Molecular cloning of a cDNA encoding mouse DNA helicase B, which has homology to Escherichia coli RecD protein, and identification of a mutation in the DNA helicase B from tsFT848 temperature-sensitive DNA replication mutant cells.

DNA helicase B is a major DNA helicase in mouse FM3A cells. A temperature-sensitive mutant defective in DNA replication, tsFT848, isolated from FM3A cells, has a heat-labile DNA helicase B. In this study, we purified DNA helicase B from mouse FM3A cells and determined partial amino acid sequences of the purified protein. By using a DNA probe synthesized according to one of the partial amino acid sequences, a cDNA was isolated, which encoded a 121.5 kDa protein containing seven conserved motifs for DNA/RNA helicase superfamily members. A database search revealed similarity between DNA helicase B and the alpha subunit of exodeoxyribonuclease V of a number of prokaryotes including Escherichia coli RecD protein, but no homologous protein was found in yeast. The cDNA encoding DNA helicase B from tsFT848 was sequenced and a mutation was found between DNA/RNA helicase motifs IV and V.

Antitumor activity of tumor necrosis factor-alpha conjugated with polyvinylpyrrolidone on solid tumors in mice.

We attempted the development of a novel polymer conjugation to further improve the therapeutic potency of antitumor cytokines compared with PEGylation for clinical application. Compared with native tumor necrosis factor (TNF)-alpha in vitro, specific bioactivities of polyvinyl-pyrrolidone (PVP)-modified TNF-alphas (PVP-TNF-alphas) were decreased by increasing the degree of PVP attachment. PVP-TNF-alpha fraction 3, Mr 101,000, had the most effective antitumor activity of the various PVP-TNF-alphas in vivo. PVP-TNF-alpha fraction 3 had >200-fold higher antitumor effect than native TNF-alpha, and the antitumor activity of PVP-TNF-alpha fraction 3 was >2-fold higher than that of MPEG-TNF-alpha (Mr 108,000), which had the highest antitumor activity among the polyethylene glycol (PEG)-conjugated TNF-alphas. Additionally, a high dose of native TNF-alpha induced toxic side effects such as body weight reduction, piloerection. and tissue inflammation, whereas no side effects were observed after i.v. administration of PVP-TNF-alpha fraction 3. The plasma half-life of PVP-TNF-alpha fraction 3 (360 min) was about 80- and 3-fold longer than those of native TNF-alpha (4.6 mm) and MPEG-TNF-alpha (122 min), respectively. The mechanism of increased antitumor effect in vivo caused the prolongation of plasma half-life and increase in stability. These results suggested that PVP is a useful polymeric modifier for bioconjugation of TNF-alpha to increase its antitumor potency, and multifunctionally bioconjugated TNF-alpha may be a potentiated antitumor agent for clinical use.

The largest isoform of platelet membrane glycoprotein Ib alpha is commonly distributed in eastern Asian populations.

Platelet membrane glycoprotein Ib alpha has at least two polymorphisms which affect phenotype. One is the dimorphism at codon 145, and the other is a molecular weight polymorphism due to variable numbers of tandem repeats (TR) in the macroglycopeptide region. These two polymorphisms are in linkage disequilibrium. The frequencies of these polymorphisms differ considerably depending on race, and the largest variant with four TR is almost exclusively present in the Japanese population. We examined the genotypes of HPA-2 and TR polymorphism in three different races from Eastern Asia; the Japanese (n = 103), Korean (n = 101) and Chinese population (n = 177). The gene frequency of HPA-2 differed significantly among these three populations. Among HPA-2b-positive individuals, the A isoform with four TR and B with three TR were present in all three populations and A dominated over B. Individuals homozygous for the A isoform were found in both Japanese and Korean populations. These findings indicate that the largest haplotype is common in the Eastern Asian region.

Serum thrombopoietin (c-Mpl ligand) levels in patients with liver cirrhosis.

To clarify the role of c-Mpl ligand (thrombopoietin: TPO) in liver cirrhosis (LC), we examined serum TPO levels (sTPO) in patients with LC (N = 44), chronic hepatitis (CH; N = 13) and healthy controls (N = 41) by an enzyme-linked immunosorbent assay. Although platelet counts of all LC patients (89 +/- 59 x 10(9)/l; mean +/- SD) were lower than those of controls and CH patients, sTPO levels in LC patients (1.23 +/- 0.51 fmol/ml) were the same as those in controls (1.22 +/- 0.37) and CH patients (1.18 +/- 0.36). Platelet counts were significantly higher in splenectomized patients than in unsplenectomized patients, but the sTPO level did not differ between these two groups. In LC patients, the sTPO level was not correlated with the platelet count, but was correlated with prothrombin time, activated partial thromboplastin time, and total bilirubin, indicating that production of TPO in the liver decreases slightly with the development of liver dysfunction. Our findings suggest that production of TPO is maintained in LC patients and their thrombocytopenia is not due to a defect in platelet production.

