Seventy genetic variations in human microsomal and soluble epoxide hydrolase genes (EPHX1 and EPHX2) in the Japanese population.

Human microsomal and soluble epoxide hydrolases (mEH and sEH) are enzymes that metabolize xenobiotic molecules. We screened DNA from 48 Japanese individuals for single-nucleotide polymorphisms (SNPs) in both genes by direct sequencing of the entire genomic regions containing EPHX1 and EPHX2, except for repetitive elements. This approach identified 33 SNPs in the EPHX1 gene; 6 of them were located in the 5' flanking region, 17 in introns, 8 in exons, and 2 in the 3' flanking region. In the EPHX2 gene, we identified 36 SNPs, including 4 in the 5' flanking region, 24 in introns, 5 in exons, and 3 in the 3' flanking region, as well as one insertion/deletion polymorphism in the 5' flanking region. These variants may contribute to a more precise understanding of the nature of correlations between genotypes and disease-susceptibility phenotypes that have been postulated in regard to human microsomal and soluble epoxide hydrolases.

Nucleotide sequences of reptile calcitonins: their high homology to chicken calcitonin.

The calcitonin genes of four species of reptiles (Reeve's turtle, rat snake, grass lizard, and spectacled caiman) were amplified from the genomic DNA, as well as from the mRNA of the ultimobranchial glands of the former three species, by the polymerase chain reaction (PCR) method, and were sequenced. Among several primer sets, only one primer set synthesized from the chicken calcitonin gene was compatible with those of the reptiles. The nucleotide sequences of the reptile calcitonin genes were highly homologous with that of chicken calcitonin (100% for turtle, 99% for caiman, 96% for lizard and 93% for snake). The products amplified from mRNA by the RT-PCR method matched completely those from genomic DNA in the turtle, snake and lizard.

Molecular genetic and immunological analysis of dystrophin of a young patient with X-linked muscular dystrophy.

We examined the nucleotide sequence of deleted part of dystrophin mRNA and its translational product with immunoblot and immunohistochemical methods in a 6-year-old boy with a deleted DMD/BMD gene. On Southern blot analysis of his genomic DNA, we found a deletion of exons 10 to 37 in the DMD/BMD gene, which was expected to preserve the translational open reading frame (ORF). Dystrophin mRNA from his biopsy sample was amplified by polymerase chain reaction (PCR) and sequenced. The mRNA lacked the sequence corresponding to the gene from exons 10-37, and the translational ORF was preserved. The transcript was expected to code a 260 kDa protein. Dystrophin expressed in this patient was investigated with immunological methods. A 260 kDa protein was detected by immunoblot analysis with antidystrophin antiserum against nondeleted regions. These observations confirmed the preservation of the reading frame and the 260 kDa protein was produced as a mutant dystrophin. All these are compatible with the diagnosis of BMD. However, the immunohistochemical pattern of his muscle cells was peculiar. With deleted-region-directed antiserum, the membrane was not stained at all as in DMD patients. In contrast, with nondeleted-region-directed antiserum, all the muscle cell membrane was stained continuously as in non-DMD/BMD individuals. These are quite different from the staining pattern in most BMD patients where muscles are stained patchily or discontinuously.

Preservation of the C-terminus of dystrophin molecule in the skeletal muscle from Becker muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive disorder of muscle in children. The DMD gene product, "dystrophin", is absent from DMD, while the allelic disease, Becker muscular dystrophy (BMD), exhibits dystrophin of abnormal size and/or quantity. But we are still uncertain about the scenario that internally deleted (or duplicated) dystrophin in BMD possesses its carboxy (C)-terminal region, and severely truncated dystrophin in DMD does not. Here we use a new monoclonal antibody directed against an peptide in the C-terminal end of the dystrophin molecule to show that the C-terminus is preserved in 30 BMD and 24 control skeletal muscles but not in 21 DMD specimens. This result, taken together with data on deletions of the dystrophin gene, emphasizes both the diagnostic and biological importance of the C-terminal domain which is required for proper function and stability of dystrophin, and substantiates the validity of the reading frame hypothesis for DMD versus BMD deletions on a biochemical level.

Expression of a dystrophin-related protein associated with the skeletal muscle cell membrane.

We previously reported that a protein which has immunological cross-reactivity with and a molecular weight similar to dystrophin, the Duchenne muscular dystrophy (DMD) gene product, is expressed on the muscle cell membrane (Tanaka et al. 1989b). To examine if this is the translation product of the autosomal transcript with homology to dystrophin mRNA identified by Love et al. (1989), we raised an antibody (PDRP) against a synthetic peptide corresponding to the putative protein (DRP) and examined its expression and cellular localization in human and murine skeletal muscle samples. In immunoblotting, PDRP stained a band with a similar molecular weight to dystrophin in samples from DMD and Becker muscular dystrophy (BMD) patients and control (non-DMD/BMD) human. PDRP was expected not to cross-react with dystrophin because the antigenic peptide was not homologous to dystrophin. In fact, PDRP did not cross-react with dystrophin present in a BMD patient. Immunohistochemically, PDRP stained the muscle cell membrane in samples from DMB and BMD patients and from mdx mice. Only a slight staining was observed in muscles from control human and wild type mice. Our results confirm the presence of DRP in human and murine skeletal muscles, and further demonstrate that it is localized on the cell membrane. The abundance of DRP in dystrophin deficient muscles might be related to some compensatory mechanisms.

