Biomedical subjects

C G Goodyer

Publications and source records attributed to C G Goodyer.

At least 37 records · Page 2Linked to original sources

PubMed1999

Role of endoplasmic reticulum, endosomal-lysosomal compartments, and microtubules in amyloid precursor protein metabolism of human neurons.

A wide interest in amyloid precursor protein (APP) metabolism stems from the fact that increased amounts of amyloid beta peptide (Abeta), arising through proteolytic processing of APP, likely play a significant role in Alzheimer's disease. As Alzheimer's disease pathology is limited almost exclusively to the human species, we established human primary neuron cultures to address the possibility of distinctive APP processing in human CNS neurons. In the present study, we investigate the role of organelles and protein trafficking in APP metabolism. Using brefeldin A, we failed to detect APP processing into Abeta in the endoplasmic reticulum. Monensin and the lysomotropic agents, NH4Cl and chloroquine, revealed a bypass pH-dependent secretory pathway in a compartment between the endoplasmic reticulum and the medial Golgi, resulting in the secretion of full-length APP. Colchicine treatment resulting in the loss of neurites inhibited processing of APP through the secretory, but not the endosomal-lysosomal, pathway of APP metabolism. The serine protease inhibitor, leupeptin, indicates a role for lysosomes in APP, Abeta, and APP C-terminal fragment turnover. These results demonstrate that the regulation of APP metabolism in human neurons differs considerably from those reported in rodent CNS primary neuron cultures or continuously dividing cell types.

Ammonium Chloride↗

PubMed1999

The baboon: a model for the study of primate growth hormone receptor gene expression during development.

In subprimates, significant onset of growth hormone receptor (GHR) expression occurs only after birth whereas, in the human, GHR mRNA and protein are widely manifest from the first trimester of fetal life. Thus, it is likely that subprimates are not the best models for studying regulation of human GHR gene transcription, especially during early stages in development. Here we have explored the potential of the baboon as a more appropriate model. Baboon GHR cDNAs were cloned from postnatal liver by reverse transcription (RT)-PCR, using human GHR-specific primers. The encoded baboon GHR precursor protein has an identical signal peptide sequence to that of human and rhesus monkey GHRs, and the mature baboon GHR is also 620 amino acids long, with 95% and 98.5% amino acid identity to the human and rhesus monkey receptors respectively. Previous studies in the human have identified eight 5' untranslated region (5' UTR) variants of the GHR mRNA (V1 to V8, numbered according to their relative abundance). We cloned the baboon V1, V3 and V4 homologues by RT-PCR: these variants have a high degree (>92%) of sequence identity with their human counterparts and also diverge at an identical point, 12 nucleotides upstream of the start of translation. The expression pattern of these three GHR mRNA isoforms in baboon liver during development was characterized. Strong expression of baboon V1 and V4 was evident by 49 days of postnatal life (n=5, 49 days and adult (18.6-19.6 kg)); very low levels of V1, but not V4, were observed in younger animals (n=2, 6 and 30 days). In contrast, V3 5' UTR variant mRNA was present in all fetal (n=4, 141-155 days gestation) and postnatal (n=7, 6-19.6 days and adult (18.6 kg)) hepatic specimens examined. Analysis of postnatal kidney and lung (n=2, 19 and 19.6 kg) revealed that V3 transcripts are present in these tissues, but not V1 and V4. Together, these data demonstrate that, as in the human, baboon V1 and V4 expression is developmentally regulated and tissue specific, while the V3 isoform is more widely expressed. Therefore, it is likely that the regulatory regions of the baboon and human GHR genes are well conserved. Our findings suggest that the baboon is an appropriate animal model in which to define the mechanisms regulating GHR gene transcription during primate development.

Adult↗

PubMed1998

Developmental upregulation of human parathyroid hormone (PTH)/PTH-related peptide receptor gene expression from conserved and human-specific promoters.

The parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR) functions in skeletal development and mediates an array of other physiological responses modulated by PTH and PTHrP. PTHR gene transcription in mouse is controlled by two promoters: P1, which is highly and selectively active in kidney; and P2, which functions in a variety of tissues. P1 and P2 are conserved in human tissue; however, P1 activity in kidney is weak. We have now identified a third human promoter, P3, which is widely expressed and accounts for approximately 80% of renal PTHR transcripts in the adult. No P3 activity was detected in mouse kidney, indicating that renal PTHR gene expression is controlled by different signals in human and mouse. During development, only P2 is active at midgestation in many human tissues, including calvaria and long bone. This strongly suggests that factors regulating well conserved P2 control PTHR gene expression during skeletal development. Our results indicate that human PTHR gene transcription is upregulated late in development with the induction of both P1 and P3 promoter activities. In addition, P2-specific transcripts are differentially spliced in a number of human cell lines and adult tissues, but not in fetal tissues, giving rise to a shorter and less structured 5' UTR. Thus, our studies show that both human PTHR gene transcription and mRNA splicing are developmentally regulated. Moreover, our data indicate that renal and nonrenal PTHR gene expression are tightly coordinated in humans.

Bone and Bones↗

PubMed1998

Protein kinase C activation increases release of secreted amyloid precursor protein without decreasing Abeta production in human primary neuron cultures.

Overexpression and altered metabolism of amyloid precursor protein (APP) resulting in increased 4 kDa amyloid beta peptide (Abeta) production are believed to play a major role in Alzheimer's disease (AD). Therefore, reducing Abeta production in the brain is a possible therapy for AD. Because AD pathology is fairly restricted to the CNS of humans, we have established human cerebral primary neuron cultures to investigate the metabolism of APP. In many cell lines and rodent primary neuron cultures, phorbol ester activation of protein kinase C (PKC) increases the release of the secreted large N-terminal fragment of amyloid precursor protein (sAPP) and decreases Abeta release (; ; ). In contrast, we find that PKC activation in human primary neurons increases the rate of sAPP release and the production of APP C-terminal fragments and 4 kDa Abeta. Our results indicate species- and cell type-specific regulation of APP metabolism. Therefore, our results curtail the use of PKC activators in controlling human brain Abeta levels.

Amyloid beta-Protein Precursor↗

PubMed1998

A functional analysis of the role of IGF2 in IDDM2-encoded susceptibility to type 1 diabetes.

Genetic studies have identified a number of loci demonstrating linkage to type 1 diabetes. One of the largest single contributors to genetic susceptibility, after the major histocompatability complex, is the IDDM2 locus, which maps to a nontranscribed variable number of tandem repeats (VNTR) minisatellite upstream of the insulin (INS) and insulin-like growth factor 2 (IGF2) genes. In a progression from population to functional studies, recent reports have shown that VNTR susceptibility alleles (class I) have different transcriptional effects on INS than protective VNTR alleles (class III) in thymus and pancreas, two tissues important in the pathogenesis of the disease. Similar VNTR transcriptional effects on IGF2 have also been proposed as a mechanism by which the IDDM2 locus confers susceptibility in addition to, or instead of, effects on INS. We evaluated this hypothesis by comparing IGF2 expression levels from chromosomes with the protective class III alleles to those with class I alleles in tissues relevant to type 1 diabetes pathogenesis. In thymus, class III alleles were associated with an IGF2 mRNA level of 4.7 +/- 0.9 (mean +/- SE, arbitrary units, n = 12) compared with 4.7 +/- 1.3 for class I alleles (n = 17). The same absence of a significant difference was found in pancreas, where class III alleles were associated with a level of 28.4 +/- 4.2 (n = 7) and class I alleles with a level of 29.5 +/- 5.2 (n = 6). There was a significant correlation between fetal age and IGF2 in both tissues, but fetal ages were not different in the genotype groups compared. We therefore did not detect any significant difference in IGF2 mRNA levels associated with the protective class of VNTR alleles as compared with the predisposing class. This is evidence against the hypotheses that have suggested IGF2 is a mediator of IDDM2-encoded susceptibility and corroborates previous studies suggesting insulin is the gene involved.

