Biomedical subjects

C G Goodyer

Publications and source records attributed to C G Goodyer.

At least 19 recordsLinked to original sources

PubMed2006

Caspase-1 activation of caspase-6 in human apoptotic neurons.

Active caspase-6 (Csp-6) induces cell death in primary cultures of human neurons and is abundant in the neuropathological lesions of Alzheimer's disease. However, the mode of Csp-6 activation is not known. Here, we show that the Csp-1 inhibitor, Z-YVAD-fmk specifically prevents activation of Csp-6 and cell death in human neurons. A transient increase in Csp-1-like activity and an increase in the p23Csp-1 subunit occur early after serum deprivation. Recombinant active Csp-1 (R-Csp-1) cleaves recombinant and neuronal pro-Csp-6 in vitro resulting in Csp-6 activity. However, R-Csp-1 does not induce cell death when microinjected in human neurons despite the inhibition of serum-deprivation induced cell death with a Csp-1 dominant negative construct. These results show that Csp-1 is an upstream positive regulator of Csp-6-mediated cell death in primary human neurons. Furthermore, these results suggest that the activation of Csp-1 must be accompanied by an apoptotic insult to induce Csp-6-mediated cell death.

Amino Acid Chloromethyl Ketones↗

PubMed2005

Cellular prion protein inhibits proapoptotic Bax conformational change in human neurons and in breast carcinoma MCF-7 cells.

Prion protein (PrP) prevents Bcl-2-associated protein X (Bax)-mediated cell death, but the step at which PrP inhibits is not known. We first show that PrP is very specific for Bax and cannot prevent Bak (Bcl-2 antagonist killer 1)-, tBid-, staurosporine- or thapsigargin-mediated cell death. As Bax activation involves Bax conformational change, mitochondrial translocation, cytochrome c release and caspase activation, we investigated which of these events was inhibited by PrP. PrP inhibits Bax conformational change, cytochrome c release and cell death in human primary neurons and MCF-7 cells. Serum deprivation-induced Bax conformational change is more rapid in PrP-null cells. PrP does not prevent active caspase-mediated cell death. PrP does not colocalize with Bax in normal or apoptotic primary neurons and cannot prevent Bax-mediated cytochrome c release in a mitochondrial cell-free system. We conclude that PrP protects against Bax-mediated cell death by preventing the Bax proapoptotic conformational change that occurs initially in Bax activation.

Apoptosis↗

PubMed2004

Developmental changes in the expression of somatostatin receptors (1-5) in the brain, hypothalamus, pituitary and spinal cord of the human fetus.

The actions of somatostatin (SST) in the nervous system are mediated by specific high affinity SST receptors (SSTR1-5). However, the role of this hormone and the distribution of its receptor subtypes have not yet been defined in neural structures of the human fetus. We have analyzed four neural tissues (CNS, hypothalamus, pituitary and spinal cord) from early to midgestation for the expression of five human SSTR mRNAs, using a reverse transcription-polymerase chain reaction and Southern blot approach. These fetal neural tissues all express mRNA for multiple SSTR subtypes from as early as 16 weeks of fetal life but the developmental patterns of expression vary considerably. Transcripts for SSTR1 and SSTR2A are the most widely distributed, being expressed in all four neural tissues. SSTR2A is often the earliest transcript to be detected (7.5 weeks in CNS). SSTR3 mRNA is confined to the pituitary, hypothalamus, and spinal cord. SSTR4 is expressed in fetal brain, hypothalamus and spinal cord but not pituitary. SSTR5 mRNA is detectable in the pituitary and spinal cord by 14-16 weeks of fetal life. This mapping of SSTR mRNA expression patterns in human fetal neural tissues is an important first step toward our goal of determining the role of SST in the nervous system during early stages in human development.

Aborted Fetus↗

PubMed2004

No evidence for mutations or altered expression of the Suppressor of Fused gene (SUFU) in primitive neuroectodermal tumours.

