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Biomedical subjects

M Buchwald

Publications and source records attributed to M Buchwald.

142 records · Page 8Linked to original sources
PubMed1979

Induction of alkaline phosphatase in cultured human fibroblasts. Comparison of normal cells and those from patients with cystic fibrosis.

The membrane glycoprotein enzyme, alkaline phosphatase was induced in cultured human fibroblasts by dibutyryl cyclic AMP, sodium butyrate, the serum glycoprotein fetuin, the Tamm-Horsfall urinary glycoprotein, and by a number of inhibitors of DNA synthesis. The uninduced basal enzyme activity increased at later stages of growth when the cells became confluent. Induction by dibutyryl cyclic AMP or fetuin was most effective when the agents were added after the cells had reached stationary phase and was maximal after at least two days of exposure. The levels of induction resulting from the addition of pairs of the agents, dibutyryl cyclic AMP, n-butyrate and fetuin were additive indicating that these have different modes of action. The inhibitors of DNA synthesis, cytosine arabinoside, hydroxyurea, and methothrexate were less effective inducers. Bromodeoxyuridine which also has non-DNA mediated effects induced to the same extent as dibutyryl cyclic AMP. Similar experiments with sex- and age-matched cell strains derived from patients with cystic fibrosis failed to detect differences in the levels of induction from those observed in normal cells. In addition, the combined inductive effects of Tamm-Horsfall glycoprotein, isoproterenol and theophylline, were similar with normal and cystic fibrosis cells.

Alkaline Phosphatase↗
PubMed1979

Specific cellular defects in patients with Fanconi anemia.

Measurements of plating efficiency, accumulation of metaphases and generation times have shown that fibroblast from patients with Fanconi anemia (FA) have decreased probability of completing a further division after successful mitosis. Thus FA cells show decreased growth rates and increased generation times. We have also measured the survival of FA fibroblasts and lymphoblasts after treatment with a variety of mutagens. All FA cells show an increased sensitivity to drugs such as MMC and psoralen plus long wave length UV which cause DNA interstrand crosslinks. FA strains show varying degrees of sensitivity to these drugs and the extent of this sensitivity seems to be characteristic of each patient. FA cells are equal to controls in their sensitivity to other alkylating agents such as ethyl methane sulfonate, N-methyl-N1-nitro-N-nitrosoguanidine and actinomycin D. Both the decreased growth and increased drug sensitivity may result from defect in DNA replication or repair.

Adolescent↗
PubMed1978

[Results of intensive care and intensive therapy of very low birth-weight infants (author's transl)].

Newborns of less than 1500 g birth-weight are thought to be especially at risk with regard to their psychomotor development. Among 71 such infants admitted to the University Childrens Clinic, Tübingen, in 1974-1975, 43 survived (38% mortality). All but two of them were followed serially and assessed for their psychomotor development. Five were found to be abnormal (12%), three of them most probably having sustained CNS damage prenatally.

Child, Preschool↗
PubMed1977

Mutagenesis at the ouabain-resistance locus in human diploid fibroblasts.

The variables affecting the frequency of ouabain-resistant mutant clones have been studied in a strain of foetal lung fibroblasts. Optimum mutant recovery was obtained when cells were selected in 10(-6) M ouabain at a cell density of 2 X 10(4) cells/cm 2 (10(6) cell per 100-mm dish). The spontaneous mutation rate was estimated to be 4 X 10(-8) per cell generation. Treatment with the mutagens ethyl methanesulfonate (EMS), N-methyl-N' -nitro-N-nitrosoguanidine, and UV light increased the frequency of mutant colonies by an order of magnitude. The maximum number of mutants after mutagenesis with EMS occurred after two population doublings of growth in non-selective medium prior to selection and depended on the dose of EMS. Ouabain-resistance is a useful marker for studies of quantitative mutagenesis in human cells.

Drug Resistance↗
PubMed1976

Human diploid fibroblast mutants with altered RNA polymerase II.

