Methods for study of mutations and mutagenesis in human lymphocytes

Detailed methods are presented for measurement and study of in vivo mutations and in vitro mutagenesis in human lymphocytes. The methods described include preparation of conditioned medium containing interleukin-2, enumeration of mutant clones, in vitro mutagenesis, and expansion of mutant clones for further study.     

Cotransfection of nucleic acid segments by Sendai virus envelopes

It is reported here that Sendai virus envelopes (SVE) can be used to transfect multiple copies of DNA segments of different varieties and size. This capability further increases the usefulness of SVE. In addition, the ability to simultaneously transfect multiple copies of different genome segments promises to be a powerful tool in the field of molecular biology. The simultaneous transfection of NEO gene and cytomegalovirus immediate early antigen gene was successfully done. Sendai virus envelopes (SVE)1 have been used successfully to study carcinogenesis of Epstein-Barr virus (1, 2). SVE have been shown to have a large carrying capacity (3) for the microinjection of macromolecules into target cells. SVE are hollow vesicles constructed from the viral proteins hemagglutinin HN and fusion factor F.     

Identification of the subunits of F1F0-ATPase from bovine heart mitochondria.

An oligomycin-sensitive F1F0-ATPase isolated from bovine heart mitochondria has been reconstituted into phospholipid vesicles and pumps protons. this preparation of F1F0-ATPase contains 14 different polypeptides that are resolved by polyacrylamide gel electrophoresis under denaturing conditions, and so it is more complex than bacterial and chloroplast enzymes, which have eight or nine different subunits. The 14 bovine subunits have been characterized by protein sequence analysis. They have been fractionated on polyacrylamide gels and transferred to poly(vinylidene difluoride) membranes, and N-terminal sequences have been determined in nine of them. By comparison with known sequences, eight of these have been identified as subunits beta, gamma, delta, and epsilon, which together with the alpha subunit form the F1 domain, as the b and c (or DCCD-reactive) subunits, both components of the membrane sector of the enzyme, and as the oligomycin sensitivity conferral protein (OSCP) and factor 6 (F6), both of which are required for attachment of F1 to the membrane sector. The sequence of the ninth, named subunit e, has been determined and is not related to any reported protein sequence. The N-terminal sequence of a tenth subunit, the membrane component A6L, could be determined after a mild acid treatment to remove an alpha-N-formyl group. Similar experiments with another membrane component, the a or ATPase-6 subunit, caused the protein to degrade, but the protein has been isolated from the enzyme complex and its position on gels has been unambiguously assigned. No N-terminal sequence could be derived from three other proteins. The largest of these is the alpha subunit, which previously has been shown to have pyrrolidonecarboxylic acid at the N terminus of the majority of its chains. The other two have been isolated from the enzyme complex; one of them is the membrane-associated protein, subunit d, which has an alpha-N-acetyl group, and the second, surprisingly, is the ATPase inhibitor protein. When it is isolated directly from mitochondrial membranes, the inhibitor protein has a frayed N terminus, with chains starting at residues 1, 2, and 3, but when it is isolated from the purified enzyme complex, its chains are not frayed and the N terminus is modified. Previously, the sequences at the N terminals of the alpha, beta, and delta subunits isolated from F1-ATPase had been shown to be frayed also, but in the F1F0 complex they each have unique N-terminal sequences.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reasons for disorders of fatty acid oxidation in isolated heart mitochondria during ischemia

The influence of malate and cytochrome c on fatty acid oxidation under control and ischemic conditions was investigated. In the medium without malate, cytochrome did not make fatty acid oxidation decreased during ischemia return to normal. Oxidation in the media containing malate and cytochrome did not differ from control only when it was measured after preliminary oxidation of endogenous substrates. The ratio of palmitoyl-CoA and palmitoyl carnitine to the respiration rates at state 3 was unchanged at 60 min ischemia. Apparently, no changes in carnitine acyltransferase playing a role in oxidation of palmitoyl-CoA took place. Thus, the decrease of fatty acid oxidation at early periods of ischemia is largely caused by a reduction in the content of cytochrome c and intermediates of Krebs cycle in the mitochondria.     

