bluuebunny/crossref_metadata_2025_split huggingface dataset:
Proinsulin processing in electrically permeabilized rat islets of Langerhans
Authors: Slee D. J. , Jones P. M. , Howell S. L. 
Published: December 2008

ABSTRACT
Proinsulin conversion to insulin was studied using electrically permeabilized rat islets of Langerhans. Using high-performance liquid chromotography separation of radiolabelled islet proteins, we have demonstrated that conversion was dependent upon temperature, sensitive to pH and regulated by MgATP. Ammonium acetate was used to collapse the granular pH gradient, over a pH range of 3·5–7·4. Conversion was optimum at pH 4·5–5·5 and was reduced, but not abolished, at pH 7·4. Ca2+ (10 μm) and 4β-phorbol 12-myristate 13-acetate (500 nm), which are insulin secretagogues in permeabilized islets, caused no significant stimulation of conversion.
β-Adrenergic regulation of β-actin mRNA abundance in mouse parotid glands by a post-transcriptional mechanism
Authors: Roberts S. G. E. , Cope G. H. , McDonald C. J. 
Published: December 2008

ABSTRACT
In the first 24 h after a single injection of the β-adrenergic agonist isoprenaline to mice, the level of β-actin mRNA in the parotid glands increased significantly above that observed in untreated mice. The increase was transient, reaching 11 times the normal level 18 h after treatment and declining thereafter. Repeated daily doses of isoprenaline did not result in any further increase in β-actin mRNA. Nuclear transcription experiments showed that there was no increase in the transcription rate of the β-actin gene 8 h after an injection of isoprenaline, although β-actin mRNA levels were increasing at this time. Immunoblotting revealed an increase in β-actin protein in parotid gland samples after isoprenaline treatment, although the increase was not to the same extent as the mRNA, perhaps indicating that degradation of β-actin had also increased. Using immunocytochemistry it was found that β-actin was located mainly in the apical cortex of the normal acinar cell. There was a significant decrease in cortical β-actin 24 h after isoprenaline treatment, suggesting that the β-actin was under the process of redistribution.
From these data we propose that isoprenaline caused an increase in β-actin synthesis by a post-transcriptional mechanism and a redistribution of β-actin in preparation for the well-known subsequent change in morphology and function of the parotid glands.
Cloning of the inhibin/activin βB subunit gene from the Booroola Merino sheep
Authors: Rodgers R. J. 
Published: December 2008

ABSTRACT
The gene encoding the inhibin/activin βB subunit was isolated from a Booroola Merino ewe which had two copies of the unidentified fertility gene. Both the DNA sequence (>87%) and amino acid sequence (>93%) were highly homologous to those of the human and rat genes. The upstream region of the gene was similar to that of the human and rat genes, having SP1 binding sites and no TATA or CAAT boxes and, like that of the rat, but unlike that of the human gene, no cyclic AMP response elements, suggesting that the βB gene of sheep and rats is regulated differently from that of man.
Protein kinase C and the regulation of insulin secretion from pancreatic B cells
Authors: Jones P. M. , Persaud S. J. , Howell S. L. 
Published: December 2008

ABSTRACT
Protein kinase C (PKC) has been identified in islets of Langerhans and insulin-secreting tumour cells. Diacylglycerols (DAGs, the endogenous PKC activators) are generated in response to insulin secretagogues, although nutrient and non-nutrient secretagogues generate DAGs of different compositions and of different potencies as PKC activators. Exogenous activators of PKC stimulate insulin secretion from B cells, but attempts to define a physiological role for PKC by using inhibitors of this enzyme have produced ambiguous results. However, in studies using PKC-depleted B cells the loss of PKC activity does not inhibit glucose-induced insulin secretion, but markedly reduces responses to cholinergic agonists. These observations are supported by measurements of PKC activation which suggest that the enzyme is activated by cholinergic agonists, but not by nutrient secretagogues. Currently available experimental evidence therefore suggests that activation of PKC is not essential for nutrient-induced insulin secretion, but is required for the expression of a normal secretory response to cholinergic neurotransmitters.
Abrogation of dominant glucose intolerance in SJL mice by a growth hormone transgene
Authors: Bchini O. , Mehtali M. , Lathe R. 
Published: December 2008

