bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of inositol lipid hydrolysis in human breast cancer cells by two classes of bombesin antagonist
Authors: Patel K. V. , Schrey M. P.
Published: December 2008

Inositol lipid hydrolysis was monitored in the human breast cancer cell line MCF-7 in response to various bombesin (BN) and substance P (SP) analogues. Both members of the BN family of peptides, i.e. BN and gastrin-releasing peptide (GRP), stimulated a dose-related increase in total inositol phosphate production, with a similar half-maximal effective dose (ED50) around 1 nm. The BN receptor antagonist [Leu13-Ψ-CH2NH-Leu14]-BN (LLBN) at 1 μm was devoid of agonist activity and displaced the BN dose—response to the right, resulting in a tenfold increase in the ED50 for BN. BN also stimulated a dose-related increase in 45Ca2+ efflux which was also inhibited by LLBN. Two SP analogues [dArg1, d-Pro2, d-Trp7,9,Leu11]-SP and [d-Arg1, d-Phe5, d-Trp7,9,Leu11]-SP ([APheTL]-SP), both antagonized BN-stimulated inositol lipid hydrolysis. [APheTL]-SP (60 and 80 μm) alone also exhibited considerable agonist activity which was not antagonized by LLBN. Indeed, a sub-threshold dose of [APheTL]-SP (40 μm) in the presence of LLBN (10 μm) potentiated the inositol lipid hydrolysis response. BN, GRP, LLBN and [APheTL]-SP all inhibited binding of 125I-labelled GRP to MCF-7 cells, to 50% of that occurring in the absence of the peptides, at concentrations of 150 pm, 150 pm, 150 nm and 600 nm respectively. These data are consistent with the presence of separate but interacting receptors or binding sites for BN and SP analogues, which are coupled to a common signal transduction pathway in human breast cancer cells.
Studies of human pituitary LH containing internally cleaved β subunit
Authors: Hartree A. Stockell , Shownkeen R. C.
Published: December 2008

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

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

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

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

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

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

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

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

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

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

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

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

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

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.
Expression of the genes for α inhibin, βA inhibin and follistatin in the ovaries of Booroola ewes which were homozygotes or non-carriers of the fecundity gene FecB
Authors: Fleming J. S. , Tisdall D. J. , Greenwood P. J. , Hudson N. L. , Heath D. A. , McNatty K. P.
Published: December 2008

Ovine cDNA probes for the α and βA inhibin subunits and for follistatin were used to investigate the mRNA species for these hormones in ovaries obtained during the luteal phase of the oestrous cycle, from Booroola ewes which were homozygous carriers (BB) or non-carriers (++) of the FecB gene. BB ewes had significantly higher concentrations of peripheral FSH and LH immunoreactivity than ++ ewes, but the peripheral inhibin immunoreactivity and ovarian inhibin and progesterone secretion rates were not significantly different between genotypes. No gene-specific differences in the number or size of mRNA transcripts detected by Northern blotting were noted for any of these genes. A single α inhibin mRNA species at 1.5 kb was observed in the follicle RNA from ++ and BB ovaries. Low amounts of α inhibin hybridization were discerned occasionally in + + and BB stroma and also in BB, but not in ++, corpora lutea. The βA inhibin gene was expressed only in the follicles from both ++ and BB ovaries. At least three βA inhibin transcripts were observed; one at 7.5kb and at least two between 1.4 and 5.0kb. The follistatin cDNA probe detected two major transcripts at 2.7 and 1.5 kb and a minor band at 0.5 kb in both follicle and corpora lutea RNA. Densitometry of the Northern blots revealed no significant gene-specific differences in the levels of α inhibin and follistatin gene mRNA transcripts. However, significantly greater amounts of total βA inhibin hybridization were detected in follicle RNA from BB compared with ++ ovaries (P<0.001) and this FecB-specific difference appeared to be associated with the 7.5 kb transcript. We conclude that the Booroola FecB gene does not influence the synthesis of the α inhibin subunit or follistatin during the luteal phase of the oestrous cycle, but may affect inhibin or activin synthesis in the ovaries of FecB carriers, by increasing the transcription or stability of the βA inhibin mRNA species.
Expression of the clusterin gene in the tissues of Booroola sheep which were homozygotes or non-carriers of the fecundity gene FecB
Authors: Fleming J. S. , Greenwood P. J. , Chen C.-L. C.
Published: December 2008