Time-dependent inhibition of platelet aggregation by dopamine and 3-methoxytyramine. Important roles of their metabolites.

The effects of dopamine (DA) and 3-methoxytyramine (3MT) on arachidonic acid (AA)-, collagen-, and adenosine 5'-diphosphate (ADP)-induced rabbit platelet aggregation were studied in relation to their metabolites in vitro. DA and 3MT did not inhibit AA- and collagen-induced platelet aggregation immediately after addition of a low concentration, but inhibition did occur, in a time-dependent and concentration-dependent manner, when DA and 3MT were incubated in platelet-rich plasma (PRP). The concentration of DA and 3MT decreased time-dependently, and the main metabolites of DA and 3MT were 3,4-dihydroxyphenylacetaldehyde (DOPAL) and 3-methoxy-4-hydroxyphenylacetaldehyde (MOPAL), respectively, as determined by high performance liquid chromatography. These aldehydes were found to have potent inhibitory activities nearly equal to those of 3,4-dihydroxyphenylethanol (DOPET) and 3-methoxy-4-hydroxyphenylethanol (MOPET) on both AA- and collagen-induced platelet aggregation. DOPET and MOPET were detected only in small concentrations. Taking into account both the anti-platelet potency and the concentrations detected in PRP of DOPAL and MOPAL, it is concluded that the time-dependent inhibition of the platelet aggregation by DA and 3MT may be due to the mediation of their corresponding main metabolites, DOPAL and MOPAL, respectively, while the concentration-dependent inhibition by DA and 3MT seems to be largely due to an innate activity.

Low prevalence of a polymorphism of platelet membrane glycoprotein Ib beta associated with neonatal alloimmune thrombocytopenic purpura in Asian populations.

Iy alloantigen system is the first polymorphism of platelet glycoprotein Ib beta reported to cause neonatal alloimmune thrombocytopenic purpura. We investigated the allelic frequency of Iy alloantigen among Japanese and Korean populations by polymerase chain reaction-restriction fragment length method to determine the possibility of alloimmunization against Iy. Two hundred and nine Japanese and 97 Korean subjects were examined. All 306 individuals were homozygous for glycine at amino acid position 15 and negative for Iy. The allelic frequency of Iy in these populations was calculated to be less than 0.0016. Alloimmunization associated with Iy antigen in Asian populations seems unlikely from these results.

Liver cirrhosis with marked thrombocytopenia and highly elevated serum thrombopoietin levels.

Three patients with liver cirrhosis (LC) and a bleeding tendency due to marked thrombocytopenia of less than 20 x 10(9)/l were admitted to our hospital for further examination. Bone marrow examination revealed megakaryocytic hypoplasia in all three patients. All patients exhibited amegakaryocytic thrombocytopenic purpura, myelodysplastic syndrome, or bone marrow hypoplasia. 111In-labeled platelet kinetic studies revealed decreased platelet production in all patients. Although serum thrombopoietin (sTPO) levels are usually within the normal level in patients with LC, the sTPO levels of our patients were about 10 times higher than the levels of normal subjects (1.22 +/- 0.37 fmol/ml): 13.34, 16.79, and 10.46 fmol/ml, respectively. These sTPO data supported our findings of decreased megakaryopoiesis. Our findings suggest that examination of sTPO levels is useful in determining the etiology of marked thrombocytopenia in LC patients.

The measurement of gastric mucus using a computer image processing system.

The measurement of gastric mucus, and the effects of aspirin and some antiulcer drugs on the content or components of gastric mucus were investigated in normal rats by using a newly devised computer image processing system. The present method enabled us to separate mucus components into PAS positive mucus (PAS+), and AB positive mucus (AB+), and to measure the amount of each mucus. Aspirin induced significant decreases in both PAS+ and AB+, teprenone increased the total amount of gastric mucus (PAS+ plus AB+), secretin tended to increase AB+, and cimetidine and omeprazole caused no remarkable changes on mucus secretion. These changes in gastric mucus could be observed on both microscopic observation and with the computer image processing system. The results indicate that the computer image processing system is very useful for the measurement of gastric mucus.

