bluuebunny/crossref_metadata_2025 huggingface dataset:
Pancreatic islet response to dicarboxylic acid esters in rats with type 2 diabetes: enzymatic, metabolic and secretory aspects
Authors: Rasschaert J , Giroix M-H , Conget I , Mercan D , Leclercq-Meyer V , Sener A , Portha B , Malaisse W J
Published: December 2008

This study aimed to compare the metabolic and secretory responses of pancreatic islets from animals with non-insulin-dependent diabetes to d-glucose with the effects of the methyl esters of succinic acid (SME) and glutamic acid (GME). The insulin secretory response to d-glucose was impaired in islets from rats with diabetes which was either inherited (Goto—Kakizaki (GK) rats) or acquired (streptozotocin-treated (STZ) rats). This coincided with a preferential alteration of oxidative relative to total glycolysis in intact islets and a selective defect of FAD-linked mitochondrial glycerophosphate dehydrogenase (m-GDH) in islet homogenates. This enzymatic defect was also found in purified B cells from STZ rats. It contrasted both with unaltered activities of glutamate dehydrogenase and succinate dehydrogenase in the islets of diabetic animals and with a normal or even increased activity of m-GDH in the livers of GK and STZ rats. The oxidation of [1,4-14C]SME and [U-14C]GME appeared decreased in islets of GK or STZ animals when compared with control rats, but no significant difference between control and diabetic rats was observed when the oxidative data were expressed relative to the rate of [U-14C]GME hydrolysis. Nevertheless, the absolute values for insulin release evoked by a non-metabolized analogue of l-leucine (BCH), by SME and by the association of BCH with either SME or GME were invariably lower in islets of GK and STZ rats than in those of control animals. These findings indicate that the enzymatic and metabolic situation in islets of GK and STZ rats could allow the expression of the insulinotropic potential of SME and GME, even if their immediate secretory effects are impaired in the islets of these diabetic animals.
Mechanism of action of γ-aminobutyric acid on frog melanotrophs
Authors: Desrues L , Vaudry H , Lamacz M , Tonon M C
Published: December 2008

We have previously demonstrated that γ-aminobutyric acid (GABA) is a potent regulator of secretory and electrical activity in melanotrophs of the frog pituitary. The aim of the present study was to investigate the intracellular events which mediate the response of melanotrophs to GABA.

We first observed that GABA (1–100 μm inhibited both basal and forskolin-stimulated cyclic AMP (cAMP) formation. The inhibitory effect of GABA on cAMP levels was mimicked by the GABAB receptor agonist baclofen (100 μm) and totally abolished by a 4-h pretreatment with pertussis toxin (01 μg/ml). In contrast, the specific GABAA agonist 3-aminopropane sulphonic acid (3APS) did not affect cAMP production. Both GABA and 3APS (100 μm each) induced a biphasic effect on α-MSH release from perifused frog neurointermediate lobes, i.e. a transient stimulation followed by an inhibition of α-MSH secretion. Administration of forskolin (10 μm) prolonged the stimulatory phase and attenuated the inhibitory phase evoked by GABA and 3APS, indicating that cAMP modulates the response of melanotrophs to GABAA agonists. Ejection of 3APS (1 μm) in the vicinity of cultured melanotrophs caused a massive increase in intracellular calcium concentration ([Ca2+]i). The stimulatory effect of 3APS on [Ca2+]i was abolished when the cells were incubated in a chloride-free medium. The formation of inositol trisphosphate was not affected by 3APS, suggesting that the increase in [Ca2+]i cannot be ascribed to mobilization of intracellular calcium stores. ω-Conotoxin did not alter the secretory response of frog neurointermediate lobes to 3APS, while nifedipine blocked the stimulation of α-MSH secretion induced by 3APS.

In conclusion, the present data indicate that, in frog pituitary melanotrophs, (i) the stimulatory phase evoked by GABAA agonists can be accounted for by an influx of calcium through L-type calcium channels, (ii) the inhibitory effect evoked by GABAB agonists can be ascribed to inhibition of adenylate cyclase activity and (iii) cAMP attenuates the inhibitory phase evoked by GABAA agonists. Taken together, these data suggest that activation of GABAB receptors may modulate GABAA receptor function.
Sexual dimorphism and growth hormone induction of murine pheromone-binding proteins
Authors: Johnson D , Al-Shawi R , Bishop J O
Published: December 2008

A number of structurally very similar pheromone-binding proteins (major urinary proteins; MUPs) are synthesized in mouse liver and rapidly excreted in the urine. Male and female inbred mice display different characteristic patterns of MUP expression. Here we present a detailed study of the RNA and protein products corresponding to specific MUP genes previously isolated from genomic DNA of the Balb/c strain. By in vitro transcription of equivalent cDNA clones, translation of the resulting RNA in the reticulocyte lysate system and isoelectric focusing, the protein products of genes BL1, BS1 and BS6 were shown to be MUP 2a, MUP 2b and MUP 4 respectively. MUPs 2a and 2b were shown to be abundant both in Balb/c male urine and among the translation products of total Balb/c male liver mRNA. Two oligodeoxynucleotide probes, oBL1A and oBS1, selective for BL1 and BS1 mRNA respectively, were chemically synthesized. mRNA that hybridized with these probes (oBL1A mRNA and oBS1 mRNA) was present at different characteristic levels in the Balb/c and C57BL/6 inbred strains. In both strains the level of expression was much higher in males than females and the male/female expression ratio of oBS1 RNA was higher than that of oBL1A RNA. Comparison of these mRNA levels with the amounts of different MUP proteins present in urine and the translation products of liver mRNA indicated that proteins other than MUP 2a and MUP 2b are coded for by the C57BL/6 oBL1A and oBS1 mRNAs.

