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Biomedical subjects

A M Hack

Publications and source records attributed to A M Hack.

11 recordsLinked to original sources
PubMed2000

Effect of the topoisomerase-II inhibitor etoposide on meiotic recombination in male mice.

Unlike other chemicals that have been tested in mammalian germ cells, the type-II topoisomerase inhibitor etoposide exhibits significant mutagenicity in primary spermatocytes. Because this is the cell stage during which meiotic recombination normally occurs, and because topoisomerases play a role in recombination, we studied the effect of etoposide on crossing-over in male mice. Exposure to those meiotic prophase stages (probably early to mid-pachytene) during which specific-locus deletion mutations can be induced resulted in decreased crossing-over in the p-Tyr(c) interval of mouse chromosome 7. Accompanying cytological studies with fluorescent antibodies indicated that while there was no detectable effect on the number of recombination nodules (MLH1 foci), there were marked changes in the stage of appearance and localization of RAD51 and RPA proteins. These temporal and spatial protein patterns suggest the formation of multiple lesions in the DNA after MLH1 has already disappeared from spermatocytes. Since etoposide blocks religation of the cut made by type II topoisomerases, repair of DNA damage may result in rejoining of the original DNA strands, undoing the reciprocal exchange that had already occurred and resulting in reduced crossing-over despite a normal frequency of MLH1 foci. Crossing-over could conceivably be affected differentially in different chromosomal regions. If, however, the predominant action of etoposide is to decrease homologous meiotic recombination, the chemical could be expected to increase nondisjunction, an event associated with human genetic risk. Three periods in spermatogenesis respond to etoposide in different ways. Exposure of (a) late differentiating spermatogonia (and, possibly, preleptotene spermatocytes) results in cell death; (b) early- to mid-pachytene induces specific-locus deletions and crossover reduction; and, (c) late pachytene-through-diakinesis leads to genetically unbalanced conceptuses as a result of clastogenic damage.

Animals↗
PubMed1987

Immunochemical studies of fibroblasts from patients with methylmalonyl-CoA mutase apoenzyme deficiency: detection of a mutation interfering with mitochondrial import.

Methylmalonyl-CoA mutase (2-methylmalonyl-CoA CoA-carbonylmutase, EC 5.4.99.2) is a mitochondrial enzyme whose deficiency in man leads to several biochemically and clinically heterogenous++ forms of methylmalonic acidemia. Intact fibroblasts from 21 patients with mutase apoenzyme deficiency have been pulse-labeled with [3H]leucine or [35S]methionine to determine how amounts of newly synthesized mutase recovered from these cells by immunoprecipitation compare with the amounts of steady-state crossreacting material previously determined. Ten lines (3 mut-, 7 mut 0 ), previously shown to have detectable steady-state crossreacting material, had amounts of newly synthesized mutase that varied from similar (7 lines) to considerably greater than (3 lines) the steady-state amounts. Of 11 lines that had no detectable steady-state crossreacting material, 6 had no detectable newly synthesized mutase, and 5 had amounts of mutase ranging from just detectable to almost half that of control. This result suggests that, at least for this latter group, one effect of the mutation in the mutase gene is to reduce the stability of the mutase protein. We examined fibroblasts from 48 patients with mutase apoenzyme deficiency to determine the sizes of the mature mutase subunit and the mutase precursor accumulated in the presence of the mitochondrial transport inhibitor rhodamine 6G. Of the 38 lines that had detectable newly synthesized mutase, only 2, lines 437 and 552, showed a pattern different from that generated by the normal precursor and mature subunits. Line 437 had two immunoprecipitable precursor proteins in the presence of rhodamine, each of which appeared to be transported and processed in the cells to produce two distinct mature proteins. Line 552 also had two anti-mutase reactive proteins in the presence of rhodamine, but each was smaller than the normal mature subunit and neither appeared to be proteolytically processed. The defect in line 552 is almost certainly an amino-terminal deletion that removes the leader peptide necessary for proper uptake and cleavage of the mutase precursor; this represents a clear example of a natural human mutation that interferes with mitochondrial transport of a protein.

Cell Line↗
PubMed1986

DNA analysis for ornithine transcarbamylase deficiency.

We have utilized the Southern blotting technique to analyse genomic DNA from males with ornithine transcarbamylase (OTC) deficiency and their families. Using a nearly full-length human cDNA probe, we have identified 3 patients with deletions at this locus and have characterized 4 different restriction fragment length polymorphisms that can be used as linkage markers for the OTC mutation. These polymorphisms occur at sufficiently high frequencies so as to enable us to distinguish the two X-chromosomes in approximately 80% of OTC carriers. As a direct consequence of these findings, prenatal diagnosis and carrier assessment can be offered to a large fraction of families at risk for OTC deficiency.

Chromosome Deletion↗
PubMed1986

Stable isotope dilution analysis of isovalerylglycine in amniotic fluid and urine and its application for the prenatal diagnosis of isovaleric acidemia.

