Biomedical subjects

J D Gitlin

Publications and source records attributed to J D Gitlin.

At least 55 records · Page 3Linked to original sources

PubMed1996

Expression of the ceruloplasmin gene in the human retina and brain: implications for a pathogenic model in aceruloplasminemia.

Aceruloplasminemia is an autosomal recessive disorder of iron metabolism characterized by progressive neurodegeneration of the retina and basal ganglia in association with inherited mutations of the ceruloplasmin gene. To begin to elucidate the pathogenesis of this disease, ceruloplasmin gene expression was examined in human brain and retinal tissue. RNA blot analysis and RNAse protection studies demonstrate ceruloplasmin-specific transcripts in multiple regions of the human brain, and biosynthetic studies reveal ceruloplasmin synthesis and secretion in these same regions. Consistent with these observations, in situ hybridization of central nervous system tissue utilizing ceruloplasmin cRNA probes reveals abundant ceruloplasmin gene expression in specific populations of glial cells associated with the brain microvasculature, surrounding dopaminergic melanized neurons in the substantia nigra and within the inner nuclear layer of the retina. Taken in the context of the clinical and pathological features observed in patients with aceruloplasminemia, these data reveal that glial cell-specific ceruloplasmin gene expression is essential for iron homeostasis and neuronal survival in the human central nervous system.

Brain↗

PubMed1996

Mechanisms of gene expression and cell fate determination in the developing pulmonary epithelium.

The pulmonary epithelium is a derivative of the foregut endoderm. Proliferation and differentiation of the primitive pulmonary epithelium result in an array of epithelial cell phenotypes that determine lung function and the response of the lung to injury, infection, or neoplastic transformation. The establishment of a cell phenotype requires the presence of transcription factors that activate or repress expression of specific genes. Members of the forkhead family of transcription factors, in particular HNF-3 alpha, HNF-3 beta, HFH-4; the homeodomain protein TTF-1; and N-myc, are all expressed in the developing pulmonary epithelium and may play important regulatory roles during development. Two genes specific to the pulmonary epithelium, the surfactant protein A and Clara cell secretory protein genes, serve as useful paradigms for understanding the mechanisms regulating cell-specific gene expression in the pulmonary epithelium.

Amino Acid Sequence↗

PubMed1995

Role of hepatocyte nuclear factor-3 alpha and hepatocyte nuclear factor-3 beta in Clara cell secretory protein gene expression in the bronchiolar epithelium.

The 5' flanking region of the Clara cell secretory protein (CCSP) gene contains two cis-acting elements which bind hepatocyte nuclear factor (HNF)-3 alpha and HNF-3 beta in vitro. To determine the role of these proteins in mediating CCSP gene expression in the bronchiolar epithelium, chimeric CCSP-reporter gene constructs containing various regions of the CCSP 5' flanking region were co-transfected into H-441 cells with HNF-3 alpha or HNF-3 beta expression plasmids. These studies indicate that each of these transcription factors positively regulates CCSP gene expression and revealed that CCSP region I (-132 to -76) is sufficient to mediate this effect. Gel-mobility-shift assays with oligonucleotides corresponding to CCSP region I, nuclear extract from bronchiolar epithelial cells and HNF-3-specific antibodies indicate that HNF-3 alpha and HNF-3 beta are the only proteins in bronchiolar epithelial cells which directly interact with this region. Consistent with these observations, HNF-3 alpha and HNF-3 beta transcripts were found to be enriched in this cell population and in situ hybridization of adult lung revealed HNF-3 gene expression in non-ciliated bronchiolar epithelial cells expressing the CCSP gene. Finally, experiments with CCSP region I and a heterologous promoter indicate that this region acts in a promoter-specific context, suggesting that additional factors interacting via the minimal CCSP promoter region are essential in determining the effects of HNF-3 on cell-specific CCSP gene expression in the bronchiolar epithelium.

Animals↗

PubMed1995

Primary structure of hepatocyte nuclear factor/forkhead homologue 4 and characterization of gene expression in the developing respiratory and reproductive epithelium.