The sonic hedgehog (Shh) and the Wnt signalling pathways are involved in the development of medulloblastomas (MBs), the most frequent malignant brain tumours in children. Components of these two developmental and cancer-associated pathways, including (Patched) PTCH, SMOH, adenomatous polyposis coli (APC), beta-catenin and AXIN1 show somatic mutations in sporadic MBs. In this study we analysed SUFU (human Suppressor of Fused), which acts as a negative regulator of both the Shh and Wnt signalling pathways and therefore represents a putative tumour suppressor gene, to find out if it is also involved in the pathogenesis of sporadic MBs. We screened 145 primitive neuroectodermal tumours (PNETs) including 90 classic MBs, 42 of the desmoplastic variant and two medullomyoblastomas as well as 11 MB cell lines for mutations using single-strand conformational polymorphism (SSCP) and sequencing analysis. 18% of the MBs exhibited allelic losses on chromosome 10q. In contrast to a previous report, in which truncating mutations of SUFU have been identified in 9% of MBs, we were not able to identify somatic mutations of SUFU in our large tumour panel. We uncovered single nucleotide polymorphisms (SNPs) in exon 4, 8, 11 and in intron 2 in the SUFU gene. Expression analysis by competitive reverse transcription-polymerase chain reaction (RT-PCR) revealed no difference in SUFU mRNA levels of both MB subtypes and normal foetal or adult cerebellar tissues. Our results indicate that genetic alterations of the SUFU gene, do not contribute significantly to the molecular pathogenesis of MBs.

Adolescent↗

PubMed2001

17-beta-estradiol induces an inhibitor of active caspases.

We have shown previously that caspase-6 activity is lethal to human neurons (LeBlanc et al., 1999; Zhang et al., 2000). Here we find that 17-beta-estradiol but not 17-alpha-estradiol prevents caspase-6-mediated neuronal cell death. 17-beta-estradiol-treated neuronal extracts directly inhibit recombinant active caspase-6, caspase-3, caspase-7, and caspase-8 in vitro. We conclude that 17-beta-estradiol induces a caspase inhibitory factor (CIF) that is preventing neuronal apoptosis. The induction of CIF occurs within 10 min of 17-beta-estradiol exposure to neurons, does not require de novo protein synthesis, and involves mitogen-activated protein kinase activation. The effect is antagonized by the estrogen receptor antagonist tamoxifen. In contrast, 17-beta-estradiol does not induce CIF or prevent caspase-mediated cell death in cultured astrocytes. CIF does not act through oxidation of the caspase active site. CIF activity copurifies with proteins of between 12 and 14 kDa in size. Our results indicate that 17-beta-estradiol induces an inhibitor of active caspases through a receptor-mediated nongenomic pathway and provide an additional mechanism for the neuroprotective action of 17-beta-estradiol that is likely highly relevant to the understanding of the role of estrogen against Alzheimer's disease.

Apoptosis↗

PubMed2001

Evidence against GRB10 as the gene responsible for Silver-Russell syndrome.

Recent evidence shows that Silver-Russell syndrome (SRS), the major functional deficit of which is limited growth, both intrauterine and postnatal, is due to a double dose of a gene within 7p11.2-p13 that is normally expressed exclusively from the maternal copy. Of the several growth-related genes in this chromosomal region, only GRB10 has been demonstrated to be imprinted; however, imprinting was limited to brain and muscle and was incomplete. Using reverse-transcript PCR, we now confirm GRB10 imprinting in these two tissues is isoform-specific and, more importantly, demonstrate absence of imprinting in growth plate cartilage, the tissue most directly involved in linear growth. Thus, it is unlikely that GRB10 is the gene responsible for SRS.

Aged↗

PubMed2001

Prion protein protects human neurons against Bax-mediated apoptosis.