Pharmacokinetics and metabolism of KW-4490, a selective phosphodiesterase 4 inhibitor: difference in excretion of KW-4490 and acylglucuronide metabolites between rats and cynomolgus monkeys.

1. The pharmacokinetics and metabolism of KW-4490, a selective phosphodiesterase 4 inhibitor, were investigated in rats and monkeys. After oral administration, KW-4490 was rapidly absorbed, and then its plasma concentrations apparently declined with half-lives of approximately 5 h in rats and 3.5 h in cynomolgus monkeys; however, a number of secondary peaks were apparent in the profiles for both species. The plasma pharmacokinetics of KW-4490 were comparable between rats and monkeys. 2. After oral administration, KW-4490 was mainly eliminated by metabolism to acylglucuronides and renal excretion in the unchanged form. KW-4490 acylglucuronides were found in monkey but not rat urine. In rats, KW-4490 acylglucuronides were excreted only in bile. Although the pathway of excretion of acylglucuronides differed between rats and monkeys, cumulative excretion in the two animals was very similar, as expected from comparable hepatic clearance for glucuronidation in rat and monkey liver microsomes. 3. The glomerular filtration rate of unbound KW-4490 indicated that renal tubular secretion was significant in monkeys, whereas reabsorption was significant in rats. These species differences in urinary excretion of KW-4490 and its acylglucuronide metabolites are most likely due to substrate specificity of active transporters in rat and monkey kidney.

[A study of (Ca2+-Mg2+)-, and actomyosin-ATPase activities in pregnant myometrium and the functional modification by calmodulin].

In order to clarify the biochemical bases of myometrial contractions, (Ca2+-Mg2+)-ATPase activity, actomyosin (ACT)-ATPase activity and ACT superprecipitation were studied during pregnancy. The results indicate that; (Ca2+-Mg2+)-ATPase activity in nonpregnant ovariectomized rats was markedly increased after estradiol treatment (15 micrograms/day for 3 days) from 12.0 +/- 20.1 to 132.5 +/- 22.8 nmolePi/mg/min (p less than 0.01). The activity on days 19, 20, 21 and 22 of pregnancy was 48.0 +/- 5.0, 48.2 +/- 7.5, 112.3 +/- 18.5 and 27.3 +/- 8.8 nmolePi/mg/min, respectively, showing marked prepartum increase and rapid decrease after delivery. In human myometrium, basal (Ca2+-Mg2+)-ATPase activity showed no significant change but at term the activity was decreased (79.4 +/- 9.8 to 60.7 +/- 7.4 nmolePi/mg/min, p less than 0.05) in the presence of calmodulin (CMD). Human myometrial ACT-ATPase activity, on the other hand, was stimulated with CMD (1st trimester, term without labor and with labor: 295 to 1,134, 550 to 1,243 and 897 to 4,735 pmolePi/mg/min, p less than 0.05). Myometrial CMD concentrations, however, showed no change during pregnancy. ACT prepared from rabbit uterus showed an enhanced interaction of contractile proteins in the presence of CMD. These data indicate that CMD stimulates (Ca2+-Mg2+)-ATPase activity in early pregnancy but inhibits at term and increases ACT-ATPase activity. Since the myometrial CMD concentration remains unchanged during pregnancy, there may exist a function which alters CMD action on ATPase activity as pregnancy advances.

Transformation of thebaine to oripavine, codeine, and morphine by rat liver, kidney, and brain microsomes.

Thebaine, an intermediate of morphine biosynthesis in the poppy plant, Papaver somniferum, was transformed to oripavine, codeine, and morphine by rat liver, kidney, and brain microsomes in the presence of an NADPH-generating system. The formation of morphine, codeine, and oripavine was identified by a specific RIA, HPLC, and GCMS. Thebaine also gave rise to four other compounds, which for the moment are unidentified. NADH dramatically increased the formation of both codeine and morphine when used together with an NADPH-generating system, especially in liver microsomes. NADPH is essential in the formation of oripavine from thebaine and morphine from codeine, while NADH is critical in the conversion of thebaine to codeine and from oripavine to morphine. Carbon monoxide or SKF 525A inhibited the conversion, indicating a role of cytochrome P-450. These results provide evidence for the enzymatic in vitro conversion by mammalian tissues of thebaine to morphine. The pathway is similar to that which exists in plants.

