bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differential accumulation of insulin-like growth factor-I in kidneys of pre- and postpubertal streptozotocin-diabetic rats
Authors: Phillip M , Werner H , Palese T , Kowarski A A , Stannard B , Bach L A , LeRoith D , Roberts C T 
Published: December 2008

ABSTRACT
Nephropathy, one of the major complications of diabetes mellitus, is characterized by an early increase in kidney size. In experimental models of diabetes, this event is preceded by a rapid and transient rise in kidney IGF-I levels, at least in adult animals. Since diabetes-associated renal changes are uncommon in young patients, we investigated the early changes in the components of the IGF system following induction of diabetes in prepubertal and postpubertal rats. The rationale for this study was the evaluation of potential differences which could lead to kidney complications only at adult stages.
Unlike the situation in the postpubertal kidney, in which there was a transient accumulation of extractable IGF-I 24–48 h after streptozotocin (STZ) administration, there was a decrease of ∼12-fold in the level of IGF-I in the prepubertal kidney over the same period of time. Paradoxically, kidney IGF-I mRNA levels were reduced by ∼50% in the postpubertal rat 24 h after STZ treatment, whereas in the prepubertal kidney IGF-I mRNA levels were unaltered. Furthermore, the levels of IGF-I receptor mRNA and 125I-labelled IGF-I binding to kidney membranes of postpubertal diabetic rats were similar to the levels in control kidneys. On the other hand, both the levels of IGF-I receptor mRNA and 125I-labelled IGF-I binding were increased (∼2·5-fold (after 24 h) and ∼ 3-fold (after 48 h) respectively) in prepubertal animals. In addition, increased expression of IGF-binding protein (IGFBP)-1 mRNA was seen early in diabetes in both pre- and postpubertal rats.
The results of this study suggest that the transient accumulation of IGF-I in the kidney of the postpubertal diabetic rat may not be due to an increase in the local synthesis of IGF-I, but rather to an increase in IGF-I uptake from the circulation due to non-membrane-associated IGFBP-1. The lack of accumulation of IGF-I in the prepubertal kidney probably reflects the ∼ 10-fold lower levels of circulating IGF-I in young as compared with adult diabetic rats.
Differential regulation of cholesterol side-chain cleavage (P450scc) and aromatase (P450arom) enzyme mRNA expression by gonadotrophins and cyclic AMP in human granulosa cells
Authors: Yong E L , Hillier S G , Turner M , Baird D T , Ng S C , Bongso A , Ratnam S S 
Published: December 2008

ABSTRACT
The co-ordinated biosynthesis of progesterone and oestradiol in the human ovary is critical for reproductive cyclicity and eventual pregnancy. The crucial regulatory enzymes for progesterone and oestradiol biosynthesis in granulosa cells are the cholesterol side-chain cleavage (P450scc) and aromatase (P450arom) enzymes respectively. We utilized the cDNA sequences encoding P450arom and P450scc to examine the roles of FSH and LH, and their intracellular second messenger, cyclic AMP (cAMP), in regulating steroidogenic gene expression. Mature granulosa cells (aspirated before the onset of the endogenous LH surge) and granulosa lutein cells (obtained after an ovulatory dose of human chorionic gonadotrophin) were cultured for 4 days with FSH, LH or dibutyryl cAMP (dbcAMP). After the period of culture, total RNA was extracted from granulosa cells and Northern analyses were performed utilizing 32P-labelled cDNAs encoding P450arom and P450scc. Spent culture media were analysed for steroid and cAMP content.
Both FSH and LH strongly stimulated P450arom mRNA expression and oestradiol production in mature granulosa cells. On the other hand, P450scc mRNA expression and progesterone biosynthesis were weakly induced by FSH; maximal synthesis occurred only in the presence of LH. With both gonadotrophins at equivalent concentrations, LH generated a 30-fold higher level of cAMP than FSH. Furthermore, the differential effects of FSH and LH on P450 mRNA expression were reproduced by the presence of low and high concentrations of dbcAMP respectively. LH (and high levels of dbcAMP) increased P450arom mRNA expression in mature granulosa cells but inhibited its accumulation in granulosa lutein cells. In contrast, it stimulated P450scc mRNA expression and progesterone synthesis in both mature granulosa and granulosa lutein cells. Therefore, FSH/low cAMP levels stimulated P450arom gene expression and oestradiol production, while LH/high cAMP levels maximally induced P450scc gene expression and function, in a development-related manner consistent with steroid production in vivo. These findings support the hypothesis that one set of genes (like P450arom) in human granulosa cells is regulated by FSH/low cAMP levels and another (like P450scc) by LH/high cAMP levels.
Distribution and developmental pattern of neuromedin U expression in the rat gastrointestinal tract
Authors: Austin C , Oka M , Nandha K A , Legon S , Khandan-Nia N , Lo G , Bloom S R 
Published: December 2008