Alleles↗

PubMed1997

The promoting effects of bFGF and astrocyte extracellular matrix on process outgrowth by adult human oligodendrocytes are mediated by protein kinase C.

Process extension by oligodendrocytes (OLs) is a critical early step in myelin formation. We have previously reported that the basal- or phorbol ester-enhanced process outgrowth by adult human OLs is mediated by oligodendroglial protein kinase C (PKC). Recently, we demonstrated that astrocytes facilitated process outgrowth by adult human OLs through the interaction between astrocyte-derived basic fibroblast growth factor (bFGF) and astrocyte extracellular matrix (ECM). If PKC is central to the signal transduction cascade that leads to process formation by OLs, then the effects of bFGF and astrocyte ECM should also involve PKC. In the current study, we have addressed the involvement of PKC in the bFGF- and astrocyte ECM- enhanced process formation by adult human OLs by using a selective inhibitor of PKC, calphostin C. The results show that calphostin C dose-dependently reduced process extension elicited by bFGF and astrocyte ECM, at IC50 concentrations of 24.5 and 26.6 nM, respectively. At the concentrations of calphostin C that inhibited process extension by adult human OLs, necrosis (measured by lactate dehydrogenase release) and apoptosis (determined by using a fluorescent terminal deoxynucleotidyl transferase assay) of OLs did not occur. Finally, we demonstrate that another specific inhibitor of PKC, CGP 41251, also reduced process formation that is elicited by bFGF and astrocyte ECM. Thus, all process-extending agents for adult human OLs identified to date signal through PKC, further implicating PKC of OLs as being central to the production of process extension, an early event in myelinogenesis.

Astrocytes↗

PubMed1997

Pex/PEX tissue distribution and evidence for a deletion in the 3' region of the Pex gene in X-linked hypophosphatemic mice.

PEX, a phosphate-regulating gene with homology to endopeptidases on the X chromosome, was recently identified as the candidate gene for X-linked hypophosphatemia. In the present study, we cloned mouse and human Pex/PEX cDNAs encoding part of the 5' untranslated region, the protein coding region, and the entire 3' untranslated region, determined the tissue distribution of Pex/PEX mRNA, and characterized the Pex mutation in the murine Hyp homologue of the human disease. Using the reverse transcriptase/polymerase chain reaction (RT/PCR) and ribonuclease protection assays, we found that Pex/PEX mRNA is expressed predominantly in human fetal and adult mouse calvaria and long bone. With RNA from Hyp mouse bone, an RT/PCR product was generated with 5' but not 3' Pex primer pairs and a protected Pex mRNA fragment was detected with 5' but not 3' Pex riboprobes by ribonuclease protection assay. Analysis of the RT/PCR product derived from Hyp bone RNA revealed an aberrant Pex transcript with retention of intron sequence downstream from nucleotide 1302 of the Pex cDNA. Pex mRNA was not detected on Northern blots of poly (A)+ RNA from Hyp bone, while a low-abundance Pex transcript of approximately 7 kb was apparent in normal bone. Southern analysis of genomic DNA from Hyp mice revealed the absence of hybridizing bands with cDNA probes from the 3' region of the Pex cDNA. We conclude that Pex/PEX is a low-abundance transcript that is expressed predominantly in bone of mice and humans and that a large deletion in the 3' region of the Pex gene is present in the murine Hyp homologue of X-linked hypophosphatemia.

Amino Acid Sequence↗

PubMed1997

Insulin expression in human thymus is modulated by INS VNTR alleles at the IDDM2 locus.