Members of the winged helix/forkhead family of transcription factors are believed to play a role in cell-specific gene expression. A cDNA encoding a member of this family of proteins, termed hepatocyte nuclear factor/forkhead homologue 4 (HFH-4), has been isolated from rat lung and rat testis cDNA libraries. This cDNA contains an open reading frame of 421 amino acids with a conserved DNA binding domain and several potential transactivating regions. During murine lung development, a single species of HFH-4-specific transcript (2.4 kb long) is first detected precisely at the start of the late pseudoglandular stage (embryonic day 14.5) and, by in situ hybridization, is specifically localized to the proximal pulmonary epithelium. The unique temporal and spatial pattern of HFH-4 gene expression in the developing lung defines this protein as a marker for the initiation of bronchial epithelial cell differentiation and suggests that it may play an important role in cell fate determination during lung development. In addition to expression in the pulmonary epithelium, RNA blot analysis reveals 2.4-kb HFH-4 transcripts in the testis and oviduct. By using mice with genetic defects in spermatogenesis, HFH-4 expression in the testis is found to be associated with the appearance of haploid germ cells and in situ hybridization studies indicate that HFH-4 expression is confined to stages I-VII of spermatogenesis. This pattern of HFH-4 gene expression during the early stages of differentiation of haploid germ cells suggests that HFH-4 may play a role in regulating stage-specific gene expression and cell-fate determination during lung development and in spermatogenesis.

Aging↗

PubMed1995

Aceruloplasminemia: molecular characterization of this disorder of iron metabolism.

Ceruloplasmin is an abundant alpha 2-serum glycoprotein that contains 95% of the copper found in the plasma of vertebrate species. We report here on the identification of a genetic defect in the ceruloplasmin gene in a patient previously noted to have a total absence of circulating serum ceruloplasmin in association with late-onset retinal and basal ganglia degeneration. In this patient T2 (transverse relaxation time)-weighted magnetic resonance imaging of the brain revealed basal ganglia densities consistent with iron deposition, and liver biopsy confirmed the presence of excess iron. Although Southern blot analysis of the patient's DNA was normal, PCR amplification of 18 of the 19 exons composing the human ceruloplasmin gene revealed a distinct size difference in exon 7. DNA sequence analysis of this exon revealed a 5-bp insertion at amino acid 410, resulting in a frame-shift mutation and a truncated open reading frame. The validity of this mutation was confirmed by analysis of DNA from the patient's daughter, which revealed heterozygosity for this same 5-bp insertion. The presence of this mutation in conjunction with the clinical and pathologic findings demonstrates an essential role for ceruloplasmin in human biology and identifies aceruloplasminemia as an autosomal recessive disorder of iron metabolism. These findings support previous studies that identified ceruloplasmin as a ferroxidase and are remarkably consistent with recent studies on the essential role of a homologous copper oxidase in iron metabolism in yeast. The clinical and laboratory findings suggest that additional patients with movement disorders and nonclassical Wilson disease should be examined for ceruloplasmin gene mutations.

Amino Acid Sequence↗

PubMed1994

Expression of the Wilson disease gene is deficient in the Long-Evans Cinnamon rat.

Long-Evans Cinnamon rats develop a necrotizing hepatitis characterized by excessive hepatic copper accumulation, defective holoceruloplasmin biosynthesis and impaired biliary copper excretion. To elucidate the molecular basis of this defect, a cDNA clone encoding the rat Wilson disease gene was isolated and used to examine gene expression in selected tissues from normal and Long-Evans Cinnamon rats. Although this cDNA readily detects Wilson transcripts in liver and other tissues from normal rats, such transcripts are entirely absent from tissues derived from the Long-Evans Cinnamon rat strain. These data therefore identify the Long-Evans Cinnamon rat as the first bona fide animal model of Wilson disease and suggest that this rat strain may be a valuable resource in the study of this genetic disorder.

Adenosine Triphosphatases↗

PubMed1994

5' flanking region of the Clara cell secretory protein gene specifies a unique temporal and spatial pattern of gene expression in the developing pulmonary epithelium.