ABSTRACT
This study has quantified, for the first time, the relative levels of neuromedin U (NmU) mRNA in the rat gastrointestinal tract using Northern blot analysis. NmU message was detected in all regions of the gastrointestinal tract from the oesophagus to the rectum. The greatest levels were found in the duodenum and jejunum, the principal sites for absorption, which were 2·5- and 3-fold respectively above ileal levels.
Quantification of NmU mRNA and peptide contents in the duodenum, jejunum and ileum during postnatal development of the rat showed message and peptide levels to be greater in the maturing rat than in neonates. Message levels in the duodenum, jejunum and ileum showed 14-, 7- and 4-fold increases respectively between 1 and 56 days after birth, whilst the corresponding peptide levels in the duodenum, jejunum and ileum showed 33-, 14- and 25-fold increases respectively.
Food deprivation caused a small, but significant, decrease in message levels in the jejunum and colon, but there was no change in the duodenum or ileum. This shows that the presence of food has little effect on NmU mRNA levels in the gut.
Insulin-like growth factor-I gene expression in human granulosa-lutein cells
Authors: Huang Z H , Clayton P E , Brady G , Morris I D 
Published: December 2008

ABSTRACT
IGF-I is an important local regulator of ovarian function, stimulating follicular growth and steroidogenesis in human granulosa cells. However, it is not known whether ovarian IGF-I is derived from the circulating serum pool or from local production. IGF-I peptide has only been detected in human thecal cells and not in granulosa cells. This study has used the sensitive technique of reverse transcription of mRNA followed by PCR amplification (RT/PCR) to examine IGF-I gene expression in human preovulatory granulosa cells.
Granulosa-lutein cell (GLC) samples were obtained by follicular puncture of seven women enrolled in an ovulation induction programme. Treatment had included buserelin acetate, human menopausal gonadotrophin to stimulate follicular growth and human chorionic gonadotrophin to induce ovulation. Total RNA (TRNA), extracted from the GLCs, was amplified by RT/PCR, using combinations of leader and 3′ IGF-I exon-specific primers, to yield four IGF-I gene products: IGF-IA (exons 1, 3, 4, 6), IGF-IB (exons 1, 3, 4, 5), IGF-IA′ (exons 2, 3, 4, 6) and IGF-IB′ (exons 2, 3, 4, 5). As controls from other tissues, an identical procedure was undertaken on TRNA from peripheral blood monocytes and liver. All four mRNAs were expressed in GLCs, monocytes and liver. However the pattern of IGF-I mRNA expression differed between the tissues; in liver and GLCs, the IGF-IA transcript was dominant, but in monocytes the IGF-IA′ species was the most prominent. Quantitative RT/PCR using standardization to the house-keeping gene for glyceraldehyde-3′-phosphate dehydrogenase revealed that IGF-IA mRNA was 300-fold more abundant in liver than GLCs.
This study has indicated that: i) RT/PCR can be used to detect multiple IGF-I mRNA species in small amounts of TRNA from human tissue, ii) the IGF-I gene is expressed at low level in human preovulatory GLCs and iii) there is preferential use of leader exon 1 in human GLCs and leader exon 2 in monocytes. Further studies of IGF-I gene expression by this method in other ovarian cell types and granulosa cells from the developing follicle may help to clarify the local role of IGF-I in the human ovary.
Directed expression of simian virus 40 T-antigen in transgenic mice using the epidermal growth factor gene promoter
Authors: Pascall J C , Surani M A , Barton S C , Vaughan T J , Brown K D 
Published: December 2008