The function of the cellular prion protein (PrP) is still poorly understood. We present here an unprecedented role for PrP against Bax-mediated neuronal apoptosis and show that PrP potently inhibits Bax-induced cell death in human primary neurons. Deletion of four octapeptide repeats of PrP (PrPDeltaOR) and familial D178N and T183A PrP mutations completely or partially eliminate the neuroprotective effect of PrP. PrP remains anti-apoptotic despite truncation of the glycosylphosphatidylinositol (GPI) anchor signal peptide, indicating that the neuroprotective form of PrP does not require the abundant cell surface GPI-anchored PrP. Our results implicate PrP as a potent and novel anti-apoptotic protein against Bax-mediated cell death.

Apoptosis↗

PubMed2001

GT repeat polymorphism in the 5' flanking region of the human growth hormone receptor gene.

A polymorphic GT dinucleotide repeat sequence has been identified in the 5' flanking region of the human growth hormone receptor (hGHR) gene on chromosome 5p13.1-p12, within the promoter region of the V9 5'UTR exon. Thirteen alleles have been identified in 50 non-related individuals, with an observed heterozygosity of 52%. The major allele contains 24 repeats, although a range of 19-32 repeats has been observed. Codominant segregation was demonstrated in five two-generation and two three-generation families. This marker may be useful in analysing the role of the hGHR gene in pre- and postnatal growth disorders.

5' Untranslated Regions↗

PubMed2001

Alternative promoter usage and splicing of ZNF74 multifinger gene produce protein isoforms with a different repressor activity and nuclear partitioning.

We have previously shown that ZNF74, a candidate gene for DiGeorge syndrome, encodes a developmentally expressed zinc finger gene of the Kruppel-associated box (KRAB) multifinger subfamily. Using RACE, RT-PCR, and primer extension on human fetal brain and heart mRNAs, we here demonstrate the existence of six mRNA variants resulting from alternative promoter usage and splicing. These transcripts encode four protein isoforms differing at their N terminus by the composition of their KRAB motif. One isoform, ZNF74-I, which corresponds to the originally cloned cDNA, was found to be encoded by two additional mRNA variants. This isoform, which contains a KRAB motif lacking the N terminus of the KRAB A box, was devoid of transcriptional activity. In contrast, ZNF74-II, a newly identified form of the protein that is encoded by a single transcript and contains an intact KRAB domain with full A and B boxes, showed strong repressor activity. Deconvolution immunofluorescence microscopy using transfected human neuroblastoma cells and nonimmortalized HS68 fibroblasts revealed a distinct subcellular distribution for ZNF74-I and ZNF74-II. In contrast to ZNF74-I, which largely colocalizes with SC-35 in nuclear speckles enriched in splicing factors, the transcriptionally active ZNF74-II had a more diffuse nuclear distribution that is more characteristic of transcriptional regulators. Taken with the previously described RNA-binding activity of ZNF74-I and direct interaction with a hyperphosphorylated form of the RNA polymerase II participating in pre-mRNA processing, our results suggest that the two ZNF74 isoforms exert different or complementary roles in RNA maturation and in transcriptional regulation.

Alternative Splicing↗

PubMed2001

Characterization of the growth hormone receptor in human dermal fibroblasts and liver during development.

Human tissues express growth hormone receptors (hGHR) by the 3rd mo of gestation. We assessed developmental changes in hGHR function in fibroblasts and liver, testing binding and hormonal response. Fetal cells showed low but reproducible hGH binding. No age-related changes occurred in fibroblasts (9 wk-34 yr). In contrast, there was a fourfold increase in hGH binding in postnatal liver, with a sixfold increase in hGHR mRNA. Both full-length and truncated hGHR mRNAs were detected in all livers. Cross-linking revealed a larger hGH/receptor complex in fetal liver. Fetal hepatocytes produced 10 times more insulin-like growth factor (IGF)-II than IGF-I, and responded to hGH (150 ng/ml) with a significant increase in IGF-II. Fetal hepatocytes secreted three IGF-binding proteins (IGFBPs), including IGFBP1, but not IGFBP3. hGH did not alter fetal hepatocyte IGFBPs but stimulated glucose uptake. Exposure of fibroblasts to hGH decreased hGH binding only in >1-yr postnatal fibroblasts, whereas treatment with dexamethasone (100-400 nM) increased binding only in postnatal cells. Thus, although fetal hepatocytes and fibroblasts possess functional hGHR, these receptors (and/or their signaling pathways) are immature or have adapted to the in utero environment.

