Increased expression of a novel leukocytic factor in patients with hemolytic anemia.

Misprimed polymerase chain reaction (PCR) products were generated from the crude leukocytic DNA extract of four of five patients with hereditary hemolytic anemia, during the course of exon 6 pyruvate kinase L gene amplification. These by-products, which originated from abundant mRNA templates, were not observed in seven healthy individuals. Two markers were cloned (GenBank accession numbers M64700 and M64701). We focused further studies on one of the human markers associated with hereditary hemolytic anemia, human DNA marker B (HUMDNAMB). HUMDNAMB is a 451-bp open reading frame that has never been described previously. The nucleic acid sequence region 303-416 is 63% homologous to a coding region of the bovine interferon alpha-A gene. Matrix analysis of the amino acid sequences reveals a structural similarity between the two proteins. The HUMDNAMB protein is expected to be larger than interferons, based on the size of its 2-kb messenger RNA detected by Northern blot in human leukocytes, fetal liver, and fetal intestine.

The enhancing effect of adrenocorticotropin on adrenal 3-hydroxy-3-methylglutaryl coenzyme A reductase messenger ribonucleic acid level is inhibited by aminoglutethimide but not by cycloheximide.

3-Hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase activity and reductase mRNA level were determined in adrenals from hamsters treated with ACTH, with or without cycloheximide or aminoglutethimide. Both reductase activity and reductase mRNA level were similarly enhanced by ACTH administration compared to levels in NaCl-treated animals. The administration of cycloheximide with ACTH resulted in a 73% decrease in reductase activity compared to control values, but did not prevent the enhancing effect of ACTH on the reductase mRNA level. Furthermore, the administration of cycloheximide alone diminished HMG-CoA reductase activity, but enhanced by 1.1- to 1.6-fold the reductase mRNA level. Coadministration of aminoglutethimide with ACTH also resulted in a decrease (65%) in reductase activity compared to that in NaCl-treated animals. However, coadministration of aminoglutethimide, in contrast to cycloheximide, with ACTH not only prevented the reductase mRNA level increase produced by ACTH, but also resulted in a 30% decrease in the reductase mRNA level compared to that in controls injected with 0.15 M NaCl. In addition, aminoglutethimide alone resulted in 50% and 54% decreases in reductase mRNA level and reductase activity, respectively. Thus, we have shown that both cycloheximide and aminoglutethimide can prevent the enhancing effect of ACTH on HMG-CoA reductase activity, but their modes of action differ. It is likely that the aminoglutethimide inhibition could be the result of a diminution of specific reductase gene transcription, whereas cycloheximide would result in inhibition of the synthesis of specific proteins, including HMG-CoA reductase. In this respect, since the adrenal free cholesterol content was increased in groups treated with ACTH-aminoglutethimide, we postulate that free cholesterol could be one of the important components involved in the regulation of HMG-CoA reductase gene transcription. As for the ACTH-cycloheximide-treated groups, the adrenal free cholesterol content was also increased, but the effect of ACTH on the reductase mRNA level was not prevented, presumably because this drug blocked the synthesis of a putative sterol regulatory protein that is required to repress HMG-CoA reductase gene transcription.

The halophilic properties of pyruvate kinase from Vibrio costicola, a moderate halophile.

Pyruvate kinase from Vibrio costicola, a moderate halophile, appears to be adapted to functioning in the presence of salt. Its stability depends on the ionic strength of the medium. The amino acid composition resembles that of other halophilic enzymes. It is proposed that the halophilic pyruvate kinase utilizes preferentially the Mn++ cofactor which forms more stable complexes in the presence of physiological concentrations of salt.

Porcine chymotrypsin A-pi, a more acidic chymotrypsin.

A kinetic study of procine chymotrypsin A-pi revealed two characteristic properties of this type of chymotrypsin: 1. Porcine chymotrypsin A-pi, like bovine chymotrypsin B-pi does not bind proflavin, which is a competitive inhibitor of bovine trypsin and chymotrypsin A-alpha. 2. The pH profiles of the steady-state parameters show the two usual important pK's. The basic one, pK2 = 9.6, affects both Km and kcat/Km and probably controls the binding conformation of chymotrypsin. The acidic one, pK1 = 5.7, affects kcat and kcat/Km and plays a role in the catalytic process. The value of pK1 is unusually low.

Hemochromatosis and pyruvate kinase deficiency. Report of a case and review of the literature.

Hemochromatosis has been reported in several patients with chronic hemolytic anemia due to pyruvate kinase deficiency. We describe here a further patient with such an association and review the literature on the subject. We hypothesize that iron overload may occur in patients with pyruvate kinase deficiency who are also carriers of the hereditary hemochromatosis gene.