A stable isotope dilution method was developed to measure accurately small amounts of isovalerylglycine in amniotic fluid and urine for the prenatal diagnosis of isovaleric acidemia. [4,4,4-D3]Isovalerylglycine was synthesized and used as an internal standard. Samples were extracted, methylated, and analyzed by chemical ionization gas chromatography-mass spectrometry operated in the selected ion monitoring mode. This method is very sensitive (lower limit approximately 5 ng/ml), linear over three orders of magnitude above 10 ng/ml up to at least 10 micrograms/ml and reproducible. No isovalerylglycine was detected at all in amniotic fluids from eleven normal pregnant women with an exception of a single case which contained 6 ng/ml. Amniotic fluids from five pregnancies at risk were analyzed. Two of these samples had isovalerylglycine concentrations of 957 and 556 ng/ml. Three others contained 18, 18, and 17 ng/ml of isovalerylglycine. Postpartum diagnostic tests and/or in vitro assay of isovaleryl-CoA dehydrogenase of [1-14C] isovaleric acid oxidation using amniocytes confirmed that the first two fetuses were affected by isovaleric acidemia, whereas the latter three were unaffected. The method described in this report provides a highly accurate and reliable technique for the prenatal diagnosis of isovaleric acidemia.

Amino Acid Metabolism, Inborn Errors↗
PubMed1986

Identification and application of additional restriction fragment length polymorphisms at the human ornithine transcarbamylase locus.

Two additional restriction fragment length polymorphisms (RFLPs) have been identified at the human ornithine transcarbamylase (OTC) locus. Approximately 11% of women are heterozygous for an RFLP characterized by polymorphic bands at 3.7 and 3.6 kilobasepairs (kbp) observed after DNA digestion with TaqI. Twenty-nine percent of women are heterozygous for an RFLP characterized by polymorphic bands at 18.0 and 5.2 kbp observed after digestion with BamHI. Thus, in combination with the previously reported RFLPs identified using MspI, the X chromosomes in approximately 80% of women at risk for having a son with OTC deficiency are distinguishable by RFLPs at the OTC locus. Furthermore, we show that these RFLPs will be useful in families for prenatal diagnosis of OTC deficiency, carrier detection, and carrier exclusion.

Chromosome Mapping↗
PubMed1984

Biogenesis of the mitochondrial enzyme methylmalonyl-CoA mutase. Synthesis and processing of a precursor in a cell-free system and in cultured cells.

Methylmalonyl-CoA mutase (EC 5.4.99.2; mutase), a cytoplasmically synthesized mitochondrial matrix enzyme, is translated in a cell-free system programmed with rat liver RNA as a larger precursor polypeptide, designated pre-mutase, which appears to be 3-4 kDa larger than the subunit of purified mutase (77.5 kDa). When pre-mutase is incubated with intact rat liver mitochondria, it is taken up by them and proteolytically processed to the size of the mature subunit. The overall reaction is inhibited by compounds such as dinitrophenol which disrupt mitochondrial energy metabolism. The final, proteolytic step can be carried out by the mitochondrial matrix in the presence of added Zn2+ and is inhibited by metal ion chelators and by certain protease inhibitors (e.g. leupeptin and p-aminobenzamidine). Newly synthesized mutase was also detected in intact, cultured Buffalo rat liver cells labeled with [3H] leucine in the presence of dinitrophenol. When dinitrophenol is removed in a pulse-chase protocol, the accumulated pre-mutase is rapidly (t1/2 = 6-9 min) converted to mutase. On the other hand, when the chase is performed in the presence of the inhibitor, the labeled pre-mutase persists for greater than 5 h. This long term stability of pre-mutase contrasts sharply with the instability previously reported for unprocessed precursors of two other mitochondrial enzymes.

Animals↗
PubMed1979

meoA is the structural gene for outer membrane protein c of Escherichia coli K12.

The isolation and characterization of two mutants of Escherichia coli K12 with an altered outer membrane protein c is described. The first mutant, strain CE1151, was isolated as a bacteriophage Me1 resistant strain which contains normal levels of protein c. Mutant cells adsorbed the phage with a strongly decreased rate. Complexes of purified nonheat modified wild type protein c and wild type lipopolysaccharide inactivated phage Me1, indicating that these components are required for receptor activity for phage Me1. When wild type protein c was replaced by protein c of strain CE1151, the receptor-complex was far less active, showing that protein c of strain CE1151 is altered. The second mutant produces a protein c with a decreased electrophoretic mobility, designated as protein c. An altered apparent molecular weight was also observed for one or more fragments obtained after fragmentation of the mutant protein with cyanogen bromide, trypsin and chymotrypsin. Alteration of protein c was not accompanied by a detectable alteration in protein b or its fragments. Both mutations are located at minute 48 of the Escherichia coli K12 linkage map. The results strongly suggest that meoA is the structural gene for protein c.

Bacterial Proteins↗
PubMed1979

Simple method for identification of plasmid-coded proteins.

Proteins encoded by plasmid DNA are specifically labeled in UV-irradiated cells of Escherichia coli carrying recA and uvrA mutations because extensive degradation of the chromosome DNA occurs concurrently with amplification of plasmid DNA.

Bacterial Proteins↗