Adult↗

PubMed2001

Organization and evolution of the human growth hormone receptor gene 5'-flanking region.

Previous studies have identified eight variant human GH receptor (hGHR) messenger RNA (mRNAs; V1-V8), that differ in their 5'-untranslated regions (5'UTRs) but splice into the same site just upstream of the translation start site in exon 2; thus, they encode the same protein. Here we report a novel variant, V9, and describe the mapping of all nine 5'UTR sequences within 40 kb upstream of exon 2. A cluster of three sequences, V2-V9-V3 (termed module A), lies furthest 5', and approximately 16 kb downstream is a second cluster of four exons, V7-V1-V4-V8 (module B). V6 is midway between modules A and B. Module B is about 18 kb upstream of V5, which lies adjacent to exon 2. hGHR expression is under developmental- and tissue-specific regulation, and expression of the variant mRNAs is related to their position within the 5'-flanking region; whereas module A (V2,V9,V3) and V5 variants are widely expressed, module B (V7,V1,V4,V8) and V6 variant mRNAs are detectable only in postnatal liver. Transcriptional start sites for V1 and V9 (representing the two different modules) were identified, showing that postnatal liver-specific expression of V1 is driven from two TATA boxes, whereas the ubiquitous V9 transcript has a single start site and a TATA-less promoter. V9 promoter activity was shown by in vivo and in vitro transfection assays, and an NF-Y binding site was demonstrated by electromobility shift assay. Thus, the regulatory regions of the hGHR gene are complex, and the clustering of seven 5'UTR exons within two modules with distinctly different mRNA expression patterns is the most striking feature.

5' Untranslated Regions↗

PubMed2001

Galphas transcripts are biallelically expressed in the human kidney cortex: implications for pseudohypoparathyroidism type 1b.

Pseudohypoparathyroid type 1b patients are characterized by renal resistance to PTH in the absence of Albright's hereditary osteodystrophy or other endocrine abnormalities. Kindred studies have suggested that the cause of this resistance is a specific decrease in Galphas activity in renal proximal tubules due to paternal imprinting of Galphas. To test this, allelic expression of Galphas was analyzed in human fetal kidney cortex samples by RT-PCR assays. The results showed that, in contrast to the parent-specific expression of exon 1A and XLalphas (paternal) or NESP (maternal) mRNAs, Galphas transcripts are biallelically expressed in human kidney cortex. These data implicate abnormal imprinting of alternative regions within the GNAS1 locus as a more likely cause of pseudohypoparathyroid type 1b.

GTP-Binding Protein alpha Subunits, Gs↗

PubMed2001

Class III alleles of the variable number of tandem repeat insulin polymorphism associated with silencing of thymic insulin predispose to type 1 diabetes.

Type 1 diabetes results from autoimmune destruction of the insulin-producing pancreatic beta cells. The insulin gene (INS) is also expressed in human thymus, an ectopic expression site likely involved in immune tolerance. The IDDM2 diabetes susceptibility locus maps to a minisatellite composed of a variable number of tandem repeats situated 0.5 kb upstream of INS. Chromosomes carrying the protective long INS variable number of tandem repeats alleles (class III) produce higher levels of thymic INS mRNA than those with the predisposing, short class I alleles. However, complete silencing of thymic INS transcripts from the class III chromosome was found in a small proportion of heterozygous human thymus samples. We hypothesized that the specific class III alleles found on these chromosomes silence rather than enhance thymic insulin expression. To test the prediction that these alleles are predisposing, we developed a DNA fingerprinting method for detecting two putative "silencing" alleles found in two thymus samples (S1, S2). In a set of 287 diabetic children and their parents we found 13 alleles matching the fingerprint of the S1 or S2 alleles. Of 18 possible transmissions, 12 of the S1-S2 alleles were transmitted to the diabetic offspring, a frequency of 0.67, significantly higher than the 0.38 seen in the remaining 142 class III alleles; P = 0.025. This confirms our prediction and represents an additional level of correlation between thymic insulin and diabetes susceptibility, which supports a thymic enhancer effect of the INS variable number of tandem repeats as the mechanism of IDDM2 and refines the contribution of IDDM2 genotyping to diabetes risk assessment.

