Search PubMed⌕ Search

Biomedical subjects

M Buchwald

Publications and source records attributed to M Buchwald.

At least 55 records · Page 3Linked to original sources
PubMed1996

Genetic complementation of radiation response by 3' untranslated regions (UTR) of RNA.

The molecular basis of radiosensitivity was studied using a cDNA complementation approach to correct radiosensitivity in cells. Four cDNAs of sizes 1.6, 2.0, 2.2 and 2.5 kb were isolated that corrected several aspects of the phenotype of cells from patients with the human genetic disorder ataxia-telangiectasia, characterized by hypersensitivity to ionizing radiation. The criteria used to assess correction included cell viability, induced chromosome aberrations, G2 phase delay and induction of p53 after exposure to radiation. One cDNA (2.5 kb) was identified as the complete sequence of the RNA helicase p68, which was capable of correcting radiosensitivity based on two of the above four criteria, with p53 induction post irradiation being partially corrected. The 2.2 kb cDNA was shown to correspond to the complete sequence of arginyl tRNA synthetase and the other two cDNAs were identical to the 3' untranslated regions (UTR) of the transcription factor TFIIS (1.6 kb) and phospholipase A2 (2.0 kb) respectively. Additional transfections with the 3'UTR (198 nucleotides) of p68 RNA helicase and its inverse sequence revealed that the 3'UTR had the same complementation capacity as the full-length cDNA, whereas the inverse construct failed to complement radiosensitivity. These data provide additional support for a novel role for 3'UTRs in the regulation of gene expression.

Ataxia Telangiectasia↗
PubMed1996

Developmental expression of the Fac gene correlates with congenital defects in Fanconi anemia patients.

Fanconi anemia (FA) is a genetically heterogeneous, autosomal recessive disorder characterized by a variety of congenital and skeletal malformations, progressive pancytopaenia and predisposition to malignancies. While the basic defect in this disease is not known, the cloning of the gene defective in FA group C patients (FAC) allows analysis of its expression pattern, which may provide clues about the functional properties of the protein. This paper describes the distribution of Fac transcripts during murine development (8-19.5 days p.c.), using RNA in situ hybridization. Fac is initially expressed (8-10 days p.c.) in the mesenchyme and its derivatives with osteogenic potential. The transcript is also apparent at later stages of bone development (13-19.5 days p.c.), localized to cells of the inner perichondrium, periosteum and zone of endochondral ossification. In the latter, Fac transcripts are seen in cells from both osteogenic and hematopoietic lineages. Fac mRNA is also seen in intramembranous cranial and facial bones. In addition, Fac signal is detected in non-skeletal tissues: brain, whisker follicles, lung, kidney, gut and stomach. Fac expression is high in progenitor cell populations but is downregulated in differentiating cells that give rise to connective tissue. The pattern of Fac expression is consistent with the skeletal and non-skeletal congenital abnormalities in FA patients. As well, expression in rapidly dividing progenitors is consistent with hypotheses regarding the nature of the basic defect in FA: a role of the protein in DNA repair or protection from oxygen toxicity.

Animals↗
PubMed1996

Correction of the spontaneous and DEB-induced chromosomal aberrations in Fanconi anemia cells of the FA(C) complementation group by the FACC gene.

Fanconi anemia (FA) cells are hypersensitive to the cytotoxic and clastogenic effects of DNA cross-linking agents. Four complementation groups have been identified to date. The gene (FACC) that corrects the hypersensitivity of one of them, FA(C), has been cloned. In the present study, both the increased spontaneous and diepoxybutane (DEB)-induced chromosomal instability in FA(C) lymphoblastoid cells were corrected by transfection of FACC.

Cell Cycle Proteins↗
PubMed1996

Phenotypic abnormalities in long-term surviving cystic fibrosis mice.

Mouse models for cystic fibrosis (CF) with no CFTR function (Cftr-/-) have the disadvantage that most animals die of intestinal obstruction shortly after weaning. The objective of this research was to extend the lifespan of CF mice and characterize their phenotype. Weanlings were placed on a nutrient liquid diet, and histologic and functional aspects of organs implicated in the disease were subsequently examined. Approximately 90% of Cftr-/- mice survived to 60 d, the majority beyond 100 d. Cftr-/- mice were underweight and had markedly abnormal intestinal histology. The intestinal epithelia did not respond to challenges with agents that raised intracellular cAMP, consistent with the absence of functional CFTR. No lesions or functional abnormalities were evident in the lungs. Liquid-fed Cftr-/- mice were infertile, although some males weaned to a solid diet were fertile before they died. Thus, we have succeeded in using dietary means to prolong the lives of Cftr-/- mice.

