Molecular cloning, sequencing, and expression of cDNA for rat liver microsomal aldehyde dehydrogenase.

The cDNA clone for rat liver microsomal aldehyde dehydrogenase (msALDH) was isolated and sequenced. The deduced amino acid sequence consisting of 484 amino acid residues revealed that the carboxyl-terminal region of msALDH has a hydrophobic segment, which is probably important for the insertion of this enzyme into the endoplasmic reticulum membrane. COS-1 cells transfected with the expression vector pcD containing the full-length cDNA showed that the active enzyme was expressed and localized mainly on the cytoplasmic surface of the endoplasmic reticulum membranes. It has been proposed that ALDH isozymes form a superfamily consisting of class 1, 2, and 3 ALDHs (Hempel, J., Harper, K., and Lindahl, R., (1989) Biochemistry 28, 1160-1167). Comparison of the amino acid sequence of rat liver msALDH with those of rat other class ALDHs showed that msALDH was 24.2, 24.0, and 65.5% identical to phenobarbital-inducible ALDH (variant class 1), mitochondrial ALDH (class 2), and tumor-associated ALDH (class 3), respectively. Several amino acid residues common to the other known ALDHs, however, were found to be conserved in msALDH. Based on these results, we proposed to classify msALDH as a new type, class 4 ALDH.     

Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells.

Bafilomycin A1 is known as a strong inhibitor of the vacuolar type H(+)-ATPase in vitro, whereas other type ATPases, e.g. F1,F0-ATPase, are not affected by this antibiotic (Bowman, E.M., Siebers, A., and Altendorf, K. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7972-7976). Effects of this inhibitor on lysosomes of living cultured cells were tested. The acidification of lysosomes revealed by the incubation with acridine orange was completely inhibited when BNL CL.2 and A431 cells were treated with 0.1-1 microM bafilomycin A1. The effect was revealed by washing the cells. Both studies using 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine and fluorescein isothiocyanate-dextran showed that the intralysomal pH of A431 cells increased from about 5.1-5.5 to about 6.3 in the presence of 1 microM bafilomycin A1. The pH increased gradually in about 50 min. In the presence of 1 microM bafilomycin A1, 125I-labeled epidermal growth factor (EGF) bound to the cell surface at 4 degrees C was internalized normally into the cells at 37 degrees C but was not degraded at all, in marked contrast to the rapid degradation of 125I-EGF in the control cells without the drug. Immunogold electron microscopy showed that EGF was transported into lysosomes irrespective of the addition of bafilomycin A1. These results suggest that the vacuolar type H(+)-ATPase plays a pivotal role in acidification and protein degradation in the lysosomes in vivo.     

Molecular basis for the interaction of histamine with the histamine H2 receptor.

We undertook these studies to characterize the molecular basis of the interaction of histamine with the H2 receptor. Key areas of homology in the structures of the histamine H2 and beta 2 adrenergic receptor suggested specific transmembrane amino acids that might be important for binding of histamine. A third transmembrane aspartic acid of the histamine receptor (Asp98), thought to serve as a counter anion that interacts with the cationic amine moiety of histamine, was mutated to Asn98, and the mutated receptor was expressed in Hepa cells. Removal of the negatively charged amino acid abolished both binding of the H2 receptor antagonist [methyl-3H]tiotidine and histamine stimulated increases in cellular cAMP content. Mutation of a fifth transmembrane aspartic acid (Asp186) to Ala186 or Asn186 by itself or in conjunction with mutation of another fifth transmembrane amino acid (Thr190 to Ala190) resulted in a loss of [methyl-3H] tiotidine binding, although the generation of cAMP in response to histamine was maintained. The histamine receptor with only a Thr190 to Ala190 or Cys190 mutation retained the ability to bind [methyl-3H]tiotidine, but both the affinity and efficacy of binding were reduced. These data lead us to propose a model for histamine binding in which Asp98 is essential for histamine binding and action, Asp186 defines H2 selectivity, and Thr190 is important in establishing the kinetics of histamine binding, but is not essential for H2 selectivity.     

Molecular cloning of the human histamine H2 receptor.

We utilized the technique of polymerase chain reaction with oligonucleotide primers based upon the nucleotide sequence of the canine H2 histamine receptor gene which we recently isolated to clone its human homologue. Transfection of a construct of this gene in Colo-320 DM cells led to the expression of a receptor that bound to [methyl-3H] tiotidine and was linked to 3',5'cyclic adenosine monophosphate (cAMP) generation in response to histamine. Both cAMP generation and [methyl-3H] tiotidine binding were inhibited with the H2 histamine receptor selective antagonist cimetidine but not diphenhydramine or thioperamide which are, respectively, H1 and H3 histamine receptor antagonists. These data confirm that we have successfully cloned a novel gene encoding the human H2 histamine receptor.     

Expression of mRNA for activin-binding protein (follistatin) during early embryonic development of Xenopus laevis.

Follistatin is a specific activin-binding protein and is supposed to control activin functions. During Xenopus embryonic development, activin is thought to act as a natural mesoderm-inducing factor. We isolated here the Xenopus follistatin cDNA from Xenopus ovary cDNA library and studied the expression of Xenopus follistatin gene during the course of early embryonic development. The Xenopus follistatin has an 84% homology at the level of deduced amino acid sequence with human and porcine follistatin. Its 3.5 kb mRNA is first expressed at the gastrula stage, when the expression of activin mRNA becomes first detectable, and increased thereafter. Another species of 2 kb mRNA become detectable from early neurula and also increased dramatically in tadpole. These results suggest that the follistatin acts also as a regulator of activin in inductive interactions during amphibian embryonic development.     