Expression of a transgene containing 2.25 kb of the 5' flanking region of the rat Clara cell secretory protein gene and the human growth hormone gene was examined in developing mice. Despite an absolute preservation of tissue specificity based on RNA blot analysis, transgene-specific transcripts were detectable as early as 12.5 days of gestation, at least 4 days prior to endogenous Clara cell secretory protein gene expression. As differentiation proceeded, in situ hybridization revealed an increasingly restricted pattern of transgene expression in the developing pulmonary epithelium, such that by day 16.5 of gestation endogenous and transgene expression were confined to identical cells within the bronchiolar epithelium. The temporal discordance in transgene expression suggests the presence of unique cis-acting elements within the Clara cell secretory protein gene, not present in the transgene, which transduce developmental timing within pulmonary epithelium by actively repressing Clara cell secretory protein gene expression during early development. The unique expression of this transgene serves as a lineage marker in the respiratory epithelium and unmasks a temporal and spatial pattern of gene expression not observed in any pulmonary genes.

Animals↗

PubMed1993

Isolation and characterization of a human liver cDNA as a candidate gene for Wilson disease.

The putative copper and ATP-binding domains of the human Menkes disease gene were used as probes to screen a human liver cDNA library at reduced stringency. Sixty-five clones which remained positive after tertiary screening were subcloned and sequenced. One of these cDNA clones contains an open reading frame with 65% amino acid homology to the Menkes protein. Southern blot analysis localizes this cDNA to the region of the Wilson disease locus on chromosome 13. This cDNA detects a 7.5 kB transcript which is present in human liver and cell lines devoid of the Menkes transcript and which is absent in liver from a patient with Wilson disease. These data suggest that this cDNA is a candidate gene for Wilson disease and that the protein encoded at this locus is a member of the P-type ATPase family.

Adenosine Triphosphatases↗

PubMed1993

Identification of hepatocyte nuclear factor-3 binding sites in the Clara cell secretory protein gene.

To determine the mechanisms of cell-specific gene expression in the developing pulmonary epithelium the Clara cell secretory protein (CCSP) gene promoter was analysed by DNAase I footprinting. A prominent site of protein-DNA interaction was detected from nucleotides -132 to -76 using nuclear extract from mouse lung and human H441 cells. Mobility shift analysis revealed that an oligonucleotide corresponding to this region interacted with multiple proteins from lung and H441 cell nuclear extracts. Analysis of the nucleotide sequence of this region identified two potential binding sites for hepatocyte nuclear factor 3 (HNF-3), and consistent with this finding binding to this CCSP oligonucleotide was specifically competed for by an oligonucleotide corresponding to the HNF-3-binding site from the mouse transthyretin gene. Mobility shift of the CCSP oligonucleotide was supershifted using antisera specific to HNF-3 alpha and HNF-3 beta, and HNF-3 alpha and HNF-3 beta translated in vitro were found to bind specifically to this same oligonucleotide. Co-transfection of HNF-3 alpha- and HNF-3 beta-expression plasmids increased cell-specific reporter gene activity in H441 cells transfected with a CCSP-CAT gene chimeric construct containing this -132 to -76 region. Taken together, these results suggest a role for HNF-3 in mediating cell-specific CCSP gene expression within the bronchiolar epithelium. These findings support the hypothesis that members of the HNF-3 'forkhead' family of transcription factors determine gene expression and cell fate in multiple cell lineages derived from the primitive gut endoderm.

Animals↗

PubMed1993

Interaction of CCAAT/enhancer-binding protein alpha and beta with the rat caeruloplasmin gene promoter.