ABSTRACT
A transgenic mouse line (EGF/Tag) has been established in which expression of SV40 T-antigen is directed by a 5·5 kb fragment of the 5′-flanking region of the mouse epidermal growth factor (EGF) gene. Of the two principal sites of EGF expression in mice, submaxillary gland and kidney, T-antigen mRNA and protein were detected in the former but not in the latter tissue of the EGF/Tag animals. T-antigen expression in the submaxillary gland was restricted to the EGF-producing cells of the granular convoluted tubules, and the oncoprotein induced hyperplasia of these cells. T-antigen levels were markedly higher in the submaxillary glands of male compared with female transgenic mice, suggesting that expression of the transgene was androgen-regulated, like the endogenous EGF gene. These results indicate that the 5·5 kb fragment upstream of the mouse EGF gene contains the DNA enhancer elements required for hormonally regulated expression in the submaxillary gland. Since the hyperplastic submaxillary glands of the EGF/Tag mice continue to synthesize EGF, these glands provide a tissue source from which it may prove possible to establish EGF-secreting cell lines for further in vitro studies of the mechanisms regulating expression of the EGF gene.
Human relaxins in normal, benign and neoplastic breast tissue
Authors: Tashima L S , Mazoujian G , Bryant-Greenwood G D 
Published: December 2008

ABSTRACT
Immunoreactive relaxin is present in human breast cyst fluid and postpartum milk without concurrent detectable serum levels, suggesting that the breast is a site of relaxin synthesis. Monoclonal and polyclonal antibodies to human relaxin H2 have been used to immunolocalize relaxins in normal, benign and neoplastic breast tissues with the avidin—biotin immunostaining technique. In view of the similarities in amino acid sequence between HI and H2 relaxins, these antibodies to H2 relaxin are likely to detect either or both relaxins present in tissue sections. Staining patterns with these antibodies were identical and showed positive diffuse cytoplasmic staining in normal, lobular and ductal epithelium and in myoepithelial cells in breast tissues from normal prepubertal, cyclic, gestational, lactational and postmenopausal females. Relaxin staining was also present in epithelial and myoepithelial cells of ducts and lobules in benign breast disease as well as in metaplastic epithelium of apocrine microcysts. All breast carcinomas (infiltrating ductal, tubular, medullary, intraductal and infiltrating lobular carcinomas) had strong uniform cytoplasmic staining within the neoplastic epithelial cells. All staining was abolished in normal and neoplastic tissues when the polyclonal antibody was preabsorbed with relaxin.
It was necessary to distinguish between the possibilities of relaxins being sequestered by breast tissue and local synthesis. Therefore, the expression of the Hl, H2 or both human relaxin genes in normal and neoplastic breast tissues was studied by the isolation of RNA, synthesis of first strand cDNA and amplification by PCR using primer sets which amplified either both H1 and H2, or specifically only H1 or H2 relaxin. The coamplification of both relaxin genes was verified by Southern analysis, diagnostic restriction enzyme digestion and sequencing. The primer set for H1 relaxin detected H1 gene expression in 1 out of 8 normal and 9 out of 12 neoplastic breast RNA samples. The H2 relaxin gene was found to be expressed in 3 out of 8 of the normal samples but in all 12 of the neoplastic samples, suggesting that this gene is expressed at higher copy number in the neoplastic tissues.
This is the first demonstration of the cellular immunolocalization of relaxin and relaxin gene expression in normal and neoplastic breast. This should allow further exploration of relaxin's role(s) in normal breast physiology and in its tumorigenesis.
Effects of oestrogen on rat uterine expression of insulin-like growth factor-binding proteins
Authors: Molnar P , Murphy L J 
Published: December 2008