Adolescent↗

PubMed2001

Alu elements in human growth hormone receptor gene 5' untranslated region exons.

The human growth hormone receptor (hGHR) is encoded by exons 2-10 of the hGHR gene on chromosome 5p13.1-p12. There are several different 5' untranslated region (5'UTR) variants of hGHR mRNA (V1-V9) that all encode the same protein. We have recently mapped the V1-V9 5'UTR sequences within 40 kb of the 5' flanking region of the hGHR gene. Seven of the exons are clustered within two small modules, module A (V2-V9-V3) and module B (V7-V1-V4-V8), approximately 38 kb and approximately 18 kb respectively upstream of exon 2 of the coding region; V6 lies midway between the two modules and V5 is adjacent to exon 2. We now report the existence of two subvariant V3 exons, one upstream of module A (exon V3b) and one midway between module B and exon 2 (exon V3a/b). Both have sequences homologous to Alu elements. In addition, we determined the alternative splicing mechanisms that produce three different mRNAs from these exons: V3c (from the V3 exon in module A) or V3a and V3b (from a combination of exon V3 and the Alu-containing V3 subvariant exons). hGHR expression is under developmental- and tissue-specific regulation: module A-derived mRNAs are widely expressed in human tissues, while module B-derived mRNAs are only detectable in postnatal liver. Expression of the variant V3 mRNAs is similar to those from module A, being produced ubiquitously in human fetal and postnatal tissues, with V3c always the major variant detected. The Alu-containing mRNAs (V3a and V3b) are also detectable in baboon and rhesus tissues, in accordance with the finding of Alu elements throughout the primate genome. In summary, we have mapped the relative locations of two new 5'UTR exons within the 5' flanking region of the hGHR gene and described the derivation and expression patterns for two variant hGHR mRNAs from these primate-specific exons. The introduction of Alu elements has contributed to the evolution of the primate GHR gene as a highly complex transcriptional unit.

5' Untranslated Regions↗

PubMed2000

Interleukin-1 is a key regulator of matrix metalloproteinase-9 expression in human neurons in culture and following mouse brain trauma in vivo.

An acute trauma to the CNS rapidly results in the upregulation of inflammatory cytokines that include interleukin-1 (IL-1). We report here that the levels of several matrix metalloproteinases (MMPs) are also elevated following a corticectomy trauma injury to the mouse CNS. The delayed upregulation of MMPs compared to that for IL-1 suggests the possibility that inflammatory cytokines regulate MMP production in CNS trauma. To resolve this, we developed a method to isolate and maintain highly enriched human fetal neurons or astrocytes in culture and examined the regulation by cytokines of the activity of a subgroup of MMPs, the gelatinases (MMP-2 and -9). While both neuronal and astrocytic cultures displayed comparable MMP-2 activity, as evidenced by gelatin zymography, levels of MMP-9 were proportionately higher in neurons compared to astrocytes. Of a variety of cytokines and growth factors tested in vitro, only IL-1beta was effective in increasing the neuronal expression of MMP-9. Finally, an IL-1 receptor antagonist attenuated the increase of neuronal MMP-9 in culture and abolished the injury-induced increase of MMP-9 in the mouse brain. These results implicate IL-1beta as a key regulator of neuronal MMP-9 in culture and of the elevation of MMP-9 that occurs following mouse CNS trauma.

Animals↗

PubMed2000

Methylation patterns of human parathyroid hormone (PTH)/PTH-related peptide receptor gene promoters are established several weeks prior to onset of their function.