C57BL/6 mice homozygous for the lit mutation are GH deficient and transcribe MUP genes at a level much lower than that obtaining in normal mice of either sex, indicating that transcription is induced by GH in both males and females. When lit/lit mice were treated with GH under two different regimes, MUP gene transcription was partially induced to different degrees and the level of oBL1A mRNA was induced more highly than that of oBS1 mRNA. Thus there exists a correlation between the inducibility of these mRNAs and their level of expression in females relative to males; oBL1A mRNA is both more highly expressed in females and more readily induced by GH than oBS1 mRNA. This suggests that the male and female expression patterns are due to differential inducibility of different MUP genes together with a stronger inducing stimulus in males. GH administered continuously by infusion repressed MUP gene expression. We interpret this to mean that induction is due to intermittent GH stimulation in both sexes and that the longer interpulse interval reported to occur in males leads to more effective induction than the shorter interpulse interval observed in females.
Sequence analysis of vasotocin cDNAs of the lamprey, Lampetra japonica, and the hagfish, Eptatretus burgeri: evolution of cyclostome vasotocin precursors
Authors: Suzuki M , Kubokawa K , Nagasawa H , Urano A
Published: December 2008

We determined the nucleotide sequences of cDNAs encoding precursors of vasotocin (VT) from two cyclostomes, the lamprey Lampetra japonica and the hagfish Eptatretus burgeri, for estimation of their phylogenetic relationships. Although only 47% similarity was found between the VT cDNAs, the predicted VT precursors of the lamprey and the hagfish were both composed of a signal peptide, VT, Gly-Lys-Arg and a neurophysin, as has been shown for precursors of vasopressin (VP) family hormones, including VP, VT and molluscan conopressin.

The central region of the lamprey neurophysin was very similar to those of previously characterized gnathostome neurophysins. Conspicuously, all the positions of 14 Cys residues were conserved in the lamprey neurophysin. The C-terminal region did not have a distinctive Leu-rich core segment, which is always found in the glycopeptide (copeptin) moiety of VP precursors. In contrast, the hagfish neurophysin showed at least two insertions and one deletion in the conserved central region including 14 Cys residues, but contained a potential N-linked glycosylation site and had a high proportion of Leu residues in the C-terminal region, like the neurophysin of another hagfish, Eptatretus stouti.

The evolutionary relationships of the precursors of VP family hormones among the lamprey, hagfish, gnathostomes and a mollusc were estimated by a maximum likelihood method. The phylogenetic tree with the highest bootstrap probability showed that the lamprey VT precursor is more closely related to the gnathostome VT and VP precursors than to the hagfish VT precursors.
9-cis-retinoic acid is more effective than all-trans-retinoic acid in upregulating expression of the α-fetoprotein gene
Authors: Wan Y-J Y , Pan T , Wang L , Locker J , Wu T-C J
Published: December 2008

In McA-RH 8994 rat hepatoma cells, all-transretinoic acid (t-RA) induces expression of the α-fetoprotein (AFP) and albumin genes and results in a phenotype similar to differentiated fetal hepatocytes. The present study elucidated the mechanism involved in AFP gene regulation mediated by retinoic acid. Northern blot analyses demonstrated that 9-cis-retinoic acid (c-RA), a ligand for retinoid x receptors (RXRs), also induced expression of the AFP gene in McA-RH 8994 cells. The induction was time- and dose-dependent. Northern blots and transfection assays using the 7·3 kb full-length regulatory region of the AFP gene demonstrated that c-RA was more effective than t-RA in regulating expression of the AFP gene. At 10−7m, c-RA increased AFP mRNA 5-fold and chloramphenicol acetyltransferase (CAT) activity 2·5-fold. In contrast, t-RA at a concentration of 10−7 m exerted no significant effect; 10− 6 to 10−5 m t-RA was needed to affect AFP gene expression. These data suggested that activation of RXRs is essential for the regulation of the AFP gene. Co-transfection experiments revealed that overexpression of RXRα in McA-RH 8994 cells further enhanced the CAT activity induced by c-RA. In addition, c-RA did not alter the half-life of AFP mRNA. Thus, RXRα may play a crucial role in transcriptional regulation of the AFP gene and in controlling hepatocyte phenotype.
Cloning and characterization of the cDNA encoding the human neuromedin U (NmU) precursor: NmU expression in the human gastrointestinal tract
Authors: Austin C , Lo G , Nandha K A , Meleagros L , Bloom S R
Published: December 2008

Many peptide precursors encode more than one bioactive peptide. Recent cloning of the rat neuromedin U (NmU) precursor revealed potential proteolytic processing sites which may generate three associated peptides in addition to the NmU peptide, which is known to have potent uterine contractile effects. To assess the degree of evolutionary conservation, which often suggests conserved biological function and hence physiological importance, we have cloned and sequenced the cDNA encoding the human NmU precursor. Sequence analysis revealed a 174 amino acid human precursor containing the 25 residue NmU peptide near the C terminus of the precursor. The human message sequence was 74% homologous with that of the rat, indicating evolutionary conservation of the precursor between these two species. Four out of five of the putative proteolytic processing sites, first revealed in the rat precursor, were conserved in the human precursor, indicating a similar processing mechanism in both species. Two such processing sites flank a 33 residue peptide sequence which differed in only two amino acids compared with the rat homologue. This conservation suggests a possible biological role for this putative peptide.