Clones resistant to the cytotoxic action of alpha-amanitin have been isolated from a strain of fetal human lung diploid fibroblasts. Resistant clones were recovered at a frequencey of 5 X 10(-8) after single-step selections following mutagenesis with the mutagen ethyl methane sulfonate. Following propagation in drug-free medium, the clones retained the selected phenotype and in both growth and plating experiments showed a 10-50-fold higher resistance than wild-type cells to the cytotoxicity of 0.25 microgram/ml alpha-amanitin. The alpha-amanitin sensitivity of RNA polymerase II purified from the mutant cells suggests the presence of two forms of the enzyme, one similar to that found in wild-type cells and a second form with increased resistance to alpha-amanitin inhibition. These results are consistent with previous evidence that alpha-amanitin resistance behaves as a codominant marker in mammalian cells.

Amanitins↗
PubMed1976

Abnormal levels of 3':5'-cyclic AMP in isoproterenol-stimulated fibroblasts from patients with cystic fibrosis.

To determine if the abnormalities of exocrine secretion characteristic of cystic fibrosis could be investigated in vitro, I studied the synthesis of 3':5'-cyclic AMP after isoproterenol stimulation in skin fibroblasts derived from patients with cystic fibrosis and from normal individuals. Comparison of normal and cystic fibrosis cells showed that the latter had 2- to 5-fold greater levels of intracellular cyclic AMP after stimulation with isoproterenol. The difference between the strains was observed at every stage of the culture cycle and was specific to stimulation by beta-adrenergic agonists. It could not be accounted for by different dose- or time-response curves nor by leakage of cyclic AMP into the medium. The increased sensitivity to catecholamines may reflect an intrinsic genetic property of cystic fibrosis cells, and it may be feasible to use this system to study the biochemical basis of the genetic defect in cystic fibrosis.

Age Factors↗
PubMedDate not supplied

A polymorphic DNA marker linked to cystic fibrosis is located on chromosome 7.

Although cystic fibrosis (CF) is among the most common inherited diseases in Caucasian populations, the basic biochemical defect is not yet known. CF is inherited as an autosomal recessive trait apparently due to mutations in a single gene, whence the efforts made to identify the genetic locus responsible by linkage studies. Two markers have recently been identified that are genetically linked to CF: one is a genetic variation in serum level of activity of the enzyme paraoxonase, and the other is a restriction fragment length polymorphism (RFLP) identified with a randomly isolated DNA probe. We report here that the genetic locus DOCRI-917 defined by the cloned DNA probe is located on chromosome 7.

Chromosome Mapping↗
PubMedDate not supplied

Expression of a mutant regulatory subunit of cAMP-dependent protein kinase in the Caco-2 human colonic carcinoma cell line.

Caco-2 human colonic carcinoma cells were transfected with an expression vector encoding a mutant form of RI (regulatory subunit of the type 1 cAMP-dependent protein kinase), driven by the metallothionein 1 promoter. A stable transformant was isolated that expressed the mutant RI gene in a Zn(2+)-inducible manner. The consequences of the RI mutation on cAMP-dependent protein kinase activity, cell division, and regulation of chloride efflux were examined. When grown in the absence of ZnSO4, protein kinase activity in the transformant was stimulated 2.5-fold by cAMP and approached the levels of cAMP-dependent protein kinase activity seen in parental Caco-2 cells; when treated with ZnSO4, cAMP-dependent protein kinase activity in the transformant was inhibited by 60%. In the absence of ZnSO4 the transformant grew with the same doubling time and to the same saturation density as the untransformed parent. In the presence of ZnSO4 the transformant exhibited a cAMP-reversible inhibition of cell division, indicating that a functional cAMP-dependent protein kinase was required for the growth of these cells in culture. Induction of the mutant RI gene also abolished forskolin-stimulated chloride efflux from these cells, suggesting obligatory roles for cAMP and cAMP-dependent protein kinase in forskolin's actions on chloride channel activity. We anticipate that this transformant will be useful for further studies on the roles of cAMP and cAMP-dependent protein kinase in the regulation of intestinal epithelial cells, including regulation of cell proliferation and differentiation, and regulation of chloride channel activity by neurohormones and neurotransmitters.

Carrier Proteins↗