Animals↗
PubMed1996

Cystic fibrosis: from the gene to the dream.

The author summarizes research on cystic fibrosis carried out since the discovery of the defective gene in 1989. As a result of this work, the molecular basis of the disease is known in considerable detail. As well, the nature of the functional defects in the cells of people with cystic fibrosis has been defined. Animal models have been developed by gene targeting; their study is leading to an understanding of the pathologic processes in the disease. Initial steps are being taken toward the development of gene therapy. The field is thus poised for major advances during the coming decade, at the end of which effective treatments may be available.

Animals↗
PubMed1995

Classification of Fanconi anemia patients by complementation analysis: evidence for a fifth genetic subtype.

Fanconi anemia (FA) is an autosomal recessive disease with diverse clinical symptoms, life-threatening progressive panmyelopathy, and cellular hypersensitivity to cross-linking agents. Currently, 4 genetic subtypes or complementation groups (FA-A through FA-D) have been distinguished among 7 unrelated FA patients. We report the use of genetically marked FA lymphoblastoid cell lines representing each of the 4 presently known complementation groups to classify 13 unrelated FA patients through cell fusion and complementation analysis. Twelve cell lines failed to complement cross-linker sensitivity in fusion hybrids with only 1 of the 4 reference cell lines and could thus be unambiguously classified as FA-A (7 patients), FA-C (4 patients), or FA-D (1 patient). One cell line complemented all 4 reference cell lines and therefore represents a new complementation group, designated FA-E. These results imply that at least 5 genes appear to be involved in a pathway that, when defective, causes bone marrow failure in FA patients.

Cell Fusion↗
PubMed1995

CFTR expression and organ damage in cystic fibrosis.

The cloning of the defective gene in cystic fibrosis (CFTR) is the most important step to date toward understanding the pathogenesis of the disease and developing novel therapeutic strategies. Although many studies have provided insights into the molecular defects and knowledge of the expression and role of the gene, the basic defect and its pathogenesis are still unclear. We hypothesize that organ damage in cystic fibrosis is the result of a combination of at least three main factors: the genotype (the type of mutation that alters the function of the cystic fibrosis transmembrane regulator [CFTR]), the rate of CFTR-mediated chloride secretion in the epithelium of each organ (inferred from the level of expression of the gene), and the anatomical and physiologic characteristics of the affected organs (the size and contents of the ducts). Confirmation of this hypothesis should allow a better understanding of the pathogenesis of the disease and help prevent organ damage.

Chloride Channels↗
PubMed1995

Expression of the Fanconi anemia gene FAC in human cell lines: lack of effect of oxygen tension.

Fanconi anemia (FA) is a recessively inherited disease characterized by bone marrow failure, congenital anomalies, chromosomal instability and hypersensitivity to crosslinking agents. Some of the cellular defects of FA are known to be responsive to the ambient oxygen concentration. We examined the responsiveness of the FA complementation group C (FAC) gene to changes in oxygen concentration using two types of human cell lines, hypoxia-responsive Hep3B hepatoma cells and Epstein-Barr virus-immortalized lymphoblasts (normal and FA complementation groups B and C). Although the expression of erythropoietin in Hep3B cells was induced in response to the hypoxia-mimicking agent CoCl₂, there was no concomitant induction in FAC expression as assessed by mRNA levels and immunoprecipitable protein, and no detectable change in the cytoplasmic location of the FAC polypeptide as determined by indirect immunofluorescence. In human lymphoblasts we examined the effect of oxygen (0.1% -95% O₂) on cell proliferation and FAC expression. FA lymphoblasts had a normal sensitivity to the cytostatic effect of hyperoxia, while in both control and FA lymphoblasts FAC mRNA levels were unaffected by oxygen. Our results indicate that ambient oxygen is not a regulator of the FAC gene.

Carcinoma, Hepatocellular↗
PubMed1995

Human cDNA clones that modify radiomimetic sensitivity of ataxia-telangiectasia (group A) cells.