Clusterin or sulphated glycoprotein-2 is a major component of the rete testis fluid, synthesized by the rete testis epithelial cells and Sertoli cells. Differences in the two-dimensional polyacrylamide gel electrophoresis pattern of clusterin-like proteins have been reported in rete testis fluid from Booroola rams carrying the fecundity gene FecB, when compared with that from non-carrier rams. In order to determine whether the FecB gene influences the expression of the clusterin gene, we used a rat clusterin cRNA probe to investigate mRNA species in the tissues of homozygous (BB) or non-carrier (+ +) Booroola sheep. Northern blots of polyadenylated RNA showed hybridization to the cRNA probe in the testis, ovarian follicles, corpora lutea and stroma, pituitary and liver. A major mRNA transcript was observed at 2·3 kb and a minor transcript in some tissues at 0·8 kb. Densitometry of the autoradiographs revealed no FecB-specific differences in the densities of the hybridization signals from + + and BB testis or ovarian follicle, corpora lutea or stromal RNA. We conclude that the gene for ovine clusterin is expressed widely in the tissues of sheep and that its expression is not affected by the presence of the FecB gene.
Effects of dexamethasone on G protein levels and adenylyl cyclase activity in rat vascular smooth muscle cells
Authors: McLellan A. R. , Tawil S. , Lyall F. , Milligan G. , Connell J. M. C. , Kenyon C. J.
Published: December 2008

Dexamethasone administration in vitro has been shown to increase adenylyl cyclase activity in vascular smooth muscle cells (VSMC) from renal arteries and in non-vascular cell lines. To investigate whether G proteins are involved in this response, cultured VSMC from mesenteric arteries of Sprague—Dawley rats were incubated in the presence and absence of 10 nm dexamethasone for 24 and 48 h. Basal and stimulated adenylyl cyclase activities were increased by approximately 50% after treatment with dexamethasone. The changes were neither specifically associated with ligands which stimulate adenylyl cyclase catalytic unit via Gs (isoproterenol and prostaglandin E1) nor with guanylylimidodiphosphate (0·1 nm), which inhibits the catalytic unit via Gi. This suggests that dexamethasone enhances adenylyl cyclase activity through changes at the level of the catalytic unit, rather than through the G proteins which modulate its activity. No differences were seen in immunoblotting studies of the levels of Giα2, Gsα, Giα3 and β subunits. Similarly, dexamethasone had no effect on the expression of mRNA for Giα2 and Gsα.

The results indicate that glucocorticoid-induced increases of adenylyl cyclase activity are due to changes at the level of the adenylyl cyclase catalytic unit rather than alteration of the levels or turnover of Gsα, Giα2, Giα3 and β subunits in the membranes of VSMC.
An association between a BclI restriction fragment length polymorphism of the glucocorticoid receptor locus and hyperinsulinaemia in obese women
Authors: Weaver J. U. , Hitman G. A. , Kopelman P. G.
Published: December 2008

Obesity is likely to be a multifactorial disease with an important genetic component. Animal models of genetic and experimentally induced obesity suggest that glucocorticoid receptor (GR) activity plays a role in the aetiology and maintenance of the obese state. Glucocorticoid activity appears to be essential for the development of hyperinsulinaemia and subsequent fat deposition. In humans, glucocorticoid excess is associated with central fat distribution. We have therefore investigated the restriction fragment length polymorphisms of the human GR gene locus (GRL) and have sought associations of specific alleles with anthropometric measurements and indices of insulin secretion and resistance in obesity.