Northern blot analysis of human gastrointestinal tissues revealed a similar level of mRNA throughout the gastrointestinal tract. RIA using a porcine specific assay showed the highest levels of peptide in the jejunum samples.
Differential control of type-I iodothyronine deiodinase expression by the activation of the cyclic AMP and phosphoinositol signalling pathways in cultured human thyrocytes
Authors: Beech S G , Walker S W , Arthur J R , Lee D , Beckett G J
Published: December 2008

The effects of TSH and the activation of the cyclic AMP (cAMP) and Ca2+-phosphatidylinositol (Ca2+-PI) cascades on the activity and expression of the selenoenzyme thyroidal type-I iodothyronine deiodinase (ID-I) have been studied using human thyrocytes grown in primary culture. Stimulation of ID-I activity and expression was obtained with TSH and an analogue of cAMP, 8-bromo-cAMP. In the presence or absence of TSH, the addition of the phorbol ester, phorbol 12-myristate 13-acetate (PMA) together with the calcium ionophore A23187, caused a decrease in ID-I activity; a decrease in ID-I expression was also observed as assessed by cell labelling with [75 Se]selenite. PMA alone had no effect on ID-I activity in the presence or absence of TSH. A23187 alone produced a small but significant reduction in ID-I activity, but only in TSH-stimulated cells. These data provide evidence that the expression of thyroidal ID-I is negatively regulated by the Ca2+-PI cascade, and positively regulated by the cAMP cascade.
Identification of multiple human calcitonin receptor isoforms: heterologous expression and pharmacological characterization
Authors: Egerton M , Needham M , Evans S , Millest A , Cerillo G , McPheat J , Popplewell M , Johnstone D , Hollis M
Published: December 2008

The human breast carcinoma cell line T47D is known to express high-affinity calcitonin receptors (CTRs). PCR amplification of the CTR cDNA from T47D mRNA resulted in the identification of two different cDNAs that encode distinct receptor isoforms, hαCTR and hβCTR. The two cDNAs are identical except that the hαCTR cDNA contains a 48 bp insert sequence that encodes a 16 amino acid domain in the first cytosolic loop of the receptor. Stable transfection of each receptor cDNA into murine erythroleukaemia (MEL) cells resulted in the expression of receptors with high affinity for radiolabelled salmon calcitonin (hαCTR Kd 0·09 nm, hβCTR Kd 0·12 nm). Ligand competition binding studies did not reveal any significant pharmacological difference between the receptor isoforms. In transfected MEL cells and COS-1 cells the hβCTR isoform was expressed at tenfold higher levels than the hαCTR. A reporter gene assay that monitored the coupling of CTR to adenylate cyclase by increases in β-galactosidase activity indicated that both receptors were able to stimulate cyclic AMP production in response to ligand binding.
Microsatellite polymorphism analysis allows the individual assignment of the rat 11β-hydroxylase gene (Cyp11b1) and the rat aldosterone synthase gene (Cyp11b2) to chromosome 7 using rat × mouse somatic cell hybrids and identifies differences between and within various rat strains
Authors: Inglis G C , Kenyon C J , Szpirer C , Klinga-Levan K , Sutcliffe R G , Connell J M C
Published: December 2008

Mouse hepatoma × rat hepatocyte hybrids that segregate rat chromosomes were used to determine the chromosomal location of the rat genes encoding 11 β-hydroxylase and aldosterone synthase (Cyp11b1 and Cyp11b2 respectively). By means of species-specific restriction fragments and microsatellite markers both genes were mapped to rat chromosome 7. The Cyp11b1 microsatellite marker was subsequently found to vary in length between and within rat strains. Furthermore, we compared the sequences of Cyp11b1 markers in two genetically hypertensive strains of rat with their normotensive counterparts. Previous studies have indicated that 11β-hydroxylase activities in Milan and Lyon hypertensive strains are different from their respective genetic controls. The Cyp11b1 microsatellite regions from Lyon hypotensive and normotensive strains of rat were similar and were both shorter by 15 bases than that of the Lyon hypertensive strain. The Cyp11b1 marker in Milan hypertensive (MHS) and normotensive (MNS) strains differ from all the Lyon strains and from each other. The MHS marker is 12 bases shorter than that of MNS rats. These differences in microsatellite length may provide useful polymorphic markers in co-segregation studies of genetic hypertension in rats.
Expression, distribution, regulation and function of IGFs in the ovine fetal pituitary
Authors: Lü F , Yang K , Han V K M , Challis J R G
Published: December 2008

Activation of the fetal pituitary-adrenal axis is crucial for fetal organ maturation and the onset of parturition in sheep. Many factors including corticotrophin-releasing hormone (CRH) and arginine vasopressin secreted from the hypothalamus, and growth factors produced within the pituitary may be involved in the regulation of maturation of the fetal pituitary gland. IGFs have mitogenic and differentiation-promoting capacities in a variety of organs and are synthesized as paracrine factors within developing tissues. However, there is little information concerning the synthesis, distribution, regulation and function of IGFs in the fetal pituitary gland at different times during pregnancy. Therefore, we have localized IGF-I and IGF-II mRNAs and peptides, and determined the effect of cortisol on the level of IGF-II mRNAs in the pituitary glands of developing sheep fetuses. We examined the possible effects of IGFs on corticotroph function in cultures of adenohypophysial cells from term fetuses.

Seven species of IGF-II transcripts of 1·2–6·0 kb were identified by Northern blot analysis in the pituitary gland of fetuses between day 60 of gestation and term (day 145). The levels of IGF-II mRNAs did not change significantly during pregnancy, although there was a trend for the presence of higher levels of IGF-II mRNAs at day 60 of gestation. IGF-I mRNA was not detectable. By in situ hybridization, IGF-II mRNA was localized to non-endocrine cells and to cells lining the blood vessels of the pars distalis, to some presumed endocrine cells in the pars distalis and pars intermedia, and to clusters of cells in the pars nervosa. In contrast, IGF-I and IGF-II peptides were detected in the presumed endocrine cells in the pars distalis and pars intermedia but not in the pars nervosa. Incubation of adenohypophysial cells from term fetuses with IGF-I, but not IGF-II, for 48 h increased specific 125I-Tyr-ovine CRH binding. However, neither IGF-I nor IGF-II had any significant effects on the basal or CRH-stimulated immunoreactive (ir)-ACTH output, the level of POMC mRNA or the number of ir-ACTH positive cells. Infusion of cortisol to fetuses starting at day 96 of gestation for 100 h or at days 120–125 of gestation for 84 h did not affect the level of IGF-II mRNAs in the pars distalis but decreased the levels of POMC mRNA.