Genes responsible for genetic diseases with increased sensitivity to DNA-damaging agents can be identified using complementation cloning. This strategy is based on in vitro complementation of the cellular sensitivity by gene transfer. Ataxia-telangiectasia (A-T) is a multisystem autosomal recessive disorder involving cellular sensitivity to ionizing radiation and radiomimetic drugs. A-T is genetically heterogeneous, with four complementation groups. We attempted to identify cDNA clones that modify the radiomimetic sensitivity of A-T cells assigned to complementation group [A-T(A)]. The cells were transfected with human cDNA libraries cloned in episomal vectors, and various protocols of radiomimetic selection were applied. Thirteen cDNAs rescued from survivor cells were found to confer various degrees of radiomimetic resistance to A-T(A) cells upon repeated introduction, and one of them also partially influenced another feature of the A-T phenotype, radioresistant DNA synthesis. None of the clones mapped to the A-T locus on chromosome 11q22-23. Nine of the clones were derived from known genes, some of which are involved in cellular stress responses. We concluded that a number of different genes, not necessarily associated with A-T, can influence the response of A-T cells to radiomimetic drugs, and hence the complementation cloning approach may be less applicable to A-T than to other diseases involving abnormal processing of DNA damage.

Antibiotics, Antineoplastic↗
PubMed1995

Characterization of the 5' region of the Fanconi anaemia group C (FACC) gene.

Fanconi anaemia (FA) is an autosomal recessive disease characterised by progressive pancytopenia, chromosome instability and an increased risk of cancer. The Fanconi Anaemia Complementation Group C (FACC) gene is mutated in patients of complementation group C. Several different forms of FACC mRNA that share the same coding region have been isolated. At least two species result from the use of alternative exons at the 5' end and three result from the use of distinct polyadenylation signals. As a first step toward the characterization of this gene we have isolated the genomic clones corresponding to the 5' region, including a putative promoter and two alternate 5' exons. These exons, named -1 and -1a, were found to be separated by a small intron, with exon -1 located 5' to exon -1a. Further, these exons are flanked by consensus sequences of donor sites at the 5' ends of introns. An acceptor splice site was not evident 5' of exon -1a, suggesting that exon -1 is not spliced onto exon -1a. The sequences upstream of exons -1 and -1a have no obvious TATA or CAAT boxes but include CG-rich sequences. Functional analysis of the sequence upstream of the putative transcription start site of both alternative exons indicates that the region upstream exon -1 is sufficient to drive the expression of the luciferase reporter gene in CaCo-2 cells and that the transcriptional regulation of this gene is complex.

Base Sequence↗
PubMed1994

Phosphatase inhibitors activate normal and defective CFTR chloride channels.

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is regulated by phosphorylation and dephosphorylation at multiple sites. Although activation by protein kinases has been studied in some detail, the dephosphorylation step has received little attention. This report examines the mechanisms responsible for the dephosphorylation and spontaneous deactivation ("rundown") of CFTR chloride channels excised from transfected Chinese hamster ovary (CHO) and human airway epithelial cells. We report that the alkaline phosphatase inhibitors bromotetramisole, 3-isobutyl-1-methylxanthine, theophylline, and vanadate slow the rundown of CFTR channel activity in excised membrane patches and reduce dephosphorylation of CFTR protein in isolated membranes. It was also found that in unstimulated cells, CFTR channels can be activated by exposure to phosphatase inhibitors alone. Most importantly, exposure of mammalian cells to phosphatase inhibitors alone activates CFTR channels that have disease-causing mutations, provided the mutant channels are present in the plasma membrane (R117H, G551D, and delta F508 after cooling). These results suggest that CFTR dephosphorylation is dynamic and that membrane-associated phosphatase activity may be a potential therapeutic target for the treatment of cystic fibrosis.

1-Methyl-3-isobutylxanthine↗
PubMed1994

A functionally active retrovirus vector for gene therapy in Fanconi anemia group C.

