Gene structure of human thrombomodulin, a cofactor for thrombin-catalyzed activation of protein C

The gene coding for human thrombomodulin, a thrombin receptor on endothelial cells and a cofactor for the activation of anticoagulant protein C zymogen, was isolated from a human genomic library by employing human thrombomodulin cDNA as a probe. The nucleotide sequences of the gene and the adjacent 5' and 3' flanking regions were then determined. The nucleotide sequence of this gene with approximately 3.7 kilobase pairs was identical to that of the cDNA, indicating that the gene for human thrombomodulin is free of introns. Hybridization data showed that there is only a single thrombomodulin gene in the human genome.     

Characteristic distribution of cathepsin E which immunologically cross-reacts with the 86-kDa acid proteinase from rat gastric mucosa

The antiserum raised against the high-molecular-weight acid proteinase from rat gastric mucosa, termed 86-kDa acid proteinase, has been shown to recognize rat cathepsin E, but not cathepsin D (Muto, N. et al. (1987) J. Biochem. 101, 1069-1075). Using this specific antiserum, characteristic distribution of cathepsin E in rats was demonstrated. The enzyme was detected in a limited number of tissues, such as stomach, thymus, spleen, bladder, and erythrocyte membranes. Among them, the highest activity was observed in the stomach. In contrast, cathepsin D immunoreactive with the antiserum specific to rat gastric cathepsin D was demonstrated in all the tissues examined. Cathepsin E-type enzymes partially purified from these five tissues were precipitated in the same manner by the specific antiserum, and they had the same molecular weight, electrophoretic mobility, and resistance against denaturation by 4 M urea. These results indicate that they could be exactly classified as cathepsin E. This type of enzyme was also detectable in mice and guinea pigs, but they showed relatively weak immunoreactivities with the antiserum. Thus, it is concluded that the distribution of cathepsin E is intrinsically different from ordinary cathepsin D, suggesting that it has a different physiological role from cathepsin D.     

Maternal inheritance of deleted mitochondrial DNA in a family with mitochondrial myopathy

Skeletal muscles from a mother and her daughter both with chronic progressive ophthalmoplegia were analyzed. Histological and biochemical analyses of their muscle samples showed typical features of this type of mitochondrial myopathy. Southern blot analysis revealed that, in both patients, there were two species of mitochondrial DNA (mtDNA): normal one and partially deleted one. The sizes of the deletion were different; the mutant mtDNAs from the mother and the daughter had about 2.5- and 5-kilobase deletions, respectively. The two mutant mtDNAs shared a common deleted region of 1.2-kilobase. However, both the start and the end of deletion were different between them, implying a novel mode of inheritance. This is the first report that the mutant mtDNA is responsible for the maternal inheritance of a human disease.     

Oxidation-induced increase in activity of angiotensin converting enzyme in the rat kidney

The activity of angiotensin converting enzyme(ACE) in crude extracts of the rat renal cortex was increased when the oxidizing agent diamide was added to the extract. The maximal activity was obtained at concentrations over 1 mM, and the value was twice or more the activity in the absence of the pretreatment. The activity of ACE was also increased by the diamide-pretreatment of the isolated membrane fraction of the renal cortex, thereby indicating that the increase in activity was not due to oxidation of endogenous glutathione (GSH) that may lower the ACE activity, but rather that ACE itself was oxidized. When O2 was included in the extract for 2 h, the ACE activity also increased to about twice the original activity. Lineweaver-Burk plots analysis demonstrated that, after oxidation with diamide and O2, the Vmax was increased but the Km remained unchanged. We conclude that the action of ACE in the kidney functions may differ in relation to oxidation of the tissue.     

Cloning of cDNA encoding human H-protein, a constituent of the glycine cleavage system

A cDNA that encodes human H-protein, a constituent protein of the glycine cleavage system, was cloned with anti-rat H-protein antibody as a probe from a human liver cDNA library constructed with an expression vector, lambda gt11. The longest size of cDNA of the isolated clones was about 750 base long (lambda HH15B9). On the other hand, we determined the primary structure of human H-protein from the amino terminal Ser by the 12th Val, including a hexapeptide, -Glu-Lys-His-Glu-Trp-Val-. In addition to the finding that most cDNA inserts cloned hybridized with the synthetic DNA probe composed of the possible sequences for the hexapeptide, we confirmed that lambda HH15B9 encodes the partial primary structure of H-protein in an open reading frame.     

1H-NMR heme resonance assignments by selective deuteration in low-spin complexes of ferric hemoglobin A

The heme methyl and vinyl alpha-proton signals have been assigned in low-spin ferric cyanide and azide ligated derivatives of the intact tetramer of hemoglobin A, as well as the isolated chains, by reconstituting the proteins with selectively deuterated hemins. For the hemoglobin cyanide tetramer, assignment to individual subunits was effected by forming hybrid hemoglobins possessing isotope-labeled hemins in only one type of subunit. The heme methyl contact shift pattern has 1-methyl and 5-methyl shifts furthest downfield in both chains and the individual subunits of the intact hemoglobin in both the cyanide- and azide-ligated species, which is consistent with a dominant rhombic perturbation due to the proximal His-F8 imidazole pi bonding in the known structure for human adult hemoglobin. The individual chain and subunit assignments confirm that the detailed electronic/magnetic properties of the heme pocket are essentially unaltered upon assembling the R-state tetramer from the isolated subunits.     