Type 1 diabetes or insulin-dependent diabetes mellitus (IDDM) is due to autoimmune destruction of pancreatic beta-cells. Genetic susceptibility to IDDM is encoded by several loci, one of which (IDDM2) maps to a variable number of tandem repeats (VNTR) minisatellite, upstream of the insulin gene (INS). The short class I VNTR alleles (26-63 repeats) predispose to IDDM, while class III alleles (140-210 repeats) have a dominant protective effect. We have reported that, in human adult and fetal pancreas in vivo, class III alleles are associated with marginally lower INS mRNA levels than class I, suggesting transcriptional effects of the VNTR. These may be related to type 1 diabetes pathogenesis, as insulin is the only known beta-cell specific IDDM autoantigen. In search of a more plausible mechanism for the dominant effect of class III alleles, we analysed expression of insulin in human fetal thymus, a critical site for tolerance induction to self proteins. Insulin was detected in all thymus tissues examined and class III VNTR alleles were associated with 2- to 3-fold higher INS mRNA levels than class I. We therefore propose higher levels of thymic INS expression, facilitating immune tolerance induction, as a mechanism for the dominant protective effect of class III alleles.

Abortion, Therapeutic↗

PubMed1997

Processing of amyloid precursor protein in human primary neuron and astrocyte cultures.

Increased production of amyloid beta peptide (A beta) is highly suspected to play a major role in Alzheimer's disease (AD) pathogenesis. Because A beta deposits in AD senile plaques appear uniquely in the brain and are fairly restricted to humans, we assessed amyloid precursor protein (APP) metabolism in primary cultures of the cell types associated with AD senile plaques: neurons, astrocytes, and microglia. We find that neurons secrete 40% of newly synthesized APP, whereas glia secrete only 10%. Neuronal and astrocytic APP processing generates five C-terminal fragments similar to those observed in human adult brain, of which the most amyloidogenic higher-molecular-weight fragments are more abundant. The level of amyloidogenic 4-kDa A beta exceeds that of nonamyloidogenic 3-kDa A beta in both neurons and astrocytes. In contrast, microglia make more of the smallest C-terminal fragment and no detectable A beta. We conclude that human neurons and astrocytes generate higher levels of amyloidogenic fragments than microglia and favor amyloidogenic processing compared with previously studied culture systems. Therefore, we propose that the higher amyloidogenic processing of APP in neurons and astrocytes, combined with the extended lifespan of individuals, likely promotes AD pathology in aging humans.

Amyloid beta-Protein Precursor↗

PubMed1996

Fetal- and tumor-specific regulation of growth hormone receptor messenger RNA expression in human liver.

Eight different 5'-untranslated region variants of the human growth hormone receptor (hGHR) mRNA have been identified in adult liver (V1-V8). We have compared the expression of two of these variants (V1 and V3) in several human fetal and postnatal tissues (including liver) as well as in hepatoblastomas (HBs) and hepatocellular carcinomas (HCCs). Using reverse transcription-PCR assays, followed by Southern blotting to confirm the specificity of the amplified fragments, we found that V3 was expressed in all fetal and postnatal liver (n = 13 fetal and 5 postnatal), kidney (n = 4 fetal and 4 postnatal), lung (n = 4 fetal and 2 postnatal), intestine (n = 8 fetal and 4 postnatal), skeletal muscle (n = 1 fetal and 1 postnatal), and adrenal (n = 1 fetal and 1 postnatal) samples. In contrast, V1 was expressed only in postnatal liver. We then screened for V1 and V3 in HBs (n = 17, ages 6-36 months, including 5 with paired normal liver), and HCCs (n = 4, ages 50-75 years, with paired normal liver). V1 was undetectable in 15 of 17 HBs, including all HBs paired with (V1-expressing) normal liver; the absence of V1 did not correlate with patient age, sex, HB subtype, +/- chemotherapy, exon 3-retaining and -deficient hGHR mRNA isoform pattern, or loss of heterozygosity at 11p, 1p, and 1q. The four HCCs showed marked (>20-fold; n = 2) or complete (n = 2) suppression of V1 as compared to paired normal liver. V3 was expressed in all HBs, HCCs, and paired normal livers. Interestingly, V3, but not V1, was detected in two Wilms' tumor and paired normal kidney specimens. Our findings suggest that, in the human, there is tissue-, fetal- and tumor-specific regulation of V1 hGHR mRNA.