Fifty-six extremely obese, unrelated, nondiabetic premenopausal British Caucasian females and 43 age-matched, normal weight controls were studied. The obese subjects were characterized by fat distribution (waist to hip ratio), insulin secretion and insulin resistance (fasting insulin (FI)), an index of insulin resistance (HOMA), stimulated insulin secretion during an oral glucose tolerance test and insulin-mediated glucose disposal, steady-state plasma glucose). A BclI polymorphism (fragments of 4·5 and 2·3 kb) demonstrated significant association with indices of glucose metabolism in obesity; those subjects homozygous for the 4·5 kb fragment had elevated FI (Pc=0·012) and HOMA (Pc=0·012) values. The genotypic and allelic frequencies of the GRL BclI polymorphism were otherwise similar in obese and normal weight subjects. We postulate that the GRL BclI polymorphism may directly affect GR gene expression, or be in linkage disequilibrium with a possible mutation within one of three exons of the GR gene, and thereby modulate GR transcriptional activity on target genes involved in glucose and insulin homeostasis.
Identification of exchange mechanisms for the regulation of intracellular pH in rat thyroid FRTL-5 cells
Authors: Wood A. M. , Warhurst G. , Bidey S. P. , Soden J. , Taylor R. , Robertson W. R.
Published: December 2008

pH is maintained in cells by plasma membrane exchange mechanisms. In the absence of HCO3− ions, FRTL-5 cells regulate intracellular pH (pHi) by an Na+/H+ antiport but HCO3−-dependent exchangers cannot operate. We have investigated pHi regulation (by microfluorimetry and the pH sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6′)-carboxy-fluorescein) in small groups (five to six cells) of FRTL-5 thyroid cell monolayers held in kREBS—Ringer buffer (pH 7·4) with or without HCO3− ions. The exchangers were investigated with inhibitors (amiloride or its derivative dimethylamiloride for the Na+/H+ antiporter and the stilbene derivative disodium 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) for HCO3 −-dependent mechanisms), ionic substitution and by NH4+/NH3 (10mm) acid loading. Basal pHi was lower in the presence (7·3±0·058, mean±s.d., n= 14) than in the absence (7·59±0·078, n=10) of HCO3 ions. In HCO3 −-free media, cells recovered from acid load by 0·34±0·04 pH units in the first 2 min and finally reached a pHi of 7·35±0·06. This recovery was Na+-dependent and blocked by dimethylamiloride during the 15 min following intracellular acidification. In HCO3−-containing media, cells recovered from an acid load at a similar rate, but reached 99 ± 10% (n = 9) of the baseline pH; this recovery was also dependent on Na+ ions. Moreover, although dimethylamiloride and DIDS reduced the rate of recovery to 0·06±0·02 and 0·18±0·04 pH units respectively during the 2-min period, the cells returned to the basal pHi within 15 min. Removal of Na+ from HCO3−-containing media acidified the cells (ΔpH=–0·82±0·05, n=10) within 40 min; this acidification was partially blocked by either amiloride or DIDS. Removal of Cl− alkalinized the cells (ΔpH=+0·51 ± 0·06, n=10) after 40 min, and this alkalinization was totally prevented by DIDS. Furthermore, in the absence of Na+ and presence of amiloride, alkalinization was still seen on the removal of Cl−, albeit at a diminished rate (i.e. ΔpH = +0·25±0·05, n=8) after 40 min. In conclusion, FRTL-5 cells maintain pHi by two Na+-dependent exchangers, one sensitive to amiloride, the other to DIDS, and a Na+-independent, Cl−/HCO3− mechanism.
The mouse relaxin gene: nucleotide sequence and expression
Authors: Evans B A , John M , Fowler K J , Summers R J , Cronk M , Shine J , Tregear G W
Published: December 2008