Deduced primary structure of a Xenopus proteasome subunit XC3 and expression of its mRNA during early development

Proteasome is a non-lysosomal proteinase complex ubiquitously distributed in eukaryotic cells. We isolated here the cDNA clone for one of the proteasome subunits (XC3) from Xenopus ovary cDNA libraries using rat RC3 cDNA as a prove. The cDNA is 885 bp long and encodes 234 amino acids. The deduced amino acid sequence is highly homologous (95.3%) to those of rat RC3 and human HC3 subunits. The mRNA for XC3 is one of the maternal mRNAs and detected at all the embryonic stages investigated, but its level changes in a characteristic way especially at the gastrula stage. We suggest that the highly conserved XC3 subunit plays an essential role in proteasome function and also that during Xenopus embryogenesis mRNA for XC3 subunit is replaced from maternal to newly-synthesized one probably around the gastrula stage.     

Double-Blind Study of the Chronopharmacotherapy of Depression

To investigate the possibility of chronotherapy with antidepressants for patients with depression, we gave single daily doses of clomipramine 150 mg/day to 30 patients with depression at three different times of day, i.e., morning, noon, or before bedtime, using a double-blind method over a 4-week period. Beneficial effects differed according to the administration time of day, with the most effective result being found for the administration at noon. Time-dependent differences in side effects were observed in tremors and dryness of mouth. An additional 10 patients were administered their medications three times a day by traditional, equally divided doses, and the efficacy was inferior than daily single doses at noon. The study results showed the significance of the administration time of day for the benefits and side effects of antidepressant therapy for depression.     

The complete amino acid sequence of a major trypsin inhibitor from seeds of foxtail millet (Setaria italica)

The complete amino acid sequence of a major trypsin inhibitor (FMTI-II) from seeds of foxtail millet (Setaria italica) was determined by analysis of peptides derived from the reduced and S-carboxymethylated protein by digestion with TPCK-trypsin and Staphylococcus aureus V8 protease. FMTI-II consists of 67 amino acid residues, including 10 half-cystine residues which are involved in 5 disulfide bridges in the molecule. The established sequence had a high degree of homology to Bowman-Birk type inhibitors from leguminous and gramineous plants. The trypsin reactive-site peptide bond in FMTI-II also appears to be Lys (16)-Ser (17) by comparison with these sequences.     

Inhibition of ischemia and reflow-induced liver injury by an SOD derivative that circulates bound to albumin

Ischemia followed by reflow often results in tissue injury. Although reactive oxygens seem to play an important role in the pathogenesis of postischemic reflow-induced tissue injury, the mechanism and an efficient way to inhibit oxidative injury are not known. We studied the mechanism by which hepatic transport function was inhibited by a transient occlusion followed by reflow of the portal vein and hepatic artery by using a superoxide dismutase (SOD) derivative (SM-SOD) which circulates bound to albumin with a half-life of 6 h. Occlusion of the hepatic vessels for 20 min followed by reflow for 60 min significantly inhibited transhepatic transport of cholephilic ligands, such as bromosulfophthalein (BSP) and taurocholic acid. Intravenous administration of SM-SOD markedly inhibited the reflow-induced decrease in transhepatic transport of these ligands. Thiobarbituric acid - reactive metabolites (TBAR) in the liver and plasma remained unchanged during occlusion and reflow, while TBAR in the bile increased significantly. Intravenous injection of SM-SOD inhibited the reflow-induced increase in biliary TBAR. Xanthine oxidase activity in plasma also increased during occlusion and reflow by an SM-SOD-inhibitable mechanism. Polymorphonuclear leukocyte-dependent chemiluminescence of the peripheral blood remained unchanged during occlusion, but increased markedly with time after reflow. SM-SOD also inhibited the increase in chemiluminescence almost completely. These and other results suggested that the superoxide radical and/or its metabolite(s) might play an important role in the pathogenesis of the reflow-induced liver injury and that SM-SOD might be useful for studying the mechanism for tissue injury caused by oxygen toxicity.     

Genetic analysis of human lymphocyte proteins by two-dimensional gel electrophoresis: 3. Frequent occurrence of genetic variants in some abundant polypeptides of PHA-stimulated peripheral blood lymphocytes

Summary The 100 or so most intensely Coomassie blue-stained polypeptides from PHA-stimulated peripheral blood lymphocytes were analyzed by two-dimensional electrophoresis in combination with family and population studies. Besides polymorphic lymphocyte cytosol 64k polypeptide reported previously, genetic variants were frequently observed in three polypeptides with molecular weights of 100,000, 49,000, and 40,000. All of them occur in the cytosol. These variant polypeptides are charge variants, because they are separated in the isoelectric focusing dimension. It is indicated by family and population studies and cell distribution analysis that the polypeptide with a molecular weight of 100,000 shows a genetic polymorphism determined by two alleles at a new autosomal locus, as described in the following paper. Family and population studies also suggest that a genetic polymorphism defined by alleles at an autosomal locus is present in each of the polypeptides with molecular weights of 49,000 and 40,000. In contrast to the previous reports of the extremely restricted genetic variability of the 100 or so most abundant fibroblast polypeptides, the present data indicate that common genetic variants are present at least in four of the 100 or so most intensely Coomassie blue-stained lymphocyte polypeptides. The result also shows that careful side-by-side comparison of two-dimensional electrophoresis patterns among both parents and their children is an effective method to detect genetic variant polypeptides.