Isolation of a urinary digitalis-like factor indistinguishable from digoxin.

A digitalis-like factor has been purified to apparent homogeneity from human urine based on the inhibitory effect on [3H] ouabain binding to intact human erythrocytes. The purification scheme involved large scale adsorption followed by preparative, semipreparative and analytical high-performance liquid chromatography. The purified material showed a prominent digoxin-like immunoreactivity. The behaviour of the isolated substance was identical to that of authentic digoxin in three high-performance liquid chromatography and three thin-layer chromatography systems. Moreover, fast atom bombardment mass spectrum and proton nuclear magnetic resonance spectrum suggested that the purified material may be indistinguishable from digoxin.

Urinary sodium pump inhibitor raises cytosolic free calcium concentration in rat aorta.

We were able to purify two distinct sodium pump inhibitors to homogeneity from human urine based on [3H]ouabain-displacing activity from intact human erythrocytes. The polar and less polar compounds were eluted off the C18 reverse-phase column with 18% and 31% acetonitrile, respectively. The polar compound cross-reacted very weakly with specific antidigoxin antibody and lacked a characteristic ultraviolet absorption peak between 190 and 300 nm. The less polar compound showed a prominent digoxinlike immunoreactivity and had an ultraviolet spectrum similar to that of digoxin. We examined the effects of these compounds on cytosolic free calcium concentration in cultured rat vascular smooth muscle cells (A10 cells) using the fluorescent calcium chelator fura-2. Only the polar ouabain-displacing compound caused a significant increase, from 108 +/- 7 to 162 +/- 8 nM (n = 6, p less than 0.01), in cytosolic free calcium concentration in A10 cells. The rise in cytosolic free calcium concentration induced by the polar ouabain-displacing compound tended to be slower in onset and more sustained than that induced by arginine vasopressin. In contrast, ouabain and bufalin had no appreciable effects on cytosolic free calcium concentration in A10 cells. These results suggest that the polar ouabain-displacing compound we isolated from human urine may possess a vasoactive property and may play an important role in the modulation of vascular tone.

Existence of a polar digitalis-like factor in mammalian hypothalamus.

We were able to partially purify a polar digitalis-like factor from rat and bovine hypothalami based on the capacity to inhibit [3H]ouabain binding to intact human erythrocytes. This factor was characterized in reference to the digitalis-like factor that we have isolated and reported on. Hypothalamic factor shared digitalis-like activities and physicochemical properties with the one derived from human urine and mammalian plasma. These findings strongly suggest that a polar digitalis-like factor identical to the circulatory factor does exist in mammalian hypothalamus.

Unexpected chain-terminating side reaction caused by histidine and acetic anhydride in solid-phase peptide synthesis.

Capping is a useful technique to facilitate purification of a crude deprotected peptide in solid-phase peptide synthesis. However, we observed a serious side reaction caused by the Ac2O capping procedure, when it was applied to a synthesis of a peptide containing His. The mechanism of the side reaction was studied.

Effects of human urine-derived digitalis-like factor on cultured renal tubular epithelial cells.

We purified a polar digitalis-like factor to apparent homogeneity from human urine using inhibition of 3H-ouabain binding to intact human erythrocytes to monitor digitalis-like activity. Since endogenous digitalis-like factor may act as a natriuretic hormone, we tested the binding characteristics of this urine-derived ouabain-displacing compound to the sodium pump in intact renal epithelial cells, in order to assess its potential physiological significance. Cultured canine and porcine epithelial cell lines, MDCK and LLC-PK1, had a high sodium pump density as estimated from 3H-ouabain binding sites. Human urine-derived ouabain-displacing compound showed a dose-related inhibition of 3H-ouabain binding to both of these cells, similar to the inhibition of unlabelled ouabain. These findings indicate that the ouabain-displacing compound is capable of acting on the sodium pump in intact renal epithelial cells and may be the circulating regulator of Na+,K+-ATPase.

Purification and characterization of human urine-derived digitalis-like factor.

We were able to purify a digitalis-like factor to apparent homogeneity from human urine based on the inhibitory effect on [3H]-ouabain binding to intact human erythrocytes. This ouabain displacing compound closely resembles ouabain in its polarity, molecular weight, non-peptidic nature and mode of action except for its UV absorbance spectrum. This compound sharing many biological activities of ouabain may be the endogenous ligand for the Na+, K+-ATPase and serve as a specific regulator of the sodium pump.

Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide.