Relaxin is a polypeptide hormone that has a variety of physiological effects both on remodelling of collagen and on uterine contractility. These are most apparent during pregnancy. The sequences of relaxin cDNAs derived from ovaries of late-pregnant random-bred Swiss mice have been established. Multiple subclones obtained from three independent polymerase chain reaction experiments were found to encode relaxins which were identical except at position 11 in the A chain (Ile or Val). All mouse relaxin cDNAs expressed in the ovary during pregnancy had an extra tyrosine inserted prior to the final A chain cysteine residue, a result confirmed by direct sequencing of relaxin peptides. Whilst this tyrosine insertion must have local effects on the folding of the A chain, structure—activity studies will clarify whether it perturbs functional interaction with the relaxin receptor. We have shown that there is a single relaxin gene in the mouse genome, and that expression during pregnancy occurs in the ovary but is not detectable in the placenta, uterus or fetus.
Glucose-regulated protein-78 expression in the rat adrenal cortex
Authors: Gregoire J , Mertz L M , Pedersen R C
Published: December 2008

We have postulated that steroidogenesis activator polypeptide (SAP) is a product of glucose-regulated protein-78 (grp78) proteolysis on the basis of a number of considerations, including a striking sequence similarity between the carboxyl-terminal region of grp78 and SAP. Since ACTH stimulates the rapid intracellular accumulation of SAP, experiments were conducted to determine whether ACTH might also regulate levels of grp78 mRNA and/or protein. Using a grp78 cDNA probe, Northern analysis of total RNA isolated from hypophysectomized or dexamethasone-suppressed rats revealed that neither treatment had a measurable influence on steady-state levels of grp78 mRNA over a 4-day period. Moreover, immunoblotting with an antiserum directed against a shared grp78/SAP sequence failed to detect a significant change in the grp78 content of adrenal homogenates from dexamethasone-suppressed rats as compared with untreated controls. On the other hand, grp78 in cultured rat adrenocortical cells fell to 50% of that in time-zero controls after 72 h in the absence of ACTH, whereas inclusion of ACTH in the medium blocked this decline. We conclude that while adrenocortical grp78 may be under some measure of trophic ACTH control, the rapid fluctuations reported for SAP are not likely to be driven by large changes in the size of the grp78 pool.
Localization of progesterone-associated endometrial protein mRNA by in-situ hybridization in human pregnancy decidua, endometriosis and borderline endometrioid adenoma
Authors: Kämäräinen M , Leivo I , Julkunen M , Seppälä M
Published: December 2008

Progesterone-associated endometrial protein (PAEP) has been isolated from human decidualized endometrium. In-situ hybridization histochemistry was employed to determine the cellular localization of PAEP mRNA in decidua during pregnancy. PAEP mRNA was found to be expressed in the glandular epithelium of decidua spongiosa throughout pregnancy. Substantial variations in the amount of PAEP mRNA during the course of pregnancy were observed, and it was most abundant at the end of the first trimester. We also found that the PAEP gene was expressed in endometriosis and in a borderline endometrioid adenoma. As in decidual tissues, PAEP mRNA in endometriosis was abundant in the glandular compartment.
Specific oxytocin agonist stimulates prolactin release but has no effect on inositol phosphate accumulation in isolated rat anterior pituitary cells
Authors: Chadio S E , Antoni F A
Published: December 2008

We have previously characterized specific oxytocin receptors in the rat anterior pituitary gland, using a highly selective oxytocin receptor antagonist as radio-ligand. The aim of the present study was to examine whether occupation of these receptors by oxytocin produces a stimulation of prolactin release and a rise in the accumulation of total inositol phosphates in the rat adenohypophysis. Anterior pituitary cells harvested from randomly cycling and diethylstilboestrol (100 μg s.c.)-treated rats were perifused with Dulbecco's minimal essential medium at a rate of 0·3 ml/min. Oxytocin and the specific oxytocin agonist [Thr4-Gly7]-oxytocin (TG-OT) both stimulated a significant prolactin release at concentrations of 10-6 and 10-7 m. Oestrogen treatment did not affect the response to oxytocin, indicating that there is no straightforward correlation between receptor number and prolactin secretory response in the rat pituitary gland.

