The relationship between lip-closing force and dental arch morphology in patient with Angle Class I malocclusion.

Dental arch morphology and tooth position are affected by lip-closing force (LCF). This study aimed to quantitatively evaluate the relationships between the horizontal or vertical balance of the LCF generated during maximum voluntary pursing-like movements and dental arch length (DAL) or width (DAW) or the lingual inclination of the upper or lower 1st molars (LIUM, LILM) in patients with Angle Class I malocclusion. Sixteen subjects with Angle Class I malocclusion (median age: 23·4 ± 5·9 years) who had never undergone orthodontic treatment were randomly selected. LCF was measured in eight directions during maximum voluntary pursing-like lip-closing movements. Dental arch models were scanned and analysed to obtain DAW, DAL, LIUM and LILM measurements. Mandibular deviation was measured on posteroanterior cephalograms. A significant negative correlation was detected between maxillary DAL and upper LCF. Maxillary DAL, DAW and the DAL/DAW ratio displayed significant negative correlations with total LCF and upper LCF. However, no significant correlations were detected between any mandibular dental arch morphological parameter and LCF. The difference in the LIUM between the deviation and non-deviation sides exhibited a significant positive correlation with the difference in upper LCF between the deviation and non-deviation sides and was significantly negatively correlated with the difference in lower LCF between the deviation and non-deviation sides. These results suggest that upper LCF is related to maxillary DAL, and the horizontal balance of the LCF of the upper and lower lips is related to the LIUM during pursing-like lip-closing movements in patients with Angle Class I malocclusion.

Divergence of repetitive DNA sequences in the heterochromatin of medaka fishes: Molecular cytogenetic characterization of constitutive heterochromatin in two medaka species: Oryzias hubbsi and O. celebensis (Adrianichthyidae, Beloniformes).

The large biarmed chromosomes of Oryzias celebensis [2n = 36, fundamental arm number (FN) = 48] are considered to have resulted from fusions of acrocentric chromosomes in the ancestral karyotype of Oryzias, which is retained in O. hubbsi (2n = 48, FN = 48). To understand the molecular evolution of heterochromatin associated with karyotype reorganization in medaka fishes, we cloned 3 and 6 novel families of heterochromatin-related repetitive DNA sequences from O. hubbsi and O. celebensis, respectively, and characterized them using molecular cytogenetics. Two AT-rich repetitive sequences isolated from the genomic DNA of O. hubbsi, a 164-bp satellite DNA (OHU-RsaI-Scen) and a 177-bp telomere-specific repeat (OHU-RsaI-Stelo), were shown to be major components of the constitutive heterochromatin of centromeres and telomeres, respectively. A GC-rich 326-bp sequence, named OHU-AluI-M1, was colocalized with the 18S-28S ribosomal RNA gene cluster to a single autosomal pair of chromosomes and the W chromosome. In O. celebensis, 2 major satellite DNA sequences (the AT-rich 157-bp OCE-AluI-Scen sequence and the 186-bp OCE-HinfI-Scen sequence) were identified in the centromeric regions of almost all chromosomes. The 197-bp OCE-HinfI-S6 sequence was located in the centromeric and distal and/or interstitial heterochromatin of almost all chromosomes, and the 191-bp OCE-HinfI-S8 sequence was located in 6 pairs of chromosomes. Constitutive heterochromatin on the short arm of large submetacentric chromosome 5 was composed of at least 3 different repetitive sequences: the 171-bp OCE-AluI-S18 sequence, the 197-bp OCE-HinfI-S6 sequence and the 172-bp OCE-HinfI-S11 sequence. All families of repeated sequences showed no nucleotide sequence similarity with each other and high species-specificity among 7 different species. These results suggest that the heterochromatin of O. hubbsi and O. celebensis consists of various types of repetitive sequence and that the sequences evolved independently and were then amplified site-specifically in each lineage after karyotype reorganization occurred in the ancestral karyotype.

An autosomal locus controls sex reversal in interspecific XY hybrids of the medaka fishes.

