[Long-term survival in terms of DNA-RNA contents in breast cancer].

Fifty-nine primary breast cancers were analyzed by flow cytometric cellular DNa-RNA contents stained by acridine orange simultaneous DNA-RNA double staining. Forty-three cases (72.9%) out of 59 had abnormal stemlines (DNA aneuploidy), and 16 normal ones (DNA diploidy). RNA indices widely ranged from 1.41 to 9.02 (2.99). There was no correlation between DNA indices and RNA indices. The patients with DNA diploidy had a better prognosis than those with DNA aneuploidy, but the differences were not significant. The patients with a high RNA index of more than 4.0 had a significantly poorer prognosis than those with an index of less than 4.0. Those results suggest that RNA contents, and DNA contents are independent prognostic factors, and especially RNA contents may be a good prognostic factor in long-term survival in breast cancer.

Isolation of cDNA clones of human argininosuccinate lyase and corrected amino acid sequence.

In the present study, we isolated clones of human argininosuccinate lyase (ASL) cDNA from a liver cDNA library using a clone of rat ASL cDNA and analyzed human ASL cDNA nucleotide sequence. The results reveal that the sequence of human ASL cDNA published by O'Brien et al. in 1986 [Proc. Natl. Acad. Sci USA 83, 7211-7215] had one-base deletions at three independent positions in the coding regions near the COOH-terminus, which caused frame-shift variations in the amino acid sequence. Amino acid sequencing of peptides prepared from purified human liver ASL showed our predicted amino acid sequence to be correct.

The heterogeneous distribution of argininosuccinate synthetase in the liver of type II citrullinemic patients. Its specificity and possible clinical implications.

The authors analyzed the heterogeneous distribution of hepatic argininosuccinate synthetase of type II citrullinemia in reference to its specificity and clinical implications. The low content of the enzyme in the liver of type II citrullinemic patients is associated with two kinds of the enzyme distribution that can be visualized by means of an immunohistochemical method (Saheki and colleagues. Biomed Res 1983;4:235-238). Among the 25 cases of type II citrullinemia examined, 11 exhibited homogeneous distribution of the enzyme, as in the control livers. On the other hand, 14 presented the clustered distribution, in which the hepatocytes stained positively with antisera to argininosuccinate synthetase formed a cluster among the poorly stained cells. No clustered distribution of the enzyme was present in the liver of control patients either with or without liver diseases. No clustered distribution of arginase and aldolase B was observed even in the liver of type II citrillinemic patients. These results suggest that clustered distribution is specific to argininosuccinate synthetase in the liver of type II citrullinemic patients. From considerations concerning the heterogeneous distribution of the enzyme and certain clinical parameters as well, the authors suggest that the clustered type in type II citrullinemia has a less favorable prognosis with regard to fatality.

Properties of component X of rat heart pyruvate dehydrogenase complex.

Pyruvate dehydrogenase complex was purified from rat heart. A new component(mol.wt; 52,000) was found in the purified complex in addition to well known three component enzymes. This component(referred to as component X) was acetylated with [2-14C] pyruvate in the absence of CoA as well as lipoate acetyltransferase. The anti-lipoate acetyltransferase antibody reacted with component X and lipoate acetyltransferase, suggesting that component X shows homology with lipoate acetyltransferase in protein structure. cDNA for lipoate acetyltransferase was isolated from rat liver cDNA library in lambda gt 11. cDNA for lipoate acetyltransferase recognized two kinds of mRNAs of 3.5 Kb and 2.5 Kb.

Increased urinary excretion of argininosuccinate in type II citrullinemia.

Although argininosuccinate is a product of the catalytic action of deficient argininosuccinate synthetase in citrullinemia, its concentration was found to be elevated in the urine of patients with type II citrullinemia. Urinary argininosuccinate was identified by two methods; its conversions to anhydride by boiling in an acidic solution and to arginine by the enzymatic action of argininosuccinate lyase. Oral administration of citrulline to patients with type II citrullinemia and control subjects increased urinary argininosuccinate levels. These phenomena are consistent with our previous findings on type II citrullinemia (Adv Exp Med Biol 1983;153:63-76,J Clin Biochem Nutr 1986;1:129-142), namely that renal argininosuccinate synthetase which plays a role in arginine synthesis is not deficient in patients with type II citrullinemia; and that serum arginine levels in patients with type II citrullinemia are rather higher than the controls, and increase after the oral administration of citrulline. The organ-specific deficiency of argininosuccinate synthetase in type II citrullinemia is further confirmed by this paper.

Immunological identification of a new component of Ascaris pyruvate dehydrogenase complex.

