Bacon W. L. 
Published: December 2008

ABSTRACT
Recombinant cDNA clones that encode the α subunit of the chicken pituitary glycoprotein hormones were isolated from a pituitary library. The longer of the two cDNA clones that were sequenced was 754bp in length. It contained 81 nucleotides of the 5′-untranslated region (UTR), an open-reading frame of 360bp that encoded a 24 amino acid leader polypeptide sequence as well as the 96 amino acid mature α subunit, and 268 nucleotides of the 3′-UTR, followed by a 45 bp poly(A) tract. There was 69–79% homology between the nucleotide sequence of the coding region for the chicken and mammalian α-subunit cDNAs. Northern blot analysis revealed that the steady-state levels of an approximately 800 bp α-subunit specific transcript increased quantitatively when dispersed chicken pituitary glands were treated in culture with chicken gonadotrophin-releasing hormone-I.
The expression of a putative insulin-like growth factor-I receptor gene in the liver of the developing chick
Authors: Armstrong D. G. , Hogg C. O. 
Published: December 2008

ABSTRACT
The biological activity of insulin-like growth factor-I (IGF-I) is mediated by a transmembrane glycoprotein (type-1 IGF receptor or IGF-I receptor) that shows considerable sequence homology with the insulin receptor. In order to detect the expression of this gene in chicken liver tissue, a plasmid was constructed containing a fragment of chicken IGF-I receptor cDNA. The cDNA fragment corresponded to nucleotides 326–599 of the human IGF-I receptor cDNA and showed 86.1 and 69.3% homology at the nucleotide level and 96.7 and 80.2% homology at the amino acid level with the human IGF-I receptor and insulin receptor respectively. The construct was used to generate an antisense RNA probe for the detection of IGF-I receptor mRNA transcripts in 1- and 4-week-old chick liver tissue.
IGF-I receptor gene expression was initially detected by the reverse transcriptase polymerase chain reaction using synthetic chicken IGF-I receptor oligonucleotides. Amplified fragments of the correct size were detected in both RNA samples. Northern blots were also used to detect IGF-I receptor mRNA transcripts in the liver RNA samples. The results indicated that the amount of receptor mRNA decreased significantly between 1 and 4 weeks after hatch. In contrast, chicken β-actin gene expression remained constant over this period. A major IGF-I receptor RNA transcript (11 kb) was observed in blots from 1-week-old livers, less abundant transcripts were also observed ranging in size from 8 to 9 kb. No bands were detected in blots from 4-week-old livers.
The results indicate that the steady-state level of chicken liver IGF-I receptor mRNA decreased significantly 1–4 weeks after hatch. It is not known whether this difference was due to a decrease in gene expression and/or an increase in the rate of IGF-I receptor mRNA degradation.
Novel recombinant fusion protein analogues of insulin-like growth factor (IGF)-I indicate the relative importance of IGF-binding protein and receptor binding for enhanced biological potency
Authors: Francis G. L. , Ross M. , Ballard F. J. , Milner S. J. , Senn C. , McNeil K. A. , Wallace J. C. , King R. , Wells J. R. E. 
Published: December 2008

ABSTRACT
An efficient expression system in Escherichia coli for several biologically active insulin-like growth factor-I (IGF-I) fusion peptide analogues is described. These novel IGF-I fusion protein analogues have properties that make them very useful reagents in the investigation of IGF-I action. The analogues comprise an IGF-I sequence and the first 11 amino acids of methionyl porcine growth hormone (pGH) and include [Met1]-pGH(1–11)-Val-Asn-IGF-I, which contains the authentic IGF-I sequence, and two analogues, [Met1]-pGH(1–11)-Val-Asn-[Gly3]-IGF-I and [Met1]-pGH(1–11)-Val-Asn-[Arg3]-IGF-I, where Glu3 in the human IGF-I sequence has been replaced by Gly or Arg respectively. The three peptides are referred to as Long IGF-I, Long [Gly3]-IGF-I or Long [Arg3]-IGF-I depending on the IGF-I sequence present. Production of the purified fusion peptides was aided by folding the reduced and denatured fusion peptide sequence under conditions that gave very high yields of biologically active product. Introduction of a hydrophobic N-terminal extension peptide appears to facilitate the correct folding of the IGF-I analogues compared with that obtained previously when folding normal-length IGFs. The biological activities of the IGF-I fusion peptides were compared with authentic IGF-I and the truncated analogue, des(1–3)IGF-I. In L6 rat myoblasts, all the analogues were more potent than authentic IGF-I in their abilities to stimulate protein and DNA synthesis and inhibit protein breakdown. In H35 hepatoma cells, where the IGFs act through the insulin receptor, the Long IGF-I analogues maintained a similar potency relative to IGF-I as was observed in the L6 myoblasts. The order of biological potency in cell lines secreting IGF-binding proteins (IGFBPs) into the medium was Long [Arg3]IGF-I-des(1–3)IGF-I>Long [Gly3]-IGF-I>Long IGF-I>IGF-I. In chicken embryo fibroblasts, a cell line that does not secrete detectable IGFBPs into the medium, Long [Arg3]-IGF-I, was less potent than IGF-I. Investigation of receptor and IGFBP association by these analogues reinforced our previous findings that N-terminal analogues of IGF-I show increased biological potency due to changes in the degree of their IGFBP interactions.
Hormonal induction of malic enzyme in rat hepatocytes cultured on laminin-rich gels
Authors: Mann D. J. , Strain A. J. , Bailey E. 
Published: December 2008

ABSTRACT
The levels of malic-enzyme mRNA and activity were determined in primary cultures of adult rat hepatocytes maintained on either rat-tail collagen or a laminin-rich substratum. Cells plated on laminin-rich gels exhibited substantially improved patterns of albumin and malic-enzyme expression when compared with cells maintained on rat-tail collagen. Moreover, hepatocytes plated on the laminin-rich matrix displayed marked malic-enzyme inducibility in response to tri-iodothyronine and dichloroacetate, especially in the presence of insulin. However, Northern blot analysis revealed that the ratio of the amounts of the two major malic-enzyme mRNA species (2.0 and 3.1 kb) was reversed when compared with that found in the liver in vivo, the altered levels of these two species being closer to those found in non-hepatic tissues. These findings indicate that, although the hormonal responsiveness of isolated hepatocytes maintained on laminin-rich gels is markedly improved, and approaches the degree of induction demonstrated in the liver in vivo, the mechanisms of control differ, indicating a loss of liver-specific expression.
Ovine follistatin: characterization of cDNA and expression in sheep ovary during the luteal phase of the oestrous cycle
Authors: Tisdall D. J. , Hill D. F. , Petersen G. B. , Fleming J. S. 
Published: December 2008

ABSTRACT
We have isolated ovine follistatin cDNA from an ovarian follicle cDNA library and determined its sequence. The deduced amino acid sequence of the ovine follistatin precursor is highly homologous (>97%) to the porcine, human and rat follistatins.
Northern analysis was used to characterize follistatin gene expression in ovaries of adult ewes, collected from days 11 to 13 of the oestrous cycle. Two major (about 2.7kb and 1.5 kb) and one minor (about 0.5 kb) transcripts were detected in polyadenylated RNA extracted from ovarian follicles and corpora lutea. The degree of expression of the transcripts varied in the two ovarian compartments, with the 2.7kb species predominating in the follicles and the 1.5kb species being more abundant in the corpora lutea. No transcripts were detected in stromal tissue containing preantral follicles of <1 mm.