ABSTRACT
Glucose tolerance was studied in transgenic mice (SJL × C57BL/6) expressing human GH under the control of a housekeeping promoter. Parental SJL mice were found to harbour a dominant allele, termed here glid, determining glucose intolerance in pure-bred animals and in Fl hybrids with glucose-tolerant C57BL/6 mice. Blood glucose levels in transgenic SJL × C57BL/6 hybrid mice were well controlled following glucose challenge, whereas non-transgenic hybrids failed to control their glucose adequately. Pancreatic morphology was normal in all animals. In confirmation of a physiological role for GH in glucose regulation, GH-deficient lit/lit mice were pathologically sensitive to glucose.
Growth hormone induces expression of c-jun and jun B oncogenes and employs a protein kinase C signal transduction pathway for the induction of c-fos oncogene expression
Authors: Slootweg M. C. , de Groot R. P. , Herrmann-Erlee M. P. M. , Koornneef I. , Kruijer W. , Kramer Y. M. 
Published: December 2008

ABSTRACT
Although the structure of several members of the GH receptor family has been defined, signal transduction following GH binding to its receptor has not been elucidated. Mouse osteoblasts were used to study the effect of GH on immediate early gene expression and, subsequently, the cellular signal(s) mediating this expression were analysed. GH rapidly and transiently induced the expression of c-jun and jun B in concert with the already reported expression of c-fos. The GH-induced expression of c-fos was completely blocked by the protein kinase inhibitors staurosporine and H7, indicating that the action of GH is mediated by one or several protein kinases. We next analysed the identity of the putative protein kinases in more detail by using a more specific protein kinase inhibitor, namely the ether-lipid 1-O-alkyl-2-O-methylglycerol, understood to be an inhibitor of protein kinase C (PKC). Data obtained from these studies revealed that GH-induced expression of c-fos is mediated by PKC. In addition, we observed a profound increase in formation of the PKC activator diacyglycerol upon addition of GH, a natural activator of PKC.
In conclusion, upon binding of GH to mouse osteoblasts, the receptor-mediated cellular signal involves diacyglycerol formation and activation of PKC, leading to the induction of oncogene expression. Finally, the expression of c-fos, c-jun and jun B results in an increased binding of protein complexes to AP-1 binding sites.
Cloning the cDNA for horse growth hormone and expression in Escherichia coli
Authors: Stewart F. , Tuffnell P. P. 
Published: December 2008

ABSTRACT
A 514 bp cDNA transcript coding for 78% of horse (Equus caballus) GH has been cloned and sequenced. The deduced amino acid sequence corresponded precisely to that previously obtained by protein sequencing and, in addition, provided new sequence information for the signal peptide. The missing 3′ fragment of the cDNA was reconstructed using synthetic oligonucleotides and site-specific directed mutagenesis. The complete cDNA sequence was then inserted into an expression vector (PIN-III-lppp−5) which utilizes a bacterial signal peptide to secrete the expressed product into the periplasmic space of Escherichia coli. Western blot analysis of cell lysates and periplasmic fractions prepared from cells harbouring this construct revealed significant quantities of immunoreactive GH and indicated that the bacterial signal peptide was successfully cleaved from the fusion protein on secretion. Recombinant-derived horse GH, recovered by osmotic shock from the periplasm, was active in a heterologous radioimmunoassay and a horse liver radioreceptor assay and resulted in a recovery of 0·5–2 mg GH/l cell culture. An apparent limitation on the secretion rate of horse GH in E. coli, possibly involving a block to translocation across the cytoplasmic membrane, prevented higher levels of expression being obtained.
Dose-dependent effects of angiotensin II, acetylcholine and vasopressin on the cytosolic concentration of Ca2+ in suspension primary cultures of zona fasciculata/reticularis cells from bovine adrenal cortex
Authors: Walker S. W. , Strachan M. W. J. , Nicol M. , Williams B. C. , Bird I. M. 
Published: December 2008