Adolescent↗

PubMed1996

Imprinted and genotype-specific expression of genes at the IDDM2 locus in pancreas and leucocytes.

One of the loci encoding susceptibility to insulin-dependent diabetes mellitus (IDDM) is IDDM2, mapped to a variable number of tandem repeats (VNTR) polymorphism situated 596 bp upstream of the insulin gene (INS). The shorter alleles (class I) predispose to IDDM, while the longer class III alleles are protective. Besides INS, it is possible that transcription levels of IGF2, the nearby gene encoding the insulin-like growth factor II, may be modulated by allelic forms of the VNTR. In an effort to define the pathophysiologic mechanism of the IDDM2 effect, we examined the effect, in cis, of VNTR genotype on steady-state mRNA levels of INS in samples of human fetal pancreas, and of IGF2 in leucocytes of diabetic children. Relative levels of mRNA transcripts derived from each chromosome carrying a defined VNTR allele were measured by RT-PCR, taking advantage of transcribed polymorphisms at the 3' untranslated region of each gene. In 10 samples of human fetal pancreas, INS transcripts from chromosomes carrying a class III VNTR were slightly but significantly (P = 0.015) lower than those from class I (13% lower, 95% confidence limits 3-21%). In 10 leucocyte samples, mRNA from both IGF2 alleles was seen, indicating relaxation of the parental imprinting of IGF2 in these cells. However, this relaxation was incomplete as maternal allele mRNA was systematically at a lower level than paternal. The paternal/maternal ratio varied widely among individual subjects. Two of the most extreme cases, demonstrating almost complete repression of the maternal allele, were identical twins, suggesting that this variable relaxation of imprinting is genotype-dependent. However, this genotype-dependence cannot be accounted for by the maternal VNTR, as the mean ratios of paternal/maternal IGF2 mRNA levels were not statistically different in individuals with a maternal VNTR of class I vs. class III (3.2 +/- 1.5 vs. 3.89 +/- 0.94). Thus, we present evidence that: (a) class III VNTR alleles are associated with lower INS mRNA in fetal pancreas than class I alleles. The biologic importance of this difference remains to be determined; and (b) the variable relaxation of IGF2 imprinting seen in human leucocytes is not dependent on the presence of a class I vs. a class III VNTR.

Alleles↗

PubMed1996

Variable imprinting of H19 and IGF2 in fetal cerebellum and medulloblastoma.

Only the maternal or paternal allele of an imprinted gene is expressed in somatic cells. The gene for insulin-like growth factor II (IGF2) and the H19 gene (a putative tumor suppressor gene) are imprinted in humans with monoallelic paternal and maternal expression, respectively. Loss of imprinting (LOI) (i.e., biallelic expression) of IGF2 occurs in some tumors and may promote tumor growth. We examined imprinting of IGF2 and H19 in 6 fetal cerebella, 1 adult cerebellum, 15 medulloblastomas, and 7 medulloblastoma cell lines using polymerase chain reaction (PCR) and reverse transcription-PCR of exonic polymorphisms. Loss of imprinting of IGF2 occurred in 2 out of 3 informative fetal cerebella, 3 out of 7 informative medulloblastomas, and 1 out of 4 informative cell lines. Loss of imprinting of H19 occurred in 0 out of 4 informative fetal cerebella, 0 out of 1 informative adult cerebellum, 4 out of 8 informative medulloblastomas, and 1 out of 4 informative cell lines. The biallelic expression of H19 was only partial in two medulloblastomas, however, with one allele being significantly weaker than the other. Loss of imprinting of IGF2 occurs in medulloblastomas or medulloblastoma cell lines but can also occur in normal fetal cerebellum. Its occurrence in medulloblastomas may therefore reflect the tumors' embryonal nature rather than representing a primary pathogenetic mechanism. Our data also indicate that both genes can be imprinted and expressed independently of each another, both in normal cerebellum and medulloblastomas.