The pyruvate dehydrogenase complex was purified from Ascaris muscle both with and without MgCl2 treatment at the first stage of purification. The specific activity of complex purified with MgCl2 treatment was about 2-fold as high as that purified without it. In addition to three component enzymes, two unknown polypeptides of 46 and 41 kDa were found in the complex purified by the two procedures. The quantity of unknown polypeptide of 41 kDa was increased in the complex purified with MgCl2 treatment as compared with that without it. Antibodies against the three component enzymes were prepared. All the antibodies precipitated the two unknown polypeptides in addition to the three component enzymes in immunoprecipitation experiments. Antibody against the alpha-subunit of pyruvate dehydrogenase reacted with the 41 kDa polypeptide as well as the alpha-subunit in the immunoblotting method. The unknown polypeptide of 46 kDa did not react with any antibody. These results suggest that the unknown 41 kDa polypeptide is a derivative of the alpha-subunit and that the unknown 46 kDa polypeptide is not a proteolytic-degradative product of component enzymes but is a component of the Ascaris pyruvate dehydrogenase complex. When the Ascaris complex was incubated with [2-14C]pyruvate in the absence of CoASH, only lipoate acetyltransferase was acetylated. In rat heart pyruvate dehydrogenase complex, lipoate acetyltransferase and another protein (referred to as component x or protein x) were acetylated. These results indicate that the unknown polypeptide of 46 kDa is a new component.

Molecular cloning of cDNA for rat liver lipoate acetyltransferase. A component of pyruvate dehydrogenase complex.

One cDNA clone for lipoate acetyltransferase, a component enzyme of pyruvate dehydrogenase complex, was isolated from a rat liver cDNA library prepared in the phage expression vector lambda gt11 using immunological screening with affinity purified anti-lipoate acetyltransferase antibody. It was identified tha cDNA insert in this clone codes for lipoate acetyltransferase by immunoblotting of lysogen carrying the isolated clone. Lipoate acetyltransferase antigenic polypeptide in fusion protein was about 11,000 daltons, agreeing with the size of cDNA insert to be 300 base pairs.

Molecular basis of enzyme abnormalities in urea cycle disorders. With special reference to citrullinemia and argininosuccinic aciduria.

This paper deals with enzymological, immunochemical and molecular genetic analyses of citrullinemia and argininosuccinic aciduria. Citrullinemia has been classified by Saheki et al. [J. inher. Metab. Dis. 8: 155-156, 1985] into three types from the properties of the deficient argininosuccinate synthetase (ASS) of the patients. Analysis of hepatic mRNA coding for ASS revealed certain characteristics in type II and III citrullinemic patients whose hepatic ASS protein was low. A newly developed enzyme-linked immunosorbent assay (ELISA) of argininosuccinate lyase (ASL) protein showed that 8 out of ten cases of argininosuccinic aciduria had no detectable ASL protein in the liver, erythrocytes, cultured skin fibroblasts or cultured amniocytes.

Messenger RNA coding for argininosuccinate synthetase in citrullinemia.

Messenger RNA coding for argininosuccinate synthetase (ASS), extracted from the livers of some patients with citrullinemia, was analyzed using a cell-free translation system and dot and Northern blot hybridization with cDNA probe for ASS. In patients with quantitative-type citrullinemia, called type II here, previous studies have demonstrated that the hepatic content of the enzyme was about 10% of the control value, whereas the translatable mRNA level for the enzyme was similar to that of control livers. Here, we confirmed that the type II liver contained an almost normal amount of mRNA coding for ASS, judged by the dot-blot hybridization technique with cDNA. Northern blot hybridization of RNA indicated that there was hybridizable mRNA of approximately normal size (about 1.7 kilobase [kb]) in each, suggesting that large structural gene deletions had not occurred. These results indicate that in type II citrullinemia, the decrease in the enzyme protein is due either to increased degradation of the enzyme or to decreased or inhibited translation in the liver. Another type of citrullinemia was found and classified as type III. It is characterized by no detectable enzyme activity for ASS or translation activity for ASS mRNA. However, a smaller amount of RNA molecule hybridized for ASS cDNA was detected.

Novel structure of the 5' end region of the human argininosuccinate synthetase gene.

The structure of the 5' end region of the human argininosuccinate synthetase (AS) gene was analyzed in detail. The 5' flanking region up to 350 nucleotides from the putative transcription initiation site is unusually G+C rich (80%). Three repeats of a CCGCCC sequence and one complementary GGGCGG sequence are present in this region. It contains no CCAAT box-like sequence, but does contain one typical TATA box and a 20 bases-long inverted repeat sequence that could form a stem and loop structure just upstream from the TATA box. An octanucleotide sequence, AGAAGTGA, found in the 5' flanking region of the AS gene is also commonly present in those regions of the two other human genes expressed mainly in the liver. These structural features of the AS gene indicate that it shares common structures with two completely different groups of genes, i.e. tissue-specific genes and housekeeping ones. Moreover, two enhancer core-like sequences, alternating purine-pyrimidines tracts and two independent Alu type highly repetitive sequences are apparent in the first intron of the AS gene.