[A study on fixatives for the simultaneous histological estimation of the gastric mucous gel layer and mucous cells in rat gastric surface mucosa].

We examined the special characteristics of various kinds of fixatives and tried to find the most suitable method for simultaneous histological estimation of the mucous gel layer and mucous cells in rat gastric paraffin sections stained with Alcian blue (pH2.5)-periodic acid Schiff (AB-PAS). The tested fixatives were (group in parentheses): absolute ethanol at -80 degrees C (A), absolute methanol at -80 degrees C (B), Carnoy's solution (C), formalin-ethanol (D) and formalin-Tyrode (E). The thickness of the mucous gel layer (ML) and the numbers of AB- and PAS-positive cells (AB cells, PAS cells) of both the fundic gland area (F. area) and pyloric gland area (P. area) were measured microscopically (x 200). ML in the F. area was found in the order of (A) > (B) > (C) > (D) = (E). AB cells were in the order of (A) > (E) > (B) > (C) > (D); and PAS cells were in the order of (B) > (A) > (E) > (C) > (D). Effects of various fixatives in the P. area showed the same trend as the F. area. Apart from above mentioned procedures, we observed the effects of ethanol at various temperatures: -80 degrees C, -25 degrees C, -4 degrees C and 20 degrees C. ML, AB and PAS cells showed the highest level at -80 degrees C. Moreover, to confirm that these fixatives retain the mucus, we evaluated the hexose and hexosamine contents contained in the fixative solution after fixation. As a result of various fixations, the hexose and hexosamine values in the fixative solution were the smallest for ethanol at -80 degrees C. In conclusion, ethanol at -80 degrees C was the most suitable fixative for histological estimation of mucous gel and mucous cells in rat gastric surface mucosa.

[Effects of FRG-8813, a new histamine H2-receptor antagonist, on gastric mucus in rats].

We examined the effects of FRG-8813, a new histamine H2-receptor antagonist with potent antisecretory activity, on gastric mucus in male SD rats (7w). In this study, the effects of FRG-8813 (1, 3, 10 mg/kg), given orally twice a day for 7 days, were investigated by histochemical and biochemical methods in comparison with those of cimetidine (CM, 30 mg/kg) and famotidine (FM, 1 mg/kg) in the fundic gland area (F. area) and pyloric gland area (P. area). In the histochemical study by alcian blue (pH 2.5)-PAS (AB-PAS) or high iron diamine-alcian blue (HID-AB) staining, the CM group showed a significant decrease in PAS and tended to show decreases in HID-AB positive mucus and mucous gel layer in the F. area; the FM group also showed a decrease in AB positive mucus in the P. area. On the other hand, the AB-PAS and HID-AB positive mucus of the FRG-8813 group were not affected. In the biochemical study, FRG-8813 increased the gastric mucosal hexose, hexosamine and sialic acid composing the mucus in a dose-dependent manner in the F. area. These results suggest that FRG-8813 does not cause a decrease in gastric mucus, unlike CM or FM, and it may be able to promote mucus secretion through increasing the mucous component in the F. area.

[Effect of FRG-8813, a new histamine H2-receptor antagonist, on gastric mucus production in rats].

We examined the effect of FRG-8813, a new histamine H2-receptor antagonist, on 1% NH3-induced gastric mucosal lesions and basal gastric mucus production in rats. The effect of FRG-8813 (10 mg/kg) given orally was investigated macroscopically and histochemically compared with that of 16,16-dimethyl prostaglandin E2 (dmPGE2, 2 micrograms/kg) or capsaicin (Cap, 10 mg/kg) in the fundic gland area. FRG-8813, dmPGE2 and capsaicin inhibited the NH3-induced mucosal lesions, and stimulated the mucus secretion 5 min after the administration. Chemical deafferentation abolished the gastroprotective effect of FRG-8813 or Cap and attenuated the increase by FRG-8813, dmPGE2 or Cap in mucus secretion seen after 5 min. These results suggest that FRG-8813 exerts its effect on gastric mucus production partially through the capsaicin-sensitive afferent nerves, like the mechanism involved in gastroprotection, and that the increase in mucus production by FRG-8813 is at least in part responsible for the gastroprotection.

Effect of the antiulcer polysaccharide fraction from Bupleurum falcatum L. on the healing of gastric ulcer induced by acetic acid in rats.