Expression of the human parathyroid hormone (PTH)/PTH-related peptide receptor (PTHR) gene is controlled by three promoters, P1-P3. P1 functions specifically in kidney, whereas P2 is ubiquitously active. P3 is also widely active, although more so in kidney than other tissues. However, only P2 functions at midgestation. We examined the role of methylation in controlling PTHR promoter activity. Function of all promoters was inhibited by CpG methylation in vitro. Significantly, P1 is selectively hypomethylated in adult kidney in vivo, strongly suggesting that demethylation is required for renal P1 function. Moreover, this pattern is established by 11. 75 weeks of fetal age, several weeks prior to the onset P1 activity. P3 is unmethylated at midgestation, although it is inactive at this stage of development, and thus exhibits characteristics of both tissue-specific and ubiquitously active promoters. These results show that adult methylation patterns of P1 and P3 are established several weeks prior to their induction, indicating that their function requires factors expressed late in development.

Adult↗

PubMed2000

p57(KIP2) is not mutated in hepatoblastoma but shows increased transcriptional activity in a comparative analysis of the three imprinted genes p57(KIP2), IGF2, and H19.

Hepatoblastomas (HBs), representing malignant liver tumors of childhood, show frequent loss of heterozygosity (LOH) in the chromosomal region 11p15.5. This loss is of maternal origin suggesting the presence of a monoallelically expressed tumor suppressor gene in this region. p57(KIP2) (KIP2) located at 11p15.5 is predominantly expressed from the maternal allele and encodes a cyclin-dependent kinase inhibitor. We screened a series of 56 HB tumors and five HB cell lines for allelic loss (LOH) of the KIP2 locus by microsatellite analysis and KIP2 coding sequence mutations by single-strand conformation polymorphism analysis. Although LOH at the KIP2 locus occurred in 25% of the cases, no mutations were found. Analysis of KIP2 mRNA expression by competitive reverse transcriptase-polymerase chain reaction revealed up-regulation in nine of 12 HBs compared to matching liver samples. In contrast, mRNA levels of the putative suppressor gene H19 on 11p15.5 were decreased in 10 of 12 tumors, indicating that KIP2 and H19 are not co-regulated in HBs. IGF2 mRNA expression was increased in 11 of 12 HB samples. All HBs showed monoallelic KIP2 expression. However, the overexpression of KIP2 in HBs with maternal loss of 11p15.5 suggests a reactivation of the paternal allele in these cases. Overexpression of KIP2 in HBs argues against a role as a HB suppressor gene.

Child, Preschool↗

PubMed2000

Transcriptional properties of Ptx1 and Ptx2 isoforms.

The Ptx (Pitx) family of homeobox transcription factors comprises Ptx1, Ptx2 and Ptx3. Ptx1 and Ptx2 are expressed in the stomodeum and its derivatives including the pituitary, as well as in mesodermal derivatives, whereas Ptx3 is expressed in one neuronal lineage of the brain and in the eyes. A large set of downstream target genes have been identified for Ptx1 in the pituitary gland where it acts as a pan-pituitary regulator of transcription. In particular, Ptx1 contributes to promoter- and lineage-specific transcription by interaction with cell-restricted factors such as SF-1, Egr-1, Pit1, and the basic helix-loop-helix heterodimer NeuroD1/Pan1. We describe the cloning from pituitary cells and the characterization of a Ptx1 isoform, named Ptx1b, generated by alternative promoter usage. The two Ptx1 and two Ptx2 isoforms have similar in vitro DNA binding specificities and they all activate transcription driven by a panel of pituitary promoters, including those for proopiomelanocortin, alphaGSU, LHbeta, FSHbeta, GnRH-R, TSHbeta, PRL, and GH. Also like Ptx1, the Ptx1b, Ptx2a, and Ptx2b transcription factors synergize with the structurally unrelated factors SF-1, Egr-1, Pit1, and NeuroD1/Pan1 to activate promoter-specific transcription. In conclusion, the pituitary transcriptional activities of the four Ptx isoforms do not appear to be dependent on the variant N-termini of these factors.

Amino Acid Sequence↗