ABSTRACT
The effects of angiotensin II (AII), acetylcholine and vasopressin on the intracellular concentration of Ca2+ have been little studied in adrenocortical cells from the zona fasciculata/reticularis (ZFR).
Primary cultures of bovine ZFR cells maintained in suspension culture for 72 h produce cortisol in response to AII (0·1 μm), acetylcholine (0·1 mm) and vasopressin (1 μm). This response is accompanied by a breakdown of membrane phosphoinositides from [3H]inositol-prelabelled cells.
Using cells loaded with the Ca2+ indicator fura-2, the intracellular concentration of Ca2+ was measured in response to increasing doses of all three agonists and found to increase in a graded fashion in each case. The basal intracellular concentration of Ca2+ was 75±3 nm (mean±s.e.m., n=52), rising to a maximum 1·82±0·14-fold (n=6) for AII (0·1 μm), 1·35±0·05-fold (n=7) for acetylcholine (0·1 mm) and 1·27±0·10-fold (n=6) for vasopressin (1 μm).
In the case of AII and acetylcholine, agonists were added sequentially in medium of normal extracellular Ca2+ concentration (1·2 mm) or in medium in which the Ca2+ concentration was buffered to approximate to the intracellular concentration of Ca2+ (75–100 nm). Evidence was thereby obtained that both AII and acetylcholine mobilize a common intracellular pool of Ca2+.
Our findings suggest that these three agonists, all of which stimulate phospholipase C, increase intracellular Ca2+ through a mechanism which depends, at least in part, on the release of Ca2+ from a common intracellular pool.
Evidence for the involvement of multiple signalling pathways in C5a-induced actin polymerization and nucleation in human monocyte-like cells
Authors: Monk P. N. , Banks P. 
Published: December 2008

ABSTRACT
The signal transduction mechanisms involved in complement fragment C5a-induced recruitment of actin to the cytoskeleton have been investigated using U-937 cells differentiated by exposure to dibutyryl cyclic AMP. Two parameters of cytoskeletal activation were compared: F-actin formation and nucleation of polymerization of pyrenyl-actin in whole cell lysates. The dose dependency of these responses to C5a was clearly different to that observed for [3H]inositol phosphate formation and also markedly different from that observed for the production of reactive oxygen intermediates (ROI). Further evidence to dissociate inositol lipid hydrolysis from these cytoskeletal responses was obtained by treating cells with neomycin, phorbol myristate acetate and pertussis toxin and by modulating the levels of intracellular Ca2+ using quin 2. Inhibition of [3H]inositol phosphate and ROI production was not correlated with effects on actin recruitment or nucleation. In addition, these agents had differing effects on F-actin formation and nucleation activity. The results show that the production of inositol phosphates is not required for stimulating either F-actin formation or nucleation activity and also that ligand-induced polymerization of actin depends primarily upon an increase in the availability of G-actin rather than nucleation sites. These cytoskeletal responses are apparently controlled by different signalling pathways which diverge at an early stage.
Signal transduction alterations in GH12C1 rat pituitary tumour cells following treatment with 5-azacytidine
Authors: Parrow V. C. , Gordeladze J. O. , Paulssen E. J. , Aleström P. , Gautvik K. M. 
Published: December 2008

ABSTRACT
In GH12C1 rat pituitary cells treated with 5-azacytidine, the stimulatory effects exerted by vasoactive intestinal peptide (VIP), the GTP analogue guanyl-5′-yl imidodiphosphate (Gpp(NH)p), 12-O-tetradecanoyl phorbol 13-acetate, cholera toxin and pertussis toxin on the membrane-bound adenylyl cyclase were almost completely abolished. The corresponding inhibitory effect of somatostatin was increased. Alterations in adenylyl cyclase responsiveness began at the end of the drug treatment, and were most pronounced on day 5 after removal of 5-azacytidine. The cells subsequently and completely recovered after 10 days in the absence of the drug. Measurements of cholera toxin- and VIP-enhanced cyclic AMP levels in intact cells confirmed these results, and VIP appeared to have no stimulatory effect on GH secretion after 5-azacytidine treatment. Down-regulation of Gαs RNA also occurred on day 5 after cessation of drug treatment. ADP-ribosylation subsequent to stimulation with pertussis toxin was markedly increased, indicating an enhancement of Gαi and/or GGαo. Furthermore, both basal and Gpp(NH)p-stimulated phospholipase C activities were augmented by pre-exposure to 5-azacytidine.
Treatment of GH12C1 rat pituitary tumour cells with 5-azacytidine therefore causes a marked but temporary increase in the ratio of Gαi/Gαs protein levels.
17β-Hydroxysteroid dehydrogenases in human breast tissues: purification and characterization of soluble enzymes and the distribution of particulate and soluble forms in adipose, non-adipose and tumour tissues
Authors: Mann V. Z. , Newton C. J. , Tait G. H. 
Published: December 2008