Alternate pathways of DNA replication in Escherichia coli

We have described the pcbA1 mutation which enables E. coli cells to replicate DNA in the absence of a functional dnaE gene product if DNA polymerase I (the polA gene product) is present. The pcbA1 mutation phenotypically suppresses multiple dnaEts and dnaEam alleles. The pcbA1/PolI replication pathway differs from normal in sensitivity to certain DNA-damaging agents such as methylmethane sulfonate (MMS) and a lack of damage-directed mutagenesis. We report here cloning of the pcbA1 gene in a multicopy plasmid. The pcbA1 mutation is detected only in cis; therefore, cloning necessitated gene eviction. The pcbA1 gene lies closely- linked to gyrB. We have demonstrated the physical presence of DNA polymerase I in the replicating holoenzyme complex by immunoblotting using dnaEam strains. We conclude that E. coli has two alternate replisome structures: REP-A, in which DNA polymerase I is the functional synthetic subunit; and REP-E, in which the alpha-subunit, product of the dnaE gene, is functional. To investigate further the role of individual DNA polymerases in replication, we have isolated the polB gene on multicopy plasmids.     

A patient with hypogonadotropic hypogonadism successfully treated by long-term pulsatile administration of luteinizing hormone-releasing hormone

A male patient with hypogonadotropic hypogonadism has been treated by pulsatile administration lf luteinizing hormone-releasing hormone (LHRH) (20-25 micrograms, every 2 hours, sc) for 4 years 6 months. His plasma testosterone (T) concentration began to increase after 4 weeks of treatment and reached the normal range in week 5. He showed complete secondary sexual development after 1 year of treatment. His sperm count was normalized after 1 year of treatment. He was married after 29 months of therapy, and has a healthy male child. Blood type tests showed his paternity of the child. During the long duration of pulsatile LHRH therapy, his gonadotropin secretion has been stimulated by LHRH and his T level has been maintained with no observable side effects. There are no other reports of patients treated by pulsatile LHRH injection for such a long duration, but finding in this patient indicated that long-term pulsatile LHRH therapy is a useful and safe method for treatment of hypothalamic hypogonadotropic hypogonadism.     

The 6R-oxygenase activity of arachidonate 5-lipoxygenase purified from porcine leukocytes

Arachidonate 5-lipoxygenase purified from porcine leukocytes was incubated with (5S)-hydroperoxy-6,8,11,14-eicosatetraenoic acid. In addition to degradation products of leukotriene A4 (6-trans-leukotriene B4 and its 12-epimer and others), (5S,6R)-dihydroperoxy-7,9,11,14-eicosatetraenoic acid was produced as a major product especially when the incubation was performed on ice rather than at room temperature. The amount of the (5S,6R)-dihydroperoxy acid was close to the total amount of leukotriene A4 degradation products. Under the anaerobic condition, production of the (5S,6R)-dihydroperoxy acid was markedly reduced. 5-Hydroxy-6,8,11,14-eicosatetraenoic acid could be a substrate of the enzyme and was transformed predominantly to a compound identified as (5S)-hydroxy-(6R)-hydroperoxy-7,9-trans-11,14-cis-eicosatetraenoic acid at about 1-2% rate of arachidonate 5-oxygenation. These findings indicated that the purified 5-lipoxygenase exhibited a 6R-oxygenase activity with (5S)-hydroxy and (5S)-hydroperoxy acids as substrates. The 6R-oxygenase activity, like the leukotriene A synthase activity, was presumed to be an integral part of 5-lipoxygenase because it required calcium and ATP and was affected by selective 5-lipoxygenase inhibitors.     

Hyperlipidemia in mast cell-deficient W/WV mice

Approximately 70% of the W/WV mice lacking mast cells due to a genetic defect showed hypertriglyceridemia combined with hypercholesterolemia. Increases of various magnitudes in chylomicrons, very-low-density lipoprotein, and intermediate-density lipoprotein were observed in the plasma of W/WV mice compared to those in the plasma of congenic normal mice. The increase in these lipoproteins was seen even in normolipidemic W/WV mice. Activities of both lipoprotein lipase and hepatic triacylglycerol lipase in the plasma after heparin injection were markedly lower in the W/WV mice than in the congenic normal mice, although activities of both lipoprotein lipase in the heart and adipose tissue and hepatic triacylglycerol lipase in the liver were not decreased. These results suggest that the W/WV mice have genetic defects in one or more of the following: secretion of both lipases from their synthesising cells, transport to the endothelium, and anchoring to the endothelial surface. Heparin deficiency in these mice may be responsible for the impairment and, thereby, may partially contribute to the hyperlipidemia.