These results are consistent with IGFs having the potential to influence fetal pituitary function, although probably on cell types other than the corticotrophs. The likely sources of IGFs may be predominantly local (IGF-II) or from extrapituitary sources (IGF-I).
Multiple isoforms of the oestrogen receptor in endometrial cancer
Authors: Marsigliante S , Muscella A , Ciardo V , Puddefoot J R , Leo G , Vinson G P , Storelli C
Published: December 2008

We evaluated the presence and variability of oestrogen receptor (ER) isoforms in endometrial cancer by using [3H]oestradiol-labelled ERs and the H222 monoclonal antibody obtained from the Abbott enzyme immunoassay kit.

Using isoelectric focusing (IEF), endometrial ER was shown to be composed of four different species, with pI values of 6·1, 6·3, 6·6 and 6·8, indistinguishable from the isoforms found in normal rat uterus, and human breast and larynx carcinomas. The isoforms at pI 6·3, 6·6 and 6·8, all sedimenting at 4S by sucrose gradient fractionation, showed, on two-dimensional SDS electrophoresis, relative masses of 50, 70 and 65 kDa respectively, equal to the masses previously found in breast cancer. These isoforms did not alter their pI values during IEF fractionation performed in a linear gradient of urea, while the pI 6·1, sedimenting at 8S, generated a new isoform at about 9 mol/l urea with pI 7·2 and a relative mass of 65 kDa. The urea-dissociated isoform (pI 7·2) was able approximately to double the antibody binding with respect to the non-dissociated oligomer, which suggested that some epitopes are 'masked', i.e. not accessible to the antibodies when ER is present in its complexed form. The evidence thus suggested that the oligomer at pI 6·1 contained a single 65 kDa ER form which, as a monomer, focused at pI 7·2.

The variability in the ER isoform profile found in endometrial cancer was similar to the variability previously reported in breast and larynx carcinomas. The balance between these isoforms could be a dynamic parameter involved in the functionality of this receptor and consequently in cell transformation.
Calcium-activated proteases in the bovine parathyroid gland: potential role in degradation of parathyroid hormone to peptide fragments
Authors: Watson P H , Mortimer S T , Wang K K W , Croall D E , Hanley D A
Published: December 2008

Our studies suggest that protein kinase C is involved in low calcium (Ca2+)-stimulated secretion of parathyroid hormone (PTH) but not directly in high Ca2+-stimulated intracellular degradation of PTH to secreted carboxyl-terminal fragments (C-PTH), an important component of Ca2+-regulated PTH secretion. The present study was undertaken to determine the presence of calciumactivated proteases, 84 kDa (micro)-calpain and 80 kDa (milli)-calpain, in the bovine parathyroid, and whether they could degrade PTH to C-terminal fragments. Immunocytochemistry of bovine parathyroid tissue using antibodies raised against bovine heart micro- and milli-calpain detected both isoforms of calpain. Western blotting of total bovine parathyroid cell protein prepared from primary cell cultures confirmed the presence of both isoforms of calpain, demonstrated by specific milli- and micro-calpain bands. Purified bovine PTH (bPTH) was incubated in vitro with human erythrocyte micro-calpain and the cleavage products were separated by reverse-phase HPLC. Eluant fractions were assayed with an RIA with equimolar sensitivity to C-PTH and bPTH, and peak areas integrated. Micro-calpain produced a C-PTH peak from bPTH which co-eluted with the major C-PTH secreted by parathyroid cells in culture. C-PTH production by micro-calpain, expressed as per cent area under the curve, increased from 0% in the absence of either micro-calpain or Ca2+, to 71·5% when a 5:1 molar ratio of bPTH to calpain was used. Amino acid sequencing and analysis of the immunoreactive PTH cleavage products indicated the presence of two fragments of bPTH in the C-PTH peak, bPTH47–84 and bPTH69–84. In summary, both isoforms of calpain are present in the bovine parathyroid and calpains may play a role in the Ca2+-dependent degradation of PTH to secreted C-terminal fragments.
Evidence for alternative splicing of the chicken vasoactive intestinal polypeptide gene transcript
Authors: Talbot R T , Dunn I C , Wilson P W , Sang H M , Sharp P J
Published: December 2008

Two forms of chicken vasoactive intestinal polypeptide (VIP) mRNA have been identified by reverse transcription (RT)-PCR and RNase protection assay. The shorter form of chicken VIP mRNA encodes a protein that does not contain an analogue of rat peptide histidine isoleucine (PHI) 1–27 or human peptide histidine methionine 1–27. The larger form encodes both VIP and a chicken analogue of PHI 1–27 in the same protein product.