ABSTRACT
17β-Hydroxysteroid dehydrogenase (17β-HSD) is present in multiple forms in human breast tissue. One soluble form, with a molecular weight of approximately 35 kDa, was purified to near homogeneity from whole normal breast tissue. This form catalysed the oxidation of oestradiol and the reduction of oestrone, with NADP+ and NADPH as the preferred coenzymes. Three other soluble forms with higher molecular weights (in the range 50–80 kDa) were isolated. They catalysed the oxidation of oestradiol but not the reduction of oestrone, and all of them had properties very different from those of the low molecular weight enzyme.
Activities of 17β-HSD were measured in particulate and soluble fractions from normal breast adipose and non-adipose tissues, and from breast tumours obtained from post-menopausal women, in the oxidative direction with NAD+ and NADP+ as coenzymes and in the reductive direction with NADH and NADPH as coenzymes. Particulate fractions from tumours had much higher oxidative and reductive activities than those from normal tissues. Soluble fractions from tumours had higher oxidative activities than those from the normal tissues but similar reductive activities. The major soluble form of 17β-HSD in adipose tissue was the 35 kDa enzyme which had both oxidative and reductive activities. In contrast, the majority of the soluble activity in non-adipose tissue was due to enzymes, with molecular weights in the range 50–80 kDa, which had oxidative activity only. The soluble fractions of tumours, like those of non-adipose tissue, contained enzymes with molecular weights in the range 50–80 kDa. In addition, they contained a 35 kDa enzyme with properties different from those of the enzyme with the same molecular weight present in adipose tissue.
Lack of expression of the mRNA encoding a major protein of the mouse vas deferens after neonatal exposure to oestrogens
Authors: Pailhoux E. , Martinez A. , Jean-Faucher Ch. , Veyssière G. , Jean Cl. 
Published: December 2008

ABSTRACT
We have previously characterized an androgen-inducible secretory protein from the mouse vas deferens (MVDP), and a cDNA to its mRNA has been obtained. This report describes altered MVDP gene expression after neonatal exposure to oestrogens. As shown by immunohistochemistry and Western blot analysis, MVDP was missing in the vas deferens from adult mice neonatally exposed to oestrogens. Northern blot analysis showed that the expression of MVDP mRNA was also suppressed. Exogenous testosterone was unable to stimulate MVDP production (either message or protein) in neonatally oestrogenized males. The results suggest that the alterations in gene expression in the oestrogen-exposed vas deferens reflect changes in the programme of differentiation of the organ itself.
Genetic variation of the glucocorticoid receptor from a steroid-resistant primate
Authors: Brandon D. D. , Markwick A. J. , Flores M. , Dixon K. , Albertson B. D. , Loriaux D. L. 
Published: December 2008

ABSTRACT
The neotropical cotton-top marmoset (Saguinus oedipus) is a New World primate known to have markedly increased total and free plasma cortisol concentrations when compared with Old World primates including man. The relative end-organ 'resistance' to glucocorticoids found in various New World primates has been attributed to a glucocorticoid receptor (GR) with diminished affinity for glucocorticoids. It has been demonstrated that the marmoset GR has approximately tenfold lower binding affinity for dexamethasone when compared with the human GR. We have examined the primary structure of the marmoset GR by molecular cloning and sequencing of GR functional domains. A library of cDNA clones was constructed in the phage vector λgt10 using poly(A)+ RNA from a marmoset-derived lymphoid cell line, and screened using the human GR cDNA. DNA sequencing determined 76 individual nucleotide substitutions in the coding region of the marmoset GR. Comparison of the marmoset GR nucleotide sequence with the human GR cDNA coding region indicated an overall sequence homology of about 97%. Thirty of the nucleotide substitutions lead to alterations in the predicted amino acid sequence (28 amino acid substitutions) of the marmoset GR.
The size of the marmoset GR predicted from the 778 amino acids is approximately 90 000 which is in agreement with previous size estimates of the human and marmoset GRs. Alterations of amino acid sequence in the marmoset GR were greatest towards the amino terminus, including the τ1 domain putatively involved in transcriptional activation. The DNA-binding domain contained an additional codon (arginine). Comparison of the DNA-binding domain of the marmoset GR with other members of the steroid receptor superfamily indicates that the additional arginine occurs in the same position as other amino acid insertions within the interfinger region of the human androgen receptor and the erb-A proto-oncogene. There are only four missense substitutions within the steroid-binding domain. Two of these substitutions occur within the transducing site which has been associated with binding of the GR to a 90 kDa heat shock protein. These data suggest that diminished GR affinity for glucocorticoids in the marmoset may be due to alterations in the primary structure of one or more functional domains of the GR gene. In addition, other important regulatory functions, such as transcriptional activation, DNA binding and receptor transduction, may also be affected.
Stimulatory effect of oestradiol-17β and tamoxifen on gross cystic disease fluid protein 15 000 production and mRNA levels in T47D human breast cancer cells
Authors: Dejardin L. , Gol-Winkler R. , Collette J. , Delvenne C. , Carlisi I. , Haagensen D.E. , Franchimont P. 
Published: December 2008