Fanconi anemia (FA) is a rare genetic disorder characterized by progressive pancytopenia, congenital abnormalities, and a predisposition to malignancy. Recently, mutation in a novel gene named FACC (Fanconi anemia C complementing) has been identified as causing one type of FA. Here, we report successful functional complementation of four FA(C) cell lines using a retroviral vector to transfer a copy of the normal FACC gene. The hallmark of the FA cell phenotype is extreme sensitivity to cross-linking agents such as mitomycin C (MMC). Cell lines transduced by FACC viral vectors were distinguished by their ability to grow at concentrations of MMC several orders of magnitude higher than those concentrations inhibitory of parental controls. The genetically corrected cell lines were analyzed for susceptibility to MMC-induced chromosomal breakage and were found to have been normalized. These two different assays confirmed that our retroviral vectors were capable of transferring a functional FACC gene to lymphoid cell lines established from FA(C) patients. We next analyzed the ability of our viral vectors to functionally correct hematopoietic progenitor cells from a patient bearing a splice donor mutation. Progenitor cells were purified by an immunoaffinity column to enrich for cells with high CD34 expression. Similar to FA lymphoid cell lines, this patient's CD34-enriched cells were extremely sensitive to MMC. After infection of these progenitor cells with viral vectors bearing normal FACC, increased numbers of colonies formed both in the absence and presence of < or = 5 nmol/L MMC, but no colonies formed from uninfected cells, even in the absence of MMC. Polymerase chain amplification was used to confirm proviral DNA integration. Thus, retroviral vectors can be engineered to transfer a normal FACC gene to lymphoid cell lines and primary hematopoietic cells bearing four different FACC mutations. FA stem cells rescued by gene transduction should have a selective growth advantage within the hypoplastic FA marrow environment in vivo. These experiments suggest that gene therapy may be an effective treatment strategy for FA.

3T3 Cells↗
PubMed1994

Fanconi anemia revisited: old ideas and new advances.

This review summarizes both historical and more recent data on the clinical, cellular and genetic features of Fanconi anemia (FA), a rare autosomal recessive disorder. FA patients are characterized by pancytopenia, congenital malformations, growth delay and an increased susceptibility to the development of malignancies, particularly acute myelogenous leukemia. FA cells show chromosomal fragility, slow growth and increased sensitivity to DNA crosslinking agents. FA can be caused by defects in any one of at least four genes. Two general hypotheses have been proposed to explain the underlying defect: loss of a DNA repair function or of a step in the defense toward oxygen toxicity. After many attempts to clone the FA genes, the first one, that defective in group C, has been cloned by complementation of the increased sensitivity of FA(C) cells to mitomycin C and diepoxybutane. This gene (FACC) codes for a novel protein and is ubiquitously expressed. Mutations in various FA(C) patients that cause loss of function have been identified. The review concludes by suggesting directions for future research in FA.

Animals↗
PubMed1994

Screening cosmid libraries with oligonucleotides corresponding to splice-site consensus sequences.

To facilitate the identification of genes within genomic DNA, we have developed a method based on the use of short oligonucleotides designed from the consensus sequences of splice sites. We describe here the hybridization and washing conditions under which such oligonucleotides can be used to screen cosmid libraries. We confirm the presence of genes within cosmids identified by screening with one oligonucleotide by showing that DNA isolated from such cosmids will hybridize to another splice-site oligonucleotide.

Animals↗
PubMed1994

Regional expression of CFTR in developing human respiratory tissues.

Morbidity and mortality in cystic fibrosis (CF) patients is strongly related to their respiratory disease. We have analyzed, by means of in situ hybridization, the localization and levels of CFTR mRNA in fetal, newborn, and infant respiratory tissues. Measurable levels of CFTR transcript are present in the fetal primordial epithelium of the pseudoglandular stage lung. During the following stages of lung development, CFTR expression decreases in cells of the future alveolar spaces and is gradually limited to the epithelium of the small airways. After birth, expression decreases in the small airways and is not detected in alveolar epithelia. In trachea and large bronchi, a differential pattern of expression is also observed. No CFTR expression is found in fetal submucosal glands during fetal development, but appears gradually in the newborn period. Since CFTR codes for a secretory Cl- channel, these data probably reflect the changes that occur in the lung transition from a fluid-secreting to an absorbing organ. The pattern of expression seems paradoxical in view of the clinical-pathological manifestations of CF. Although CFTR is expressed in the normal fetus and lung development is influenced by the amount of fetal lung liquid, newborns affected with CF have normal lungs. In addition, the earliest pathologic change described in CF lungs in hyperplasia of the submucosal glands, yet expression in these structures is seen only after birth. An improved understanding of the factors that alter the expected relationship between CFTR expression and pathologic lesions in the fetal lung may provide important insights into the pathogenesis and potential treatment of lung disease in CF patients.

Child, Preschool↗