To determine the mechanisms of expression of the rat caeruloplasmin gene, the promoter region was analysed by DNAase I footprinting. Using nuclear extract from rat liver, a prominent site of protein-DNA interaction was detected from -93 to -48 upstream of the caeruloplasmin gene transcription start and sequence analysis of this region revealed three potential CCAAT/enhancer-binding protein (C/EBP) consensus elements. Mobility-shift analysis using an oligonucleotide encoding this region identified specific binding of proteins from rat liver nuclear extract, and some of these complexes were supershifted using antisera to the C/EBP alpha and beta family members. Mobility-shift studies using a polypeptide encoding the DNA-binding domain of C/EBP alpha also revealed a specific interaction with this region of the caeruloplasmin promoter, and DNAase I footprinting using this polypeptide protected the identical region from -93 to -48. Co-transfection of expression plasmids encoding C/EBP alpha or a related leucine-zipper factor D-binding protein (DBP) revealed a C/EBP-specific increase in reporter gene activity in HepG2 cells transfected with caeruloplasmin-chloramphenicol acetyltransferase containing the -93 to -48 region. A similar result was obtained when these constructs were co-transfected into mouse L cells which were shown not to express the endogenous caeruloplasmin gene. Taken together, these data indicate a role for C/EBP alpha and beta in mediating transcription from the caeruloplasmin gene promoter and suggest that this region of the promoter is not responsible for tissue-specific expression.

Animals↗

PubMed1992

Cell-specific expression of a Clara cell secretory protein-human growth hormone gene in the bronchiolar epithelium of transgenic mice.

Clara cell secretory protein (CCSP) is an abundant 10-kDa protein synthesized and secreted by nonciliated epithelial cells lining the respiratory and terminal bronchioles of the lung. CCSP gene expression is an informative developmental marker within the bronchiolar epithelium recapitulating cellular differentiation in the distal respiratory epithelium during late fetal and early postnatal life. To define the mechanisms that establish and maintain gene expression within this epithelium, CCSP-human growth hormone chimeric gene constructs were created and used to generate transgenic mice. RNA blot analysis of organs from F1 transgenic offspring and normal littermates revealed that cis-acting elements within 2.25 kilobases of the 5' flanking region of the CCSP gene were sufficient to direct lung-specific expression of human growth hormone. In situ hybridization and immunohistochemistry of individual bronchioles revealed that human growth hormone expression in the respiratory epithelium of these mice was confined to Clara cells, consistent with observations of the endogenous CCSP gene. Unexpectedly, founder animals and F1 transgenic offspring exhibited an unusual phenotype of growth retardation and delayed hair appearance, suggesting a unique effect of human growth hormone on normal intrauterine development. CCSP-human growth hormone transgenic mice provide a model to dissect the developmental mechanisms regulating gene expression during pulmonary epithelial cell growth and differentiation. Definition of the cis-acting elements determining such cell-specific expression will be of value in strategies for the somatic gene therapy of human pulmonary disease.

Animals↗

PubMed1992

Mechanisms of caeruloplasmin biosynthesis in normal and copper-deficient rats.

To examine the mechanisms of holo-caeruloplasmin biosynthesis, we measured the serum caeruloplasmin concentration and oxidase activity, hepatic caeruloplasmin mRNA content and hepatocyte caeruloplasmin biosynthesis and secretion in normal and copper-deficient rats. Copper deficiency resulted in a near-complete loss of serum caeruloplasmin oxidase activity, yet only a 60% reduction in serum caeruloplasmin concentration and no change in the abundance of hepatic caeruloplasmin mRNA or the rate of caeruloplasmin biosynthesis. Both interleukin-1 alpha and lipopolysaccharide increased hepatic caeruloplasmin mRNA content and caeruloplasmin biosynthesis in normal and copper-deficient animals, but neither mediator increased caeruloplasmin oxidase activity in the copper-deficient group. Pulse-chase studies in primary hepatocytes from normal and copper-deficient rats revealed that the secretory rates for newly synthesized caeruloplasmin were identical, despite little or no holo-caeruloplasmin synthesis in hepatocytes of copper-deficient rats. We conclude that hepatocyte copper content has no effect on hepatic caeruloplasmin-gene expression or caeruloplasmin biosynthesis and that the incorporation of copper into newly synthesized caeruloplasmin is not a rate-limiting step in the biosynthesis or secretion of the apoprotein from rat hepatocytes.