Duchenne muscular dystrophy (DMD) is a debilitating X-linked muscle disease. We have used sequence information from complementary DNA clones, derived from the gene that is deleted in DMD patients, to generate an antiserum that stains the surface membrane of intact human and mouse skeletal muscle, but not that of DMD patients and mdx mice. Here we identify the protein reacting with this antiserum as a single component of relative molecular mass 210,000 (Mr = 210K) that fractionates with a low-ionic strength extract of intact human and mouse skeletal muscle. It is therefore distinct from the 400 K protein found in the heavy microsomal fraction of normal muscle and identified as a putative product of the DMD gene. We also analyse further the disease specificity of the antiserum. Positive staining is seen in normal controls, and in samples from patients with a wide range of muscular dystrophies other than DMD. Becker muscular dystrophy, which is allelically related to DMD, was the only other exception, and gave a sporadic staining pattern. The demonstration of a specific defect in the surface membrane of DMD muscle fibres substantiates the hypothesis that membrane lesions may initiate muscle degradation in DMD.

Presence of digitalis-like factor in mammalian plasma.

We attempted to purify a digitalis-like factor from volume expanded dog plasma using an inhibitory effect on the binding of [3H]ouabain to intact human erythrocytes to monitor digitalis-like activity. A highly polar [3H] ouabain displacing compound was purified to a high degree using a combination of chromatographic procedures including reverse phase and gel filtration high performance liquid chromatography. This compound, a reversible inhibitor of [3H]ouabain binding, closely resembles ouabain in its polarity and significantly increases during saline infusion. Its molecular weight was estimated to be 343Da. Moreover, similar compound was consistently detected in other mammalian plasma.

A new orally active hypotensive peptide, N-(dibenzyloxyphosphinoyl)-L-alanyl-L-prolyl-L-proline (PAPP).

The antihypertensive effects and mechanisms of a newly developed orally active tripeptide, N-(dibenzyloxyphosphinoyl)-L-alanyl-L-prolyl-L-proline (PAPP), were investigated. When PAPP (1-30 mg/kg orally) was administered to spontaneously hypertensive rats (SHR), systolic blood pressure (SBP) reached a maximum reduction after 8 h and this effect lasted over 24 h. In two-kidney, one clip hypertensive rats (2KIC rats) and DOCA-salt hypertensive rats, PAPP also reduced SBP. In normotensive Wistar rats, however, PAPP did not have a hypotensive effect. PAPP showed low toxicity in Sprague-Dawley rats. The depressor response to bradykinin (BK) was potentiated, but the pressor response to angiotensin I (ANG I) was not inhibited by PAPP. PAPP significantly relaxed isolated SHR aortic strips treated with KC or norepinephrine. Angiotensin converting enzyme (ACE) inhibition by PAPP was relatively low in vivo and in vitro.

Novel peptides with orally active and long-lasting antihypertensive activity.

A series of N-(P-substituted phosphinoyl)peptides were synthesized and their antihypertensive activities were tested in spontaneously hypertensive rats (SHR). Among them, N-(dibenzyloxyphosphinoyl)-L-Ala-L-Pro-L-Pro-OH showed the most potent and long-lasting antihypertensive activity in SHR when administered orally. Although the inhibitory activity of this peptide against the angiotensin-converting enzyme was about one-hundredth of that of Captopril, the antihypertensive activity in SHR was significantly higher and longer-lasting than that of Enalapril which has been reported to be the most potent agent among similar converting enzyme inhibitors.

Syntheses of optically active amino acids by the combination of chemical methods and microbial techniques.

The newly developed processes combining the advantages of chemical methods and microbial techniques brought about optically active amino acids (D- or L-) from racemic compounds. In all cases, the other isomer of substrates (L- or D-) that cannot be catalyzed by hydrolyzing enzymes were easily racemized under reaction conditions. Then, optically active amino acids were produced quantitatively from the racemic compounds.

Transposition response, a cardiovascular response to change of habitat in the rat.

Aortic flow and arterial pressure were observed in conscious rats by means of an electromagnetic flowmeter probe implanted around the ascending aorta and an arterial cannula inserted into the abdominal aorta through a femoral artery. When the rat was transposed to a new box from its usual one, heart rate, cardiac output and peak flow acceleration were increased but arterial pressure remained unchanged. Total peripheral resistance was decreased. This response complex of the cardiovascular system to change of habitat was designated as "transposition response." After beta adrenoceptor blockade with propranolol, transposition no longer increased heart rate, cardiac output or peak flow acceleration but markedly increased arterial pressure and total peripheral resistance. The response was essentially unchanged by atropinization or adrenalectomy. Besides cardiac excitation and adrenergic vasoconstriction, the response includes a beta adrenergic vasodilatation, which is at least in part mediated by transmitters released at the sympathetic nerve endings and presumably equivalent to the cholinergic vasodilatation in the cat and dog.

Determination of sites in rabbit muscle glycogen phosphorylase b modified by an adenosine 5'-monophosphate analog, N6-p-bromoacetaminobenzyladenosine-5'-phosphate.

To identify the residues modified by radioactive N6-p-bromoacetaminobenzyladenosine-5'-phosphate in phosphorylase b, the peptic peptides of the modified enzyme were purified. Two radioactive peptides were isolated, each of which had a cysteine residue modified. The cysteine residue involved in the AMP site was determined to be cystein-317 and the other eactive cysteine residue was cysteine-171.