Adult↗

PubMed1996

Expression of exon 3-retaining and -deleted human growth hormone receptor messenger ribonucleic acid isoforms during development.

Recent investigations have suggested that human GH (hGH) and its receptor may have specific functions during human fetal life. To improve our understanding of the mechanisms of hGH action during gestation, we characterized the ontogenic appearance of hGH receptor messenger ribonucleic acid (mRNA) in multiple human fetal and postnatal tissues. Using RT-PCR assays, followed by Southern hybridization to confirm the specificity of the amplified fragments, we scanned the entire coding region of the hGH receptor mRNA. Transcription of the hGH receptor gene was observed in all fetal tissues studied (liver, kidney, skin, muscle, lung, adrenal, spleen, intestine, central nervous system, pancreas, and placental villi) from the earliest stage that could be examined [7-14.8 weeks fetal age (FA)]. Furthermore, we identified only 2 isoforms of the hGH receptor mRNA-coding region: exon 3 can be retained or deleted. Surprisingly, we found individual-specific, not tissue-specific, expression patterns of these two transcripts when we examined multiple tissues (n = 2-6) from 15 individuals (11.5-33 weeks FA); this individual-specific pattern of expression is maintained in cultured dermal fibroblasts for at least 12 generations (n = 2; 16 and 20 weeks FA). In addition, a cross-sectional study of 78 individuals (9 weeks FA to 43 yr postnatal age) showed that the exon 3-deleted transcript is predominantly expressed in tissues from fetuses of 9-20 weeks FA (P < 0.002). Finally, we showed that the absence of exon 3 from the mRNA is not due to genomic deletion of exon 3 by amplifying exon 3 from genomic DNA of 3 fetuses (13.3-19 weeks FA) expressing only the exon 3-deleted mRNA transcript. We conclude that 1) transcription of the hGH receptor gene occurs in multiple tissues as early as the first trimester of human fetal life; 2) the exon 3-retaining and -deleted transcripts are the only two isoforms of the hGH receptor mRNA-coding region during gestation; and 3) the pattern of expression of these transcripts is individual specific and may be developmentally regulated.

Base Sequence↗

PubMed1996

Ontogeny of growth hormone receptors in human tissues: an immunohistochemical study.

Both experimental and clinical studies suggest that human GH (hGH) may have a functional role during early stages in human development. To determine when receptors for hGH appear in specific target cells, we carried out an immunohistochemical analysis of six tissues obtained from 20 fetuses (8.5-20 weeks fetal age [FA]), two term infants, one 11-yr-old male, and one 43-yr-old male. Liver parenchymal and bile duct cells showed specific staining from the earliest FA examined (8.5 weeks); in fetal liver, the intensity of the immunoreaction was greatest in hepatocytes surrounding the central veins and portal triads, whereas the numerous hematopoietic cells exhibited little to no immunostaining. Kidney tissues also showed positive localization at 8.5-9 weeks FA. By 13 weeks, epithelial cells of most tubules were immunoreactive, with medullary tubules showing stronger staining than those in the cortex, whereas visceral epithelial cells of glomeruli were weakly positive; metanephrogenic blastemal cells were negative. A similar immunostaining pattern was observed in midgestation and postnatal kidneys, except that glomerular staining was lost by 19 weeks FA. Fetal zone cells of fetal adrenals showed weak immunostaining beginning at 13-14 weeks; glomerulosa cells stained intensely in the postnatal adrenal. Epithelial crypt cells in both small and large intestine immunostained beginning at 19-20 weeks; a similar pattern was observed in postnatal sections. Dermal fibroblasts and skeletal muscle of abdominal skin were positive from 8.5 weeks. Epidermal cells first showed immunostaining at 13-14 weeks; germinal layer cells showed the most intense reaction until 19-20 weeks, when the staining pattern became uneven throughout the epidermal layers. Epithelial cells in hair follicles and sebaceous glands were positive from 13-14 weeks; by 19-20 weeks, these cells were the most intensely immunostained in skin sections. Lung tissue was negative from 8.5-20 weeks, as well as postnatally. Vascular endothelial and smooth muscle cells routinely stained from 8.5 weeks FA in all tissues examined. We conclude that immunoreactive hGH receptors can be identified in early fetal, as well as postnatal, tissues and that the pattern of cellular immunostaining develops gradually throughout gestation in a very tissue-specific manner.