Animals↗

PubMed1992

Structural and functional analysis of the 5'-flanking region of the rat ceruloplasmin gene.

To characterize the mechanisms determining tissue-specific ceruloplasmin gene expression during development, the rat ceruloplasmin gene was isolated in a series of overlapping phage clones. The 5'-flanking region was characterized and the transcription initiation site was identified by primer extension and RNase protection. Nucleotide sequence analysis of this region revealed a typical eukaryotic promotor structure, but no obvious homology with cis-acting elements previously characterized as determining tissue-specific gene expression. Transient expression of chimeric ceruloplasmin-reporter gene constructs containing up to 5200 base pairs (bp) of the 5'-flanking region revealed that sequences 732 bp upstream of the start nucleotide were sufficient to confer hepatocyte-specific expression. The region from -393 to -348 was determined by deletion analysis to contain a positive-acting element, and includes sequence partially homologous to the rat albumin D site. Mobility shift analysis revealed that this region specifically binds a heat-labile nuclear protein from rat liver and from newborn but not adult rat lung. Binding to this region was competed by oligonucleotides corresponding to the albumin D site, but not by oligonucleotides corresponding to binding sites for the hepatocyte transcription factors HNF-1, HNF-3, HNF-4, and C/EBP. These data indicate that ceruloplasmin gene expression is determined in part by a cis-acting region 393 bp upstream of the transcription start site, which binds a previously uncharacterized nuclear protein. The tissue distribution of this nuclear protein suggests that it plays a role in directing ceruloplasmin gene expression in lung and liver during development.

Albumins↗

PubMed1992

Clara cell secretory protein gene expression in bronchiolar epithelium.

To determine the mechanisms of Clara cell secretory protein (CCSP) gene expression, a cDNA clone was isolated and used in RNA blot analysis. A single 600 bp CCSP specific transcript was detected in the developing rat lung on fetal day 18. This transcript increased in abundance during late fetal life such that adult levels were attained within 2 wk postpartum. CCSP gene expression was tissue specific, being confined to lung and trachea at all developmental stages. The abundance of CCSP mRNA in lung tissue was unchanged after the induction of lung injury in adult rats either with lipopolysaccharide or prolonged exposure to hyperoxia. In situ hybridization of lung tissue revealed that CCSP gene expression is localized to the nonciliated epithelial (Clara) cells of the bronchiolar epithelium throughout fetal and postnatal development. Taken together the results indicate that the gene for CCSP is abundantly expressed in a cell-specific fashion in the lung and suggest that analysis of such expression will be useful in elucidating the role of Clara cells in the growth and development of the bronchiolar epithelium.

Animals↗

PubMed1991

Tissue-specific ceruloplasmin gene expression in the mammary gland.

Using a ceruloplasmin cDNA clone in RNA blot analysis, a single 3.7 kb ceruloplasmin-specific transcript was detected in rat mammary gland tissue from pregnant and lactating animals. Ceruloplasmin gene expression in the mammary gland was tissue-specific, with no evidence of expression in brain, heart or other extrahepatic tissues. Ceruloplasmin mRNA was also detected in mammary gland tissue from male, virgin female and non-pregnant/multiparous animals, and the abundance of ceruloplasmin-specific transcripts in virgin female rats was independent of their stage of oestrus. In virgin female mammary gland the content of ceruloplasmin mRNA was 20% of that in hepatic tissue from these animals and approx. 2-3-fold greater than that found in mammary gland tissue of pregnant or lactating animals. Development studies revealed ceruloplasmin gene expression in male and female mammary gland by only 2 weeks of age, prior to the onset of puberty. Biosynthetic studies indicated that the ceruloplasmin mRNA in mammary gland tissue was translated into a 132 kDa protein qualitatively similar to that synthesized in liver. By in situ hybridization, ceruloplasmin gene expression was localized to the epithelium lining the mammary gland alveolar ducts, without evidence of expression in the surrounding mesenchyme. Ceruloplasmin gene expression was also detected in a human breast adenocarcinoma cell line and in biopsy tissue from women with invasive ductal carcinoma. Taken together, these data indicate that the mammary gland is a prominent site of extrahepatic ceruloplasmin gene expression and add to the evidence that ceruloplasmin biosynthesis is associated with growth and differentiation in non-hepatic tissues.