Three VIP cDNAs isolated from a chicken hypothalamic cDNA library were derived from the shorter mRNA. Sequence analysis of the longest clone identified an open reading frame that codes for a 165 amino acid preproVIP protein and contains two polyadenylation signals. In situ hybridisation with an oligonucleotide probe from the VIP cDNA sequence showed that VIP-encoding mRNA occurs in cells in the basal hypothalamus, an area of the brain known to contain VIP neurosecretory neurones. RT-PCR of total RNA from liver, kidney, gut, pancreas, pituitary, cerebellum, forebrain and hypothalamus, using primers derived from the VIP cDNA sequence, showed that the shorter form of VIP mRNA is present in all of these tissues. The sequence of the longer form of VIP mRNA was obtained by sequencing a portion of the VIP gene from genomic DNA. This revealed a potential exon that was not represented in the VIP cDNA clones analysed. RT-PCR with primers from this sequence showed that it was expressed in the gut and hypothalamus. RNase protection assays confirmed the presence of the two forms of mRNA in gut and hypothalamus. The relative proportions of the two mRNA forms were: 97·8% VIP only, 2·2% PHI/VIP in the hypothalamus and 98·5% VIP only, 1·5% PHI/VIP in the gut.

In conclusion, chicken VIP mRNA is alternatively spliced. The shortest form, which encodes a preproprotein containing only the VIP peptide, is the most abundant. The longer form of chicken VIP mRNA encodes a preproprotein containing sequences for both VIP and a chicken form of PHI.
Influence of xenobiotics on rainbow trout liver estrogen receptor and vitellogenin gene expression
Authors: Flouriot G , Pakdel F , Ducouret B , Valotaire Y
Published: December 2008

Rainbow trout hepatocyte primary culture was used to test the influence of some xenobiotics on the expression of two genes implicated in reproduction, those for the estrogen receptor (ER) and vitellogenin (Vg). We showed that chlordecone, nonylphenol, a polychlorobiphenol (PCB) mixture (Aroclor 1245) and lindane were able to induce ER and Vg mRNA accumulation. Antiestrogens, 4-hydroxytamoxifen and ICI 164,384, prevented the effects of the xenobiotics, indicating that the induction of gene expression is mediated by the ER. Among these four xenobiotics, only chlordecone and nonylphenol were able to displace the binding of [3H]estradiol to ER-enriched COS-1 extracts, and to activate an estrogen-dependent reporter gene (ERE-TK-CAT) cotransfected with an expression vector containing ER cDNA. The results suggest that chlordecone and nonylphenol are direct inducers of rainbow trout ER and Vg gene expression, whereas PCBs and lindane act through their hepatic metabolites. Moreover, pentachlorophenol acts as an antagonist of the induction by estradiol of rainbow trout ER and Vg gene expression.
Secretagogue-induced [Ca2+]i changes in single rat pancreatic islets and correlation with simultaneously measured insulin release
Authors: Martin F , Reig J A , Soria B
Published: December 2008

The effects of secretagogues (glucose, tolbutamide and phorbol esters) on simultaneously measured intracellular free calcium concentration ([Ca2+]i) and insulin release were studied in rat pancreatic islets of Langerhans. Stimulatory concentrations (11 mm) of glucose caused a transient [Ca2+]i increase followed by an almost flat second phase. Increasing glucose concentrations to 16·7 mm in steps caused a further increase in [Ca2+]i. In contrast with mouse islets, rat islets scarcely showed glucose-induced [Ca2+]i oscillations. Digital image analysis showed that [Ca2+]i changes occurred synchronously across the whole islet. As expected, simultaneously measured insulin release was biphasic with a clear second phase. This clearly indicated that in rat islets there is a lack of correlation between [Ca2+]i and insulin release. This was further explored using agents which separately promoted the first (tolbutamide, 200 μm) and second (phorbol-12-myristate-13-acetate; PMA; 5 nm) phases of insulin release. Tolbutamide induced a transient increase in [Ca2+]i paralleled by a transient increase in insulin release, whereas PMA induced a slow increase in insulin release without a clear change in [Ca2+]i. These results suggest that in rat islets the first phase of insulin release is calcium dependent, whereas the second phase is related to the activation of protein kinase C (PKC). However, the glucose-induced second phase of insulin release did not coincide with an increase in membrane-associated PKC activity. Other messengers may contribute to this late phase of insulin release.
Intrauterine injection of ovine interferon-τ alters oestrogen receptor and oxytocin receptor expression in the endometrium of cyclic ewes
Authors: Spencer T E , Ing N H , Ott T L , Mayes J S , Becker W C , Watson G H , Mirando M A , Bazer F W
Published: December 2008

This study determined the effects of intrauterine injections of recombinant ovine interferon-τ (roIFN-τ; 2 × 107 antiviral units/day) or control proteins (6 mg/day) from day 11 to day 14 post-oestrus (oestrus=day 0) on endometrial expression of receptors for oestrogen, progesterone and oxytocin in cyclic ewes. Plasma concentrations of progesterone were greater on day 15 in ewes receiving roIFN-τ compared with control proteins (P<0·02, treatment × day). Ewes injected with roIFN-τ had lower endometrial levels of oestrogen receptor mRNA (P<0·10) and protein (P<0·01) on day 15 compared with ewes receiving control proteins. In situ hybridization analysis indicated that oestrogen receptor mRNA was more abundant in the luminal and glandular epithelium of control ewes compared with roIFN-τ-treated ewes. Immunoreactive oestrogen receptor was also present in the luminal and glandular epithelium of control, but not roIFN-τ-treated ewes. Endometrial levels of progesterone receptor mRNA and protein were not different (P>0·10) between control and roIFN-τ-treated ewes. In situ hybridization analyses indicated that progesterone receptor mRNA abundance was low in endometrial epithelium and stroma of both control and roIFN-τ-injected ewes. Immunoreactive progesterone receptors were present in the endometrial stroma and epithelium of control ewes, but confined to the stroma of roIFN-τ-treated ewes. Oxytocin receptor density was lower (P<0·10) in the endometrium of ewes injected with roIFN-τ than control proteins; however, oxytocin receptor affinity was not affected (P>0·10) by treatment. Concentrations of 13,14-dihydro-15-keto-prostaglandin F2α (PGFM) were not increased by exogenous oxytocin administration in control and roIFN-τ-treated ewes on days 10 or 12 post-oestrus. However, on day 14, control ewes responded to oxytocin with increased plasma concentrations of PGFM, whereas ewes receiving roIFN-τ remained unresponsive to oxytocin. These results indicate that the antiluteolytic effects of IFN-τ are to prevent increases in endometrial oestrogen receptor mRNA and protein and oxytocin receptor density which abrogates uterine release of prostaglandin F2α during maternal recognition of pregnancy. IFN-τ may inhibit the synthesis of oestrogen receptor mRNA by a transcriptional or post-transcriptional regulatory mechanism to suppress oxytocin receptor formation during early pregnancy in ewes.
Prolactin and growth hormone mRNA and protein characterization in SMtTW rat pituitary tumours
Authors: Delhase M , Rajas F , Verdood P , Remy C , Chevallier P , Velkeniers B , Trouillas J , Hooghe-Peters E L
Published: December 2008