Regulation of genes for glycolytic enzymes in cultured rat hepatoma cell lines.

We examined the control by hormones and culture conditions of the expression of pyruvate kinase L, aldolase B, and a liver-specific 5.4-kb mRNA species [Pichard, A. L. et al. (1985) Biochem. J. 226, 637-644] in three rat hepatoma cell lines, MH1C1, Fao and Faza. The expression level of these markers ranges from 2% (for pyruvate kinase L mRNA) to 10-12% (for 5.4-kb mRNA species) of the glucose-induced mRNA values found in rat liver. The mRNAs of the three liver-specific genes strongly decrease after treatment of the hepatoma cells with cyclic 8-bromo-AMP, cyclic dibutyryl-AMP or forkolin, pyruvate kinase L mRNA being the most sensitive to this inhibiting effect. In contrast, the concentration of pyruvate kinase L mRNA nuclear precursors is not modified by the cyclic AMP analogues, indicating that these agents do not act at the transcriptional level but, instead, probably destabilize the transcripts. Glucose or fructose does not modify the expression of these three marker genes in any of the studied cell lines. Insulin is inefficient in modifying concentrations of the mRNAs for pyruvate kinase L and aldolase B, alone or in the presence of carbohydrates. In contrast, it stimulates about fivefold the expression of the 5.4-kb mRNA species in the MH1C1 cell line; this stimulation is carbohydrate-independent. The hepatoma cell lines mimic, therefore, the effect of cyclic AMP on the inhibition in vivo of the expression of genes encoding glycolytic or lipogenic enzymes [Vaulont, S. et al. (1984) Biochem. Biophys. Res. Commun. 125, 135-147]. In contrast, the effect of carbohydrates [Munnich, A. et al. (1984) J. Biol. Chem. 259, 10228-10231] is undetectable. The insulin sensitivity of the liver-specific genes is conserved for the 5.4-kb mRNA species only, especially in the MH1C1 cell line, but not for the other investigated mRNAs, which seems to reflect a fundamental difference in the in vivo effect of insulin on these genes. Finally, S1 nuclease mapping of the start-site of pyruvate kinase L gene transcription shows that the normal site used in vivo is also used in the Fao and Faza lines while, in the MH1C1 line, it coexists with multiple aberrant upstream initiation sites.

Effect of ACTH on cholesterol and steroid synthesis in adrenocortical tissues.

Previous studies have established that under normal conditions, adrenal HMG-CoA reductase activity is higher in hamsters than in rats and humans. The hamster reductase activity follows a diurnal rhythm corresponding to that of plasma ACTH and glucocorticoids [Endocrinology 107 (1980) 215] but not to that of aldosterone. ACTH treatments to hamsters increased reductase activity after a latency of 60 min; this enhancement was prevented by cycloheximide [J. steroid Biochem. 24 (1986) 325]. Immunotitration and immunoblotting studies confirmed that ACTH caused an increase in reductase protein synthesis. In rats, long-term (1-9 days) and short-term (3 h) treatments with ACTH also induced increase in adrenal HMG-CoA reductase activity and reductase protein. In the presence of iodoacetamide and inhibitors of proteolytic enzyme, a main specific band of enzyme was evinced in the area of 102 +/- 6 kDaMr, by Western blotting, for both hamster homogenate and microsomal preparations (Endocrinology, 120 (1987]. Similarly Mr values were found with rat adrenal preparations. The concentration of mRNA, analyzed using the c-DNA pRed-10 coding for the Chinese hamster ovary reductase, was increased in adrenals of hamsters treated with ACTH. The reductase mRNA levels also fluctuated during the day in parallel with those of reductase activity and reductase protein. In conclusion, these results indicate that ACTH and other conditions inducing a change in hamster adrenal HMG-CoA reductase activity provoke parallel changes in reductase mRNA and reductase protein content. ACTH acts on the adrenal reductase of species synthesizing large as well as small quantities of cholesterol, thus indicating the general importance of this hormonal control.

Hereditary nonspherocytic hemolytic anemia due to pyruvate kinase deficiency: a prevalence study in Quebec (Canada).

Fifty-eight cases of hereditary nonspherocytic hemolytic anemia due to pyruvate kinase deficiency were collected from both laboratories that have performed the assay since 1972 and from attending physicians in the province of Quebec. Using the postal addresses of the probands, a prevalence map was constructed according to the administrative regions of the province of Quebec. The prevalence was found to be higher in eastern Quebec (1/81,838) than in western Quebec (1/139,086). Fifty probands were of French Canadian origin whereas the remaining 6 were recent immigrants.