An antiulcer polysaccharide fraction (BR-2) from Bupleurum falcatum L. was examined for its effect on the healing of chronic ulcers induced by acetic acid in rats. When BR-2 was administered orally to the rats, it was shown to be effective in the healing of acetic acid-induced chronic ulcer. This result suggests that the use of herbal prescriptions containing B. falcatum L. may prove useful for the treatment of peptic ulcers.

Identification of the convulsant opiate thebaine in mammalian brain.

The convulsant opiate thebaine, an intermediate of morphine biosynthesis, was purified from bovine brain to homogeneity by gel filtration and high-performance liquid chromatography (HPLC) monitored by a radioimmunoassay. The immunoreactive material behaved identically to standard thebaine in two HPLC systems and was confirmed to be thebaine by combined gas chromatography/mass spectrometry. To our knowledge, the presence of thebaine in mammalian tissue has not been demonstrated previously. Codeine and morphine were also found to exist in ovine brain. The presence of thebaine in ovine brain provides strong evidence that morphine and codeine, in various mammalian tissues, are of endogenous origin and actually biosynthesized from a precursor.

Resistance to activated protein C and Arg 506 Gln factor V mutation are uncommon in eastern Asian populations.

We investigated the prevalence of the factor V (FV) Arg 506 Gln mutation in healthy subjects from three eastern Asian countries (Japan, n = 270; China, n = 113; and Korea, n = 93) and in 26 Japanese patients showing venous thromboembolic events. The patients were also examined for activated protein C (APC) resistance by using the Coatest APC resistance kit. The FV mutation was investigated by polymerase chain reaction and restricted enzyme digestion with MnlI RFLP assay of the FV gene. None of the patients showed APC resistance, while all subjects examined were homozygous for Arg at position 506 of the FV gene. Our results imply that FV mutation and APC resistance contribute little to venous thrombotic diseases in eastern Asia.

Rat gastric mucous gel layer contains sialomucin not produced by the stomach.

The sialylated mucus components of the normal gastric mucosa and mucous gel layer of rats were studied by using various histochemical staining methods including Maackia amurensis II (MAL-II) and Sambucus nigra (SNA) lectins, alcian blue (AB) pH 2.5 -- periodic acid Schiff (PAS) and high iron diamine (HID) -- AB pH 2.5. The acidic and neutral mucins characterized by the AB-PAS staining were abundantly present in the mucous gel layer as well as in the gastric mucosa. The sialomucin characterized by HID-AB was barely found in either the mucous gel layer or the mucosa. The sialomucin positive to MAL-II and SNA, which react with the N-acetyl neuraminic acid residue linked to galactose via an alpha-linkage, was moderately detected only in the mucous gel layer, but not in the entire mucosal layer. Furthermore, in animals given surgery to form an esophageal fistula through which saliva was excluded or in animals subjected to salivectomy, the mucous gel layer stained with MAL-II and SNA lectins was markedly decreased. These results indicate that a part of the sialomucin containing-mucous gel layer covering normal rat gastric mucosa originates from the saliva and that MAL-II and SNA lectins are useful for detecting this specific sialomucin.

Study of oxytocin receptor: II. oxytocin and prostaglandin F2 alpha receptors in human myometria and amnion-decidua complex during pregnancy and labor.

Oxytocin receptors (OXT-R) and prostaglandin F2 alpha receptors (PGF2 alpha-R) in human myometrium, amnion and decidua during pregnancy and at parturition were examined in an effort to clarify their role in the initiation and maintenance of uterine contractions. The number of binding sites for OXT in myometria showed an increase as gestation advance (Ist trimester v.s. at term; 205 +/- 90 v.s. 671 +/- 98 fmol/mg protein, N = 5, p less than 0.01), and a rapid decrease following the onset of labor (254 +/- 60 fmol/mg protein, N = 5, p less than 0.02). On the other hand the number of PGF2 alpha-R, remained unchanged throughout pregnancy and in labor. This myometrial PGF2 alpha binding capacity was approximately 1/20 to 1/30 that of the OXT binding, while binding affinity was almost equal. The OXT-R both in amnion and decidua, which was 1/6 to 1/7 that in myometrium, showed no significant changes throughout pregnancy or after the onset of labor. Binding affinity for each tissue was almost the same and appeared to increase towards term but no statistical significance was detected. Present data confirmed the presence of OXT as well as PGF2 alpha receptors in the three functionally distinct entities of pregnant human uterus; myometrium, amnion, and decidua. Among the components, the OXT binding increased only in the myometrium during pregnancy, suggesting this tissue specifically responds to OXT. In contrast, there was a constant binding in myometria for PGF2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)