ABSTRACT
Previous studies have established that the IGFs are involved in oestrogen-induced uterine proliferation. IGF-binding proteins (IGFBPs) are present in most biological fluids and tissues and may modulate the actions of the IGFs. We examined uterine, hepatic and renal expression of the IGFBPs throughout the oestrous cycle and investigated the effects of oestradiol (OE2) on IGFBP expression in ovariectomized (ovx) rats. Uterine expression of IGFBPs-1 and -3 showed a definite variation throughout the oestrous cycle with highest levels during dioestrus. In the liver and kidney the changes in IGFBP-1 and IGFBP-3 mRNA abundance were the opposite of those observed in the uterus, with the highest levels observed during oestrus. Administration of OE2 to ovx rats decreased uterine IGFBP-3 mRNA and increased IGFBP-4 mRNA levels. In these rats there were no consistent changes in renal IGFBP-1 or IGFBP-3 mRNAs; however, a significant increase in IGFBP-4 mRNA was observed in this tissue, as in the uterus. In the liver an increase in IGFBP-1 mRNA and a decrease in IGFBP-3 mRNA levels were observed in rats treated with OE2. Despite changes in uterine, hepatic and renal IGFBP mRNA levels, no significant variation was seen in serum IGFBPs as determined by ligand blotting of sera. These data demonstrate that there is a cyclical variation in the expression of the IGFBPs in the uterus, kidney and liver, and that OE2 is able to modulate differentially IGFBP expression in these tissues.
Regulators for the rat clusterin gene: DNA methylation and cis-acting regulatory elements
Authors: Rosemblit N , Chen C-L C 
Published: December 2008

ABSTRACT
Clusterin, also known as sulphated glycoprotein-2 or testosterone-repressed prostate message-2, is a ubiquitous protein found in a variety of tissues and species. In the reproductive tract of the male rat, clusterin is regulated in a complex age-dependent and cell-specific manner. It is expressed at high levels in the epididymis and testis and at very low levels in the prostate under basal conditions. The expression of this gene in the prostate and seminal vesicles is associated with androgen withdrawal, while in the testis clusterin mRNA is repressed by cyclic AMP (cAMP). To understand the mechanisms that control the expression of the clusterin gene better, we isolated and characterized the gene encoding rat clusterin, and analysed its cytosine methylation pattern in various tissues. Several putative regulatory DNA elements were identified, including a consensus AP-1 site in the 5′ flanking region. Two AP-1 sites and two transforming growth factor-β inhibitory elements, one AP-2 site and eight half-sites for glucocorticoid/androgen response elements were found within the first intron, and one cAMP response element was found in the first exon. The cytosine methylation pattern indicated that testicular or epididymal DNA in the rat is hypomethylated in the region between positions −534 and −99 of the clusterin gene, when compared with tissues with lower levels of expression such as prostate as well as liver, lung, kidney and spleen.
Importance of the extracellular domain of the human thyrotrophin receptor for activation of cyclic AMP production
Authors: Paschke R , Parmentier M , Vassart G 
Published: December 2008

ABSTRACT
The mechanism by which the TSH receptor is activated is unknown. Current knowledge leads us to consider that G protein-coupled receptors are activated by positioning of their ligand in the pocket formed by the hydrophobic transmembrane segments. Furthermore, activation of an N-terminally truncated LH receptor lacking most of the extracellular domain has been described, suggesting the existence of a mechanism involving a direct interaction between LH and the transmembrane segments. The high conservation of the transmembrane segments among G protein-coupled receptors is a strong indication for a common mechanism of receptor activation.
To test this hypothesis for the TSH receptor we have constructed four N-terminally truncated TSH receptor mutants with 5 or 69 amino acids of the extracellular domain joined to signal peptide regions consisting of the first 23 or 33 amino acids. The four fragments were amplified by PCR and subcloned into pBSK+. Sequences were confirmed after subcloning in M13. After joining the four fragments in pBSK+, the four TSH receptor constructs were subcloned in pSVL and transiently or stably expressed in COS and Chinese hamster ovary (CHO) cells respectively.
In contrast to results obtained for the LH receptor, stimulation of the transfectants with 10 μm human chorionic gonadotrophin or 350 mU TSH/ml did not increase cyclic AMP (cAMP) concentrations in cultures of transiently transfected COS cells or stably transfected CHO cells. However, mRNA for the TSH receptor could be detected by RNase protection assay in all stable transfectants used for stimulation of cAMP.
These results suggest that activation of the receptor does not implicate direct interaction of TSH with the transmembrane domains. However, our experiments could not investigate whether binding of TSH to the extracellular part of the TSH receptor can induce conformational changes of the transmembrane part, which might trigger activation of the receptor or any other role of the extracellular receptor domain as a cofactor for TSH receptor activation.
Effect of oestradiol-17β on the expression of oestrogen receptor mRNA in human tonsillar cells
Authors: Evagelatou M , Farrant J 
Published: December 2008