ABSTRACT
Oestradiol-17β and tamoxifen regulate the synthesis of a gross cystic disease fluid protein (GCDFP-15) in T47D human breast cancer cells. Dose-response curves of GCDFP-15 mRNA contents and GCDFP-15 levels in culture media and cells versus hormone or antihormone concentration have been established. Production of GCDFP-15 was increased by oestradiol-17β, tamoxifen and 4-OH tamoxifen. The effect of tamoxifen and 4-OH tamoxifen was greater than the effect of oestradiol-17β. Moreover, oestradiol-17β and 4-OH tamoxifen acted synergystically in enhancing GCDFP-15 release. The strong oestrogenic effect of the antioestrogen tamoxifen in regulating GCDFP-15 may reflect an unusual interaction between the tamoxifen-oestrogen receptor complex and the DNA oestrogen-responsive elements. As oestrogen control of GCDFP-15 depends also on the cell line studied, investigation of GCDFP-15 could extend our knowledge of the possible mechanism of action of oestrogens or antioestrogens.
Evidence that androgen-binding protein endocytosis in vitro is receptor mediated in principal cells of the rat epididymis
Authors: Guéant J.-L. , Fremont S. , Felden F. , Nicolas J.-P. , Gerard A. , Leheup B. , Gerard H. , Grignon G. 
Published: December 2008

ABSTRACT
We have studied the binding of [ I-iodo]androgen-binding protein (ABP) and of [3H]Δ6-testosterone photoaffinity-labelled ABP to receptors in the plasma membrane of rat epididymal cells in three ways: ABP binding to a Triton X-100-solubilized membrane extract, ABP binding to isolated epithelial cells in suspension and autoradiography of segments of dissected epididymides after in-vitro intraluminal injection of labelled ABP. The binding of iodinated ABP to the receptor was similar to that of photoaffinity-labelled ABP in gel filtration. The ABP-receptor complex was eluted from Superose 6 gels as an aggregate, with a molecular mass of 2000 kDa. It was separated into two peaks by sucrose gradient ultracentrifugation, with respective sedimentation coefficients of 18.4 and 9.0s. The activity of the receptor (ABP-binding capacity/mg protein) was tenfold higher in the caput than in the cauda. The binding of ABP to the receptor was pH dependent, being almost abolished at pH <4. The binding at 4°C of photoaffinity-labelled ABP to epithelial cells corresponded to two types of binding sites. The numbers of high-affinity and low-affinity sites per cell were 1600 and 7700 respectively; the association constants of these sites were 67.9 and 2.8litres/ nm respectively. The binding was decreased by treatment of the cells with trypsin or incubation in the presence of ED TA. The binding in vitro of labelled ABP to the epididymis epithelium reached a maximum after about 20 min at 4°C. In the autoradiographic study the tracer was found to be closely associated with coated pits, coated vesicles, endosomes and pale multivesicular bodies. Treatment of rats with cycloheximide significantly reduced the uptake of the tracer. Perfusion in vitro of epididymides with chloroquine produced a fourfold increase of the tracer in endosomes and multivesicular bodies.