We have combined different techniques to analyse passages of five different rat spontaneous pituitary tumours (SMtTW) that were transplanted under the kidney capsule. These tumours were secreting prolactin (PRL), GH or both hormones. RIA, immunocytochemistry (ICC) and Western blot analysis were applied to characterize the hormone(s) stored (ICC and Western blot) and secreted (RIA). mRNA content was analysed by PCR, Northern blot analysis and in situ hybridization.

The data point not only to the reliability of the techniques used at both protein and RNA levels for each tumour studied but also to the complementarity of some techniques. For example, whereas Northern blot analysis demonstrates the presence and size of hormone mRNA, in situ hybridization indicates the percentage of cells expressing a given hormone mRNA and allows the presence of one population (or more) of cells in a given tumour to be identified.

Moreover, the tumours were compared with normal rat pituitary. Although the PRL and GH mRNAs were identical in size, the amount of mRNA was lower in the tumours. At the protein level, the PRL and GH variants exhibited a different pattern of expression in tumours compared with the normal rat pituitary.

The biological significance of these differences is discussed.
Multiple alternative splice isoforms of parathyroid hormone-related peptide mRNA in human cell lines
Authors: Nakamura M , Hashimoto T , Furuyama J-i , Kakudo K
Published: December 2008

Parathyroid hormone-related peptide (PTHrP) is thought to be responsible for hypercalcemia in some patients with malignant tumors. The PTHrP gene has seven exons, giving rise to three types of PTHrP isoform through alternative splicing. We studied the expression of mRNAs in 14 human cell lines using the reverse transcription-PCR method, to examine tissue-specific expression. All the cell lines expressed at least two types of PTHrP transcript. Most cell lines expressed all four types of PTHrP mRNA isoform. However, a rhabdomyosarcoma cell line, RD, and a bladder carcinoma cell line, T24, expressed only two types. These results may suggest that PTHrP mRNA is expressed in the majority of tumor and normal tissues and that it shows less tissue- or tumor-specificity.
Expression of human insulin-like growth factor-binding protein-3 in transgenic mice
Authors: Murphy L J , Molnar P , Lu X , Huang H
Published: December 2008

Transgenic mice which expressed human IGF-binding protein-3 (hIGFBP-3) were generated by pronuclear injection of an hIGFBP-3 cDNA driven by the mouse metallothionein 1 promoter. Two of the seven founder mice had measurable levels of hIGFBP-3 in the circulation. The serum levels of hIGFBP-3 increased as the mice were bred to homozygosity and were further induced by supplementing the drinking water with 25 mm ZnCl2. While the birth weight, litter size and body weight of transgenic mice were not significantly different from non-transgenic litter mates or wild-type mice derived from the same genetic background, the transgenic mice demonstrated selective organomegaly. The spleen, liver and heart of mice derived from both founders were significantly heavier compared with organs from non-transgenic mice (P<0·05, P<0·005 and P<0·01 respectively). The weights of the brain and kidney were similar in transgenic and non-transgenic mice. Expression of the transgene was detected in the kidney, small intestine and colon by Northern blot analysis.

Western ligand blotting of serum from transgenic mice did not demonstrate any change in the abundance of the IGFBPs detected by this method. When serum from transgenic mice was incubated with 125I-labeled IGF-I and analyzed by Sephacryl S-200 chromatography under neutral conditions a significantly (P<0·05) increased amount of the radioactivity was found in the 140 kDa ternary complex compared with serum from wild-type mice. Immunoreactive hIGFBP-3 was detected in the 140 kDa ternary complex but the majority of immunoreactive hIGFBP-3 present in transgenic mouse serum eluted in later fractions indicating that it was not associated with the acid-labile subunit. These data demonstrate that modest constitutive expression of hIGFBP-3 has a selective effect on organ growth and development. The establishment of these IGFBP-3 transgenic mouse strains may provide useful models to investigate further the physiological role of IGFBP-3.
Structure and sequence of a mouse gene encoding an androgen-regulated protein: a new member of the seminal vesicle secretory protein family
Authors: Simon A M , Veyssière G , Jean Cl
Published: December 2008