Adult↗

PubMed1995

Mammosomatotroph adenoma causing gigantism in an 8-year old boy: a possible pathogenetic mechanism.

The pathophysiology of mammosomatotroph adenomas remains unclear. We studied a mammosomatotroph adenoma removed from an 8-year old boy with a 5-year history of growth acceleration and acromegalic gigantism at presentation. Elevated basal GH (mean 28 micrograms/l) and PRL (mean 120 micrograms/l) plasma levels were observed, as well as paradoxical responses of GH to L-dopa, TRH and oral glucose administration; PRL was reduced by L-dopa and slightly increased by TRH; GHRH stimulated release of both GH and PRL. Two operations were required to remove the very large tumour and the patient was treated with bromocriptine before the second. Hormonal secretion by tumour explants in culture was evaluated under basal conditions and after stimulation or inhibition. High levels of GH and PRL were secreted for up to 24 days. Furthermore, GHRH and TRH caused a dose-related stimulation of both hormones, while somatostatin and dopamine were effective in suppressing either basal or stimulated hormone release only at very high (microM) concentrations. Intracellular events were studied by determination of the guanosine triphosphate binding (G) protein levels and adenylate cyclase (AC) activity in the tumour tissue. Before bromocriptine treatment, AC activity was very high in the tumour and could be further stimulated by various agents; very high levels of the AC-stimulatory G protein alpha subunit Gs alpha and very low amounts of the AC-inhibiting G protein alpha subunit Gi3 alpha and of the phospholipase C-stimulating G protein alpha subunit Gq alpha were found in the tumour. After bromocriptine, baseline AC activity was normalized and could no longer be stimulated; Gs alpha and Gi3 alpha levels were unchanged while those of Gq alpha were normalized. Screening of tumour DNA after amplification by polymerase chain reaction followed by single-strand conformational polymorphism analysis did not reveal any mutations in the hot spots of G protein alpha subunits (alpha s, alpha i2, alpha o2 and alpha 11) genes or in the H-ras and p53 genes. Gs alpha and GH transcription factor-1 (pit-1) expression were evaluated by amplification of cDNA. While the mRNA expression of pit-1 decreased after bromocriptine treatment, that of Gs alpha increased. These data suggest the possibility of an oncogenic process involving overexpression of Gs alpha, resulting in chronic activation of adenylate cyclase. Furthermore, our results suggest that the anti-secretory and anti-proliferative effects of bromocriptine may be mediated through a decrease in Pit-1 secondary to the inhibition of adenylate cyclase activity.

Adenoma↗

PubMed1993

Functional polymorphism in the parental imprinting of the human IGF2R gene.