The involvement of phosphoinositide hydrolysis was investigated in dispersed anterior pituitary cells and uterine tissue from randomly cycling rats. Oxytocin and arginine-vasopressin stimulated a significant (P<0·05) and dose-related increase in total inositol phosphates, vasopressin being more potent. The specific oxytocin agonist TG-OT had no effect on total inositol phosphate production in pituitary cells, but when tested in uterine tissue it significantly (P< 0.05) stimulated the accumulation of total inositol phosphate at all concentrations tested (10-5 to 10-9 m). In conclusion, the data show that oxytocin has prolactin-releasing activity, acting on specific receptors in the anterior pituitary gland. Furthermore, although oxytocin receptors in the rat uterus are coupled to the inositol phospholipid cycle, it would appear that this is not a prerequisite for the stimulation of prolactin secretion when specific oxytocin receptors in the rat adenohypophysis are activated.
Up-regulation of insulin-like growth factor-I (IGF-I) receptor gene expression in patients with reduced serum IGF-I levels
Authors: Eshet R , Werner H , Klinger B , Silbergeld A , Laron Z , LeRoith D , Roberts C T
Published: December 2008

We have analysed the expression of the IGF-I receptor gene in lymphocytes of patients with low levels of circulating IGF-I (four patients with isolated GH deficiency (IGHD) and one Laron-type dwarf (LTD)) in comparison with a control group exhibiting normal serum IGF-I levels and endocrine profiles. 125I-Labelled IGF-I binding assays were performed on erythrocytes to determine the number of IGF-I binding sites per cell and their dissociation constants. Erythrocytes from patients with IGHD or LTD contained significantly (P=0·002) more receptors per cell (10·9±3·1 binding sites/cell), with a reduced affinity (Kd = 0·49±0·05 nm), than erythrocytes from controls (2·0±0·4 sites/cell; Kd = 0·14 nm). The levels of IGF-I receptor mRNA in circulating lymphocytes were determined by an RNA template-specific reverse transcription/polymerase chain reaction method. There was a statistically significant increase in IGF-I receptor mRNA levels in lymphocytes from patients with LTD or IGHD when compared with controls (3108·1±775·9 vs 576·0±465·7 arbitrary units, P=0·006). The increased level of IGF-I binding due to increased IGF-I receptor gene expression may represent a compensatory up-regulation process activated in response to the low levels of IGF-I in the circulation of patients with LTD or IGHD.
The human insulin gene-linked polymorphic region adopts a G-quartet structure in chromatin assembled in vitro
Authors: Hammond-Kosack M C U , Kilpatrick M W , Docherty K
Published: December 2008

The insulin gene-linked polymorphic region (ILPR), located 363 bp upstream of the human insulin gene, is composed of tandem repeats of the consensus sequence ACAGGGGT(G/C)(T/C)GGGG. It has previously been shown that an insulin gene fragment containing the ILPR adopts an altered DNA structure in vitro. Furthermore, oligonucleotides containing the consensus repeat sequence exhibit multiple quadriplex DNA structures. The present study was undertaken to determine whether such altered DNA structures existed within the ILPR when the insulin gene was assembled into chromatin in vitro. Chromatin assembly was achieved using histones and an extract from unfertilized eggs from Xenopus laevis. The presence of altered DNA conformations within the 5′ region of the human insulin gene was investigated using the structural probe nuclease P1. Nuclease P1 recognized multiple distinct sites in the 5′ flanking region of the human insulin gene in naked DNA. Most of these sites disappeared when the recombinant plasmid DNA was treated with histones and unfertilized egg extract. In the assembled DNA, the ILPR appeared as the major site of nuclease P1 hypersensitivity. Fine-mapping of the multiple reactive sites within the ILPR showed a pattern characteristic of G-quartet foldback structures similar to those that have been observed for telomeric DNA.
Changes in progesterone and oestrogen receptor mRNA and protein and oxytocin receptors in endometrium of ewes after intrauterine injection of ovine trophoblast interferon
Authors: Mirando M A , Harney J P , Zhou Y , Ogle T F , Ott T L , Moffatt R J , Bazer F W
Published: December 2008