The gene encoding MSVSP99 (mouse seminal vesicle secretory protein of 99 amino acids), an androgen-dependent protein specifically expressed in the mouse seminal vesicle, was isolated and sequenced. A mouse genomic library constructed in the λEMBL12 vector was screened using a full length cDNA probe. One genomic clone was selected, 7·4 kb of which were shown to contain the whole MSVSP99 gene. The complete sequence of the MSVSP99 gene (1·7 kb), plus 0·8 and 0·3 kb of the 5′ and 3′ flanking regions respectively, has been determined. The gene is composed of four exons interrupted by three introns. The size range for the four exons is 47–217 bp, while that of introns is 87–615 bp. The transcription start site was identified as an adenine residue located 21 nucleotides upstream from the ATG start codon. Putative TATA and CAAT boxes were identified, along with a number of regions that shared homologies with known regulatory sequences. These included androgen-responsive elements located in the promoter as well as in the gene sequence. Sequence comparisons with other androgen-responsive genes showed strong homologies between the MSVSP99 gene and the seminal vesicle secretory protein (SVS) family genes (rat SVS II, IV, V and VI). Moreover, some regions were found to be conserved between the MSVSP99 gene and the human semenogelin I and II genes.
Peptide and immunochemical mapping of the ectodomain of the porcine LH receptor
Authors: Couture L , Naharisoa H , Grebert D , Remy J-J , Pajot-Augy E , Bozon V , Haertle T , Salesse R
Published: December 2008

The LH/hCG receptor is a G protein-coupled receptor with an N-terminal extracellular domain involved in hormone—receptor interaction. The recombinant porcine receptor, stably expressed in Chinese hamster ovary (CHO) cells, has the same characteristics (Kd and cAMP production) as in Leydig cells. Six synthetic peptides derived from the receptor ectodomain and two polyclonal anti-peptide sera were tested in the homologous system porcine LH and porcine LH receptor. Their ability to inhibit hormone binding and signal transduction on CHO cells expressing the recombinant receptor was evaluated. Peptides 25–40 and 107–121 exhibited a high transduction inhibition as compared with hormone binding, peptides 21–36, 102–111, and 102–121 inhibited hormone binding more efficiently than signal transduction, and peptide 7–24 exhibited inhibition of both hormone binding and hormone-induced cAMP production. Immuno-globulins against peptides 21–36 and 102–111 inhibited both hormone binding and receptor activation suggesting that these sequences are located on the receptor surface.

The data suggest that multiple, discontinuous regions of the extracellular domain of porcine LH receptor are involved in hormone binding and signal transduction. Two minimum critical sequences, 21–24 and 102–107, are involved in hormone binding and vicinal segments may be implicated in signal transduction.
Nature of ligand affinity and dimerization of corticotrophin-releasing factor-binding protein may be detected by circular dichroism
Authors: Lowry P J , Koerber S C , Woods R J , Baigent S , Sutton S , Behan D P , Vale W , Rivier J
Published: December 2008

As the association of corticotrophin-releasing factor (CRF) with its binding protein (BP) to form a dimer complex (CRF2/BP2) appears to be dependent on the nature of the ligand we have compared the circular dichroism difference spectra after association of the BP with ovine (o) CRF, human (h) CRF and the α-helical CRF(9–41) antagonist. All three ligands caused a negative change in molar ellipticity above 210 nm, with oCRF having the least and hCRF the greatest effect. Below 210 nm there was a marked divergence of difference spectra, with the reaction with the natural peptides, hCRF and oCRF, resulting in a positive change in ellipticity, whilst that with the antagonist produced a negative change. In view of the BP spectrum indicating predominantly β-sheet and the peptides showing mainly α-helix these results were interpreted as the changes above 210 nm being due to dimerization and below 210 nm to a change in the conformation of ligand on binding. The opposite change in α-helicity of the antagonist observed on binding compared with the two natural CRF peptides could have fundamental pharmacological implications.
A monoclonal antibody to mammalian angiotensin II AT1 receptor recognizes one of the angiotensin II receptor isoforms expressed by the eel (Anguilla anguilla)
Authors: Marsigliante S , Muscella A , Vilella S , Nicolardi G , Ingrosso L , Ciardo V , Zonno V , Vinson G P , Ho M M , Storelli C
Published: December 2008

Using labelled ligand-binding methods, previous studies have identified specific angiotensin II receptors (Ang II-Rs) in eel liver, kidney and intestine membranes. Isoelectric focusing on polyacrylamide gels also showed that there are two Ang II-R isoforms in eel liver, focusing at isoelectric points (pI) 6·5 and 6·7. These may have different functions. In contrast, eel enterocyte plasma membrane and renal brush border membranes contain only the pI 6·5 form.

To characterize the eel receptors more fully, a newly developed monoclonal antibody (6313/G2) which selectively recognizes the AT1 subtype of mammalian Ang II-R was used. In ligand-binding experiments, the preincubation of eel liver membranes with 6313/G2 antibody eliminated the specific [3,5-3H]Tyr4-Ile5-Ang II binding. Moreover, Ang II—receptor complexes from solubilized liver membranes, which were immunoprecipitated by 6313/G2-coated beads, had a pI of 6·5. In immunoblotting experiments, the antibody recognized the isoform focusing at pI 6·5 in eel intestine and liver preparations, but not the liver pI 6·7 isoform. Immunoblotting of SDS gels showed that the antibody bound to a single protein of molecular mass of 75 kDa in eel liver, gill and kidney and to a doublet of molecular mass of about 74 and 75 kDa in intestinal membrane preparations. Immunocytochemistry of paraffin-embedded and cryostat sections of eel liver, kidney, intestine and gill showed that antibody 6313/G2 bound to uniformly distributed intracellular sites and cell surface membranes in proximal tubular cells, absorptive intestinal cells, hepatocytes and chloride cells. It also stained endothelium and both the longitudinal and circular layers of smooth muscle cells in the intestine.