Aging↗

PubMed1991

Tissue-specific regulation of dipeptidyl peptidase IV expression during development.

The patterns of dipeptidyl peptidase (DPP) IV activity and protein amount in different rat organs during development were compared. In order to elucidate the molecular basis for these patterns, total RNA was isolated from lung and kidney at different stages of development and analysed by Northern-blot hybridization using an oligonucleotide derived from the DPP IV cDNA sequence. This oligonucleotide hybridized to two distinct mRNAs of approx. 3.2 and 4.8 kb respectively. During kidney development, the pattern for DPP IV mRNA paralleled that of DPP IV activity and protein amount, suggesting that, in kidney, the expression of DPP IV is primarily controlled at the transcriptional level. In contrast, the magnitude of DPP IV activity during lung development compared with that of DPP IV mRNA in lung suggests that post-transcriptional mechanisms are involved in regulating the expression of DPP IV in lung. Organ-specific regulation of DPP IV expression may provide a useful model for further comparative studies of transcriptional and post-transcriptional mechanisms of DPP IV expression within the same organism.

Aging↗

PubMed1991

Hepatic caeruloplasmin-gene expression during development in the guinea-pig. Correlation with changes in hepatic copper metabolism.

Decreased amounts of the serum copper-binding protein caeruloplasmin (hypocaeruloplasminaemia) are one of the characteristic abnormalities of copper metabolism that are found in all neonatal mammals. In the present study we have investigated the mechanism responsible for hypocaeruloplasminaemia found in neonatal guinea pigs. Northern-blot analysis of guinea-pig liver RNA revealed the presence of two caeruloplasmin mRNA species. A marked developmental change in expression was observed, with no detectable mRNA before birth, when hepatic copper is elevated. Expression was initiated soon after birth, when copper levels in the liver are falling, and was closely linked to the rise in enzymic activity and protein levels in serum. There was no differential regulation of the two mRNA species; however, a marked inter-animal variation in mRNA levels was observed. Using gel-filtration chromatography we were able to show that, before birth, serum copper was associated with a low-molecular-mass species and that, with the advent of expression of the caeruloplasmin gene after birth, copper was increasingly associated with caeruloplasmin.

Adult↗

PubMed1991

Mechanisms of copper incorporation during the biosynthesis of human ceruloplasmin.

To examine the mechanisms of copper incorporation during ceruloplasmin biosynthesis, we developed methods to resolve and identify apo and holoceruloplasmin. The identity of holoceruloplasmin was confirmed by oxidase activity staining, immunoblotting, 67Cu-ligand exchange, and 67Cu-ligand blotting. Following metabolic labeling of human liver and lung cell lines with 67Cu, newly synthesized holoceruloplasmin was detected in the culture media as two species with apparent molecular masses of 84 and 79 kDa. Pulse-chase studies demonstrate that exogenous copper is readily available for incorporation into newly synthesized ceruloplasmin and that the kinetics of apo and holoceruloplasmin synthesis and secretion are identical. Inhibition of N-linked glycosylation did not affect the rate or amount of copper incorporated into newly synthesized ceruloplasmin but did result in the secretion of a single 68-kDa holoceruloplasmin moiety. Despite differences in the kinetics of copper uptake between cell lines a linear rate of copper incorporation into newly synthesized ceruloplasmin was observed with no evidence of copper exchange following biosynthesis. Under the conditions studied, holoceruloplasmin accounted for less than 5% of the total ceruloplasmin synthesized and secreted by each cell line. The data indicate that copper is incorporated into newly synthesized ceruloplasmin early in the course of biosynthesis by a process independent of N-linked carbohydrate addition. This process of copper incorporation results in an apparent conformational change in the ceruloplasmin molecule which does not affect the secretory rate of the protein.

Apoproteins↗