Mechanistic studies on carboxypeptidase a from goat pancreas — part II: Evidence for carboxyl group

Studies with substrate analogues and the pH optimum indicated the involvement of carboxyl group in the active site of goat carboxypeptidase A. Chemical modification of the enzyme with 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide methoI -p-toluene sulphonate, a carboxyl specific reagent, led to loss of both esterase and peptidase activities. Protection studies showed that this carboxyl group was in the active site and was protected by Βp-phenylpropionic acid and glycyl-L-tyrosine. Kinetic studies also confirmed the involvement of carboxylic group because the enzyme modification with water soluble carbodiimide was a two step reaction which excluded the possibility of tyrosine or lysine which are known to give a one step reaction with this reagent     

Chromosomal assignment of amplified genes in hydroxyurea-resistant hamster cells

We have shown previously that cDNAs for the M1 and M2 subunits of ribonucleotide reductase, ornithine decarboxylase (ODC), and p5-8, a 55,000-Dalton protein, hybridize to amplified genomic sequences in a highly hydroxyurea-resistant hamster cell line. We have extended these observations to include two additional, independently isolated, hydroxyurea-resistant cell lines: SC8, a single-step hamster ovary cell line, and KH450, a multistep human myeloid leukemic cell line, have also undergone genomic amplification for sequences homologous to ODC and p5-8 cDNAs. However, neither SC8 nor KH450 contains amplified genomic sequences homologous to an M1 cDNA probe. A panel of mouse-hamster somatic cell hybrids was used to map sequences homologous to M1, M2, ODC, and 5-8 cDNAs in the hamster genome. The M2, ODC, and p5-8 cDNAs hybridized to DNA fragments that segregated with hamster chromosome 7. In contrast, M1 cDNA hybridized to DNA fragments that segregated with hamster chromosome 3. These data suggest that the genes RRM2, (M2), ODC, and p5-8, but not RRMI (M1), are linked and may have been co-amplified in the selection of the hydroxyurea-resistant hamster and human cell lines.     

Effect of etmozin on thrombocyte aggregation capacity

It was shown in in vitro experiments that etmozin at a concentration of 100 micrograms/ml significantly suppressed (by 21%) platelet aggregation induced by ADP, but it had no effect on platelet aggregation induced by arachidonic acid. In in vivo experiments etmozin was found to cause a marked suppression of tendon collagen-induced platelet aggregation in the doses 2-5 mg/kg having antiarrhythmic activity. Under suppressed platelet aggregation induced by indomethacin, the prostaglandin biosynthesis blocker etmozin displayed no antiaggregation effect. It is suggested that etmozin effects on ADP release from platelets play the main role in the mechanism of its antiaggregation action.     

Stereospecific binding of 3H-dopamine in neostriatal membrane preparations: inhibitory effects of sodium ascorbate

It has been pointed out by several different groups of investigators in the past several years that ascorbic acid was a potent inhibitor of the binding of dopamine (DA) agonists including 3H-DA itself and 3H-ADTN, 3H-apomorphine and 3H-norpropylapomorphine to neostriatal membrane preparations. However, the significance of this effect of ascorbic acid has been controversial. For example, it has recently been claimed that the stereospecific binding of DA agonists is facilitated by ascorbic acid and can be measured only in its presence. In the present study in neostriatal membrane preparations in the absence of ascorbic acid, the binding of 3H-DA was very potently inhibited by potent DA agonists (DA, ADTN, apomorphine). Considerably weaker effects were obtained with norepinephrine, isoproterenol, serotonin, catechol and pyrogallol. Stereospecific effects were clearly observed in that the binding of 3H-DA was inhibited to a much greater extent by several biologically active enantiomers than by their less active counterparts. For example, (-)-2-hydroxyapomorphine and (-)-norpropylapomorphine were much more potent inhibitors than their corresponding (+) isomers. This binding of 3H-DA was also very strongly inhibited by sodium ascorbate and several other reducing agents. In control experiments in the neostriatal membrane preparation in the absence of ascorbic acid, there was no detectable decomposition of 3H-DA. The data suggest that 3H-DA can, in the absence of sodium ascorbate, bind stereospecifically to a site that has the properties of a DA receptor. Furthermore, sodium ascorbate is a potent inhibitor of this stereospecific binding.