ABSTRACT
We have investigated the expression of oestrogen receptor (ER) mRNA in Ficoll-separated tonsillar cells and the changes that occur with the addition of oestradiol (OE2) both in the presence and the absence of the T cell mitogen phytohaemagglutinin (PHA). The amounts of ER mRNA and β-actin mRNA in the samples were determined by slot blotting and hybridization and quantified by densitometry. The levels of ER mRNA were normalized against the β-actin mRNA content. In the presence of OE2 (7×10−8m) after a 10-h culture there was a significant decrease (P<0·05) to about 66% of the control (0-h culture) ER mRNA levels. Stimulating the cultures with PHA (1 μg/ml, without the presence of OE2, had no effect on the expression of ER mRNA. However, when OE2 was present in a 10-h culture of PHA-stimulated cells, the ER mRNA level was significantly decreased (P<0·05) to about 60% of control levels. In 24-h cultures, the presence of OE2 and/or PHA had no effect. When separated T cell preparations from the tonsils were used, no significant effects of OE2 were seen in either the 10-h or 24-h cultures. In conclusion, OE2 downregulates the ER mRNA content in a tonsillar mononuclear cell system in vitro as it does in many primary oestrogen target cells.
High-level expression of recombinant porcine LH receptor in baculovirus-infected insect cells or caterpillars
Authors: Pajot-Augy E , Couture L , Bozon V , Remy J-J , Biache G , Severini M , Huer J-C , Pernoller J-C , Salesse R 
Published: December 2008

ABSTRACT
Porcine LH receptor ectodomain was overexpressed in insect cells and lepidopteran larvae using the recombinant baculovirus expression system. A low multiplicity of infection yielded the largest active production, of approximately 107 receptors/cell or 3 μg active receptor/mg total protein in infected cells. The truncated ectodomain solubilized with Triton X-100 bound its ligand with a high affinity which was comparable with that of the native membrane receptor. Increasing the multiplicity of infection resulted in an optimum protein production of 0·6 mg receptor/mg total protein in infected cells. This receptor was largely inactive, probably trapped within aggregation pools. Active receptor could be recovered by dilution of the samples. No secretion of recombinant receptor was ever observed whatever the conditions of infection. Expression of the recombinant receptor in insect larvae was also tested. This low-cost system failed both to increase the amount of active receptor and to induce secretion into the haemolymph. Two methods remain for producing sizeable amounts of active receptor with this baculovirus/insect cell system. One relies on immunoaffinity purification of the active protein and requires large-scale production, and the other is based on the purification of overexpressed inactive receptor followed by renaturation.
A novel cyclic AMP response element-binding protein-1 (CREB-1) splice product may down-regulate CREB-1 activity
Authors: Ellis M J C , Hurst H C , Goodbourn S 
Published: December 2008

ABSTRACT
In this report we identify novel spliced forms of cyclic AMP (cAMP) response element-binding protein-1 (CREB-1) mRNA. These forms contained an additional 17 nucleotide insert, which we refer to as the β exon, located between exons 4 and 7 of the Δ, and 5 and 7 of the α forms of CREB-1 transcript (nomenclature of Ruppert et al. 1992; EMBO Journal 11, 1503-1512). The inclusion of the β exon led to the generation of mRNAs in which the frame of CREB-1 sequences 3′ to the exon was shifted such that the encoded proteins terminate after the transactivation domain, but before the target serine for cAMP-dependent protein kinase. The β exon-containing CREB-1 mRNAs were more abundant in tissues that respond poorly to cAMP, suggesting that the generation of βCREB-1 mRNAs may contribute to the down-regulation of CREB-1 activity and cAMP responsiveness.