The data suggest that the previously described Ang II-R from eel liver, kidney and intestine may be similar to the mammalian AT1 subtype.
Molecular cloning and characterization of the yellowtail GH gene and its promoter: a consensus sequence for teleost and avian Pit-1/GHF-1 binding sites
Authors: Ohkubo T , Araki M , Tanaka M , Sudo S , Nakashima K
Published: December 2008

The gene and 5′ flanking promoter region for yellowtail (Seriola quinqueradiata) GH (yGH) have been cloned, sequenced and characterized. The yGH gene spans approximately 4·6 kb and consists of six exons and five introns, as has been observed with rainbow trout, Atlantic salmon and tilapia GH genes. This result suggests that the structure of six exons and five introns is a dominant form in fish GH genes. A typical TATA box exists 26 bp upstream from the transcription start site, and Pit-1/GHF-1 (Pit-1) binding site-homologous regions were found in the promoter region of the yGH gene. In a gel shift assay, however, a single shifted band was detected with the fragments containing a region from −128 to −90 of the yGH 5′ flanking region when they were incubated with yellowtail pituitary nuclear extracts. The bound fragments contained an octamer base sequence similar, but not identical, to mammalian consensus Pit-1 binding element. A consensus octamer sequence is also proposed in this report for the binding of teleost and avian Pit-1 transcription factors.
Cloning of a gar (Lepisosteus osseus) GH cDNA: trends in actinopterygian GH structure
Authors: Rubin D A , Youson J H , Marra L E , Dores R M
Published: December 2008

A cDNA containing the sequence of GH was cloned and sequenced from a pituitary cDNA library for the holostean fish Lepisosteus osseus (common name: gar). The gar GH cDNA contained an open reading frame of 633 nucleotides and a 3′ untranslated region (including the terminal codon TAG) of 1058 nucleotides. The overall length of the gar GH cDNA including leader sequence, signal sequence, hormone sequence and 3′ untranslated region was 1713 nucleotides. Thus, the gar GH cDNA is the largest vertebrate GH cDNA yet cloned. A comparison of GH sequences from ancient (holostean fishes — gar and bowfin; one chondrostean fish — the Russian sturgeon) and more modern (27 species of teleosts) members of class Actinopterygii indicate that members of this class have maintained many of the invariant residues deemed necessary for GH folding motifs (intramolecular relationships) observed in mammals.
The roles of adenosine 3′,5′-cyclic monophosphate-dependent protein kinase A and protein kinase C in stimulus-secretion coupling in AtT-20 cells
Authors: McFerran B W , Guild S B
Published: December 2008

The ACTH-secreting mouse AtT-20/D16–16 anterior pituitary tumour cell line was used to study adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) and protein kinase C (PKC) involvement in stimulus-secretion coupling pathways. In permeabilised AtT-20 cells under calcium ion-free conditions, forskolin (10 μm), CRH-41 (100 nm), guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-S; 100 μm) but not mastoparan (10 μm) stimulated cAMP accumulation. Measurement of ACTH secretion under identical incubation conditions revealed that GTP-γ-S and mastoparan significantly stimulated ACTH secretion but forskolin and CRH-41 did not. This dissociates cAMP accumulation from ACTH secretion under calcium ion-free conditions and indicated that the effects of mastoparan and GTP-γ-S on ACTH secretion are not mediated by cAMP production. Calcium ions (1 nm to 1 mm) stimulated ACTH secretion from electrically permeabilised cells in a concentration-dependent manner. cAMP (100μm) and phorbol 12-myristate 13-acetate (PMA; 100 nm) synergistically enhanced the response to calcium ions. cAMP did not stimulate ACTH secretion in the absence of calcium ions nor did it alter the concentrations at which calcium stimulated ACTH secretion. This suggests that stimulation of ACTH secretion via the calcium-dependent pathway is necessary before any cAMP-mediated enhancement of secretion is manifest. PMA, however, did stimulate ACTH secretion in the absence of calcium ions, indicating distinct mechanisms for PKC-evoked secretion. Co-incubation with cAMP and PMA did not exceed the secretory response obtained with the combination of PMA and calcium ions. CRH-41 (1 pm to 100 nm) and forskolin (1 nm to 100μm) stimulated ACTH secretion from intact cells in a concentration-dependent manner. Co-incubation with PMA (100 nm) further enhanced the ACTH response to CRH-41 and forskolin; the effects were simply additive. The present study indicates that there are distinct roles for PKA and PKC in stimulussecretion coupling in AtT-20 cells. The PKA-dependent pathway, acting in concert with the calcium messenger system, serves as part of the stimulus-secretion coupling pathway by which activation of CRH-41 receptors control ACTH secretion. The PKC-dependent pathway, in contrast, seems to be independent of the calcium messenger system and may represent a separate control mechanism of ACTH secretion.
Insect cells infected with a recombinant baculovirus express both O- and N-glycosylated forms of the rat glycoprotein hormone α-subunit
Authors: Delahaye R , Berreur P , Salesse R , Counis R
Published: December 2008

Glycoprotein hormones LH, FSH, TSH and chorionic gonadotrophin are heterodimers composed of two non-covalently associated subunits, a common α- and a specific β-subunit. A recombinant baculo-virus containing a cDNA encoding the α-subunit of rat glycoprotein hormones was constructed. Viral-infected cells expressed, 48 h post infection, 7–10mg immunoreactive α-glycopolypeptide/6×108 cells, of which 65·6% was able to associate with native LHβ and formed a biologically active heterodimeric hormone that bound to testicular receptors. The treatment with specific glycanases showed that the recombinant α-subunit was produced as two differently glycosylated forms; an Mr 23 000 form which contained exclusively N-linked carbohydrate units and another of Mr 25 000 which appeared to contain additional O-linked carbohydrate. Data demonstrated that the α-subunit was expressed by insect cells in a manner similar to that by mammalian pituitary gonadotropes producing both the N- and O-glycosylated forms although only the N-glycosylated α-subunit is known to be capable of associating with the β-subunit.