This study determined whether twice-daily intrauterine injections of ovine conceptus secretory proteins (oCSP) containing type-I trophoblast interferon (25 μg/uterine horn) from day 11 to day 15 post-oestrus (oestrus = day 0) could alter the binding capacities of endometrial receptors for oxytocin, progesterone and oestrogen in cyclic ewes when compared with control ewes receiving serum protein (SP) injections. Injections of oCSP on days 11–15 post-oestrus decreased concentrations of oestrogen receptors (P<0·06), oestrogen receptor mRNA (P<0·05) and progesterone receptors (P<0·08) in endometrium on day 16 when compared with SP-infused control ewes, which were undergoing corpus luteum regression on days 14–16. Injection of oCSP also decreased the number (P<0·10) and affinity (P<0·06) of oxytocin receptors. Inositol phosphate formation induced in the endometrium on day 16 by 100 nm oxytocin in vitro was highly correlated with the concentration (r≥0·93, P<0·001) and Kd (r= –0·91, P<0·01) of oxytocin receptors in SP-infused ewes, but was not as highly correlated with concentration (r≤0·83, P<0·06) and Kd (r≤ –0·40, P>0·40) of oxytocin receptors in oCSP-infused ewes. This indicates that oCSP disrupted the relationship between oxytocin receptor binding and oxytocin-induced activation of its second messenger system. These results indicate that antiluteolytic type-I trophoblast interferon may prevent oxytocininduced luteolytic pulsatile secretion of prostaglandin F2α during maternal recognition of pregnancy in sheep, by reducing the synthesis and affinity of endometrial oxytocin receptors. Inhibition of other components of the oxytocin receptor—phospholipase C system by ovine trophoblast interferon may also be involved in reducing endometrial responsiveness to oxytocin. Ovine trophoblast interferon may inhibit the synthesis of endometrial oestrogen receptors to inhibit responsiveness to oxytocin during early pregnancy in ewes.
Expression of the β3-adrenergic receptor in human white adipose tissue
Authors: Revelli J-P , Muzzin P , Paoloni A , Moinat M , Giacobino J-P
Published: December 2008

The results of this study suggest that the atypical β-adrenergic receptor (β3 subtype) is expressed in human white adipose tissue. A β3-adrenergic receptor mRNA could be detected in human omental fat poly(A)+ RNA by polymerase chain reaction amplification with appropriate primers. The expression of the β3-adrenergic receptor in human fat was confirmed by Northern blot analysis; however, the level of its mRNA was lower than those of the β1- and β2-adrenergic receptors. Two populations of ((−)-4-(3-t-butylamino-2-hydroxypropoxy)-[5,7-3H]benz-imidazole-2-one) ([3H]CGP 12177)-binding sites were identified in human omental fat membranes, one with a high affinity (Kd= 1·7 nm) and the other with a low affinity (Kd = 22 nm). The low affinity binding site population, which should represent the β3-adrenergic receptor, was predominant (75% of the total binding sites).
Effects of free fatty acids on somatostatin secretion, content and mRNA levels in cortical and hypothalamic fetal rat neurones in monolayer culture
Authors: Señarís R M , Lewis M D , Lago F , Domínguez F , Scanlon M F , Diéguez C
Published: December 2008

The effects of free fatty acids on somatostatin secretion, content and mRNA levels in fetal rat hypothalamic and cortical cell cultures were investigated. Somatostatin secretion and content were quantified by radioimmunoassay. Somatostatin mRNA levels were measured by Northern blot hybridization using a cRNA probe. Treatment with either caprylic acid (5×10-3m) or oleic acid (5× 10-5 m) for 90 min inhibited basal somatostatin secretion in both hypothalamic and cortical cell cultures. In addition, the increase in somatostatin secretion induced by incubation with veratridine (10-4m) or carbachol (10-4m) for 90 min was significantly reduced by the addition of caprylic acid, but somatostatin release stimulated by 5·6×10-2m KCl was not affected. Furthermore, treatment with these free fatty acids for 90 min markedly decreased somatostatin mRNA levels in both types of neurone culture. These inhibitory effects were transient, being observed after 90 min, but not after 5 h. These results support the probability that there is a role for free fatty acids in the regulation of somatostatin mRNA levels and somatostatin secretion in both hypothalamic and cortical cell cultures.