The murine genes coding for insulin-like growth factor II (Igf2) and its specific receptor (Igf2r) are parentally imprinted, with exclusive expression from the paternal (Igf2) or maternal (Igf2r) gene copy. We have demonstrated that the human IGF2 gene is imprinted, like its murine homologue. To examine whether the human IGF2R is also imprinted, we used CA repeat polymorphisms of the cDNA sequence to distinguish expression from each copy. Unlike the mouse, most subjects equally expressed both gene copies. However, two out of 14 informative fetuses showed exclusive expression from the maternal gene copy. We conclude that the human IGF2R gene is parentally imprinted, like its murine homologue, but only in a minority of individuals. This is the first direct demonstration of imprinting as a polymorphic trait, a property that had been observed in transgene methylation and predicted from the behavior of certain human tumors.

Animals↗

PubMed1993

Parental genomic imprinting of the human IGF2 gene.

The mouse igf2 gene, coding for the insulin-like growth factor II (IGF-II) is parentally imprinted, only the gene copy derived from the father is expressed. To know whether IGF2, the human homologue, is also imprinted, we used an ApaI polymorphism at the 3' untranslated region in order to distinguish between mRNA derived from each copy of the gene in placentae from heterozygote human fetuses, studied after careful removal of the decidua. Six term and two pre-term placentae of heterozygotes were studied, and in each case the cDNA contained only one of the two alleles present in the genomic DNA. In three cases the mother was homozygous for the non-expressed allele, allowing assignment of paternal origin to the transcribed gene copy. We conclude that, as in the mouse, human IGF2 is parentally imprinted.

Alleles↗

PubMed1993

Effects of growth hormone (GH)-releasing factor and somatostatin on GH secretion from early to midgestation human fetal pituitaries.

Using explant cultures of human anterior pituitary glands (9-19 weeks fetal age) and an acute (3-h) test protocol, we investigated fetal somatotrope responsiveness to human GH-releasing factor [hGRF-(1-44)] and somatostatin [SRIF-(1-14) and SRIF-(1-28)] as a function of age. Ontogenic data were analyzed using three age groups: 9-10, 12-13, and 15-19 weeks fetal age. Both daily (24-h) and acute test (3-h) basal GH secretion increases as a function of fetal age, with the greatest increase occurring after 12-13 weeks; however, the 3 h/24 h secretion ratio remains unchanged, at approximately 12%. GRF (0.1-10 nM) stimulates GH release in a dose-related fashion, regardless of fetal age; there is a significant increase in the response to both 1 nM (P < 0.05) and 10 nM (P < 0.01) GRF between 9-10 and 15-19 weeks fetal age and to 10 nM GRF (P < 0.05) between 12-13 and 15-19 weeks. Pretreatment of cultures (9-19 weeks) with 1 or 10 nM GRF for 24 h does not alter basal 3-h GH secretion, but significantly decreases subsequent responses to 1 or 10 nM GRF, respectively (P < 0.01). Pretreatment with 1 nM GRF does not alter a subsequent 3-h response to 10 nM GRF. SRIF-(1-14) (1-100 nM) causes a dose-related inhibition of basal GH secretion from as early as the ninth week of fetal life; there is a small age-related increase in the somatotrope response to 100 nM SRIF-(1-14) between 12-13 and 15-19 weeks fetal age (P < 0.05). In a group of 11- to 14-week-old fetal pituitaries, SRIF-(1-28) had a significantly (P < 0.05) greater inhibitory effect than SRIF-(1-14) at both 1 and 10 nM; the two peptides decreased basal GH secretion to a similar extent at 100 nM (52.8 +/- 4.0% of control value; P < 0.01). SRIF-(1-14) (10 and 100 nM) does not significantly alter 10 nM GRF-stimulated GH release from 9- to 10-week-old fetal pituitaries. However, by 12-13 weeks, 10 nM SRIF-(1-14) reduces GRF-stimulated GH secretion by 60% (P < 0.01), while 100 nM SRIF-(1-14) decreases it by 80% (P < 0.01); similar inhibitory effects are observed with 15- to 19-week-old fetal somatotropes.(ABSTRACT TRUNCATED AT 400 WORDS)

Cells, Cultured↗