Although the two medaka species Oryzias latipes and O. curvinotus share the sex-determining gene Dmy, XY sex reversal occurs in interspecific hybridization between O. latipes females of the Hd-rR inbred strain and O. curvinotus males. In this Hd-rR-curvinotus mating, all XX and XY hybrids developed as females. In this study, we used another O. latipes inbred strain (HNI) for the mating, and found that 23% of XY hybrids developed as males, although all XX and the remaining XY hybrids developed as females. Linkage analysis using 236 XY hybrid males obtained from (Hd-rR × HNI) F(1) females showed that a single major locus, Hybrid maleless (Hml), on autosomal linkage group 17, contributed to the strain difference in the XY sex reversal. Furthermore, we found that crossing females of a different O. latipes inbred strain, HO4C, did not cause XY sex reversal in the interspecific hybrids, and that the XY hybrids from (Hd-rR × HO4C) F(1) females showed a 1:1 sex ratio. XY hybrid males had the HO4C allele at sequence-tagged site loci around the Hml locus whereas XY females had the Hd-rR allele, confirming the strong contribution of this locus to XY sex reversal. Reverse transcriptase PCR analysis showed a reduced expression of Dmy(curvinotus) in XY fry of the Hd-rR-curvinotus hybrids at hatching. These results suggest that the Hd-rR allele at the Hml locus interfere with the function of Dmy(curvinotus) on a hybrid background, thus resulting in XY sex reversal.

A sex-determining region on the Y chromosome controls the sex-reversal ratio in interspecific hybrids between Oryzias curvinotus females and Oryzias latipes males.

Oryzias latipes and Oryzias curvinotus are closely related medaka species that have the common sex-determining gene, DMY, on their homologous Y chromosomes. We previously reported that sex-reversed XY females were produced in hybrids between O. curvinotus females and O. latipes males (Hd-rR inbred strain). In this study we used HNI inbred strain males of O. latipes for mating with O. curvinotus females, and found that all the XY hybrids developed as males. To map the factor responsible for this strain-specific XY sex reversal, O. curvinotus females were mated with two Y-congenic strains (HNI.Y(Hd-rR) and Hd-rR.Y(HNI)) and a recombinant congenic strain (Hd-rR.Y(HNI)rr). HNI.Y(Hd-rR) produced sex-reversed females in the XY hybrids, whereas no sex-reversed females were obtained in the XY hybrids from Hd-rR.Y(HNI) and Hd-rR.Y(HNI)rr, demonstrating that a small region on the Y chromosome, which includes DMY, is responsible for the XY sex reversal. Sex-reversed hybrids were only produced in the presence of the Y-chromosomal region derived from the Hd-rR strain, suggesting that missense or regulatory mutations specific to the Hd-rR Y-chromosomal region induce the sex reversal.

Identification of the sex-determining locus in the Thai medaka, Oryzias minutillus.

A sex-determining gene, DMY, which is comparable to the SRY gene in mammals, has been identified in the medaka, Oryzias latipes. Although Oryzias curvinotus, a closely related species to O. latipes also has DMY, this gene has not been found in other Oryzias fishes. It has recently been demonstrated that the sex chromosomes of Oryzias dancena and Oryzias hubbsi differ from those of O. latipes and these species have XX/XY and ZZ/ZW systems, respectively. This may suggest that Oryzias species have evolved different sex-determining genes on different sex chromosomes. In the present study, we investigated the sex determination mechanism in Oryzias minutillus, which is closely related to O. dancena and O. hubbsi. Linkage analysis using 14 isolated sex-linked DNA markers showed that this species has an XX/XY sex determination system. These sex-linked markers were located on linkage group 8 of O. latipes, suggesting that the sex chromosomes of O. minutillus are homologous to the autosomes of other Oryzias species. Furthermore, fluorescence in situ hybridization using a tightly sex-linked marker demonstrated that the XY sex chromosomes of O. minutillus and O. dancena were not homologous. These findings provide additional evidence for independent origins of sex chromosomes and sex-determining genes in these closely related species.

Suppression of laser-induced choroidal neovascularization by oral tranilast in the rat.

PURPOSE: To determine whether tranilast administered to pigmented rats inhibits formation of choroidal neovascularization induced by diode-laser photocoagulation. METHODS: Female Brown Norway rats were used. On day 0, choroidal neovascularization was induced by diode-laser photocoagulation, using a setting of 75 microm spot size, 0.1 second's duration, and 100 mW intensity. Tranilast (200 or 600 mg/kg per day) was administered orally twice daily for 14 days. Indomethacin (1 and 5 mg/kg per day) was administered orally once a day for 14 days. Choroidal neovascularization was evaluated on days 7 and 14 by fundus photography and fluorescein angiography. Late-phase fluorescein angiography was scored according to four grades. The animals were killed on day 14, and the lesions were evaluated histologically. RESULTS: In the vehicle-treated group, 34 of 35 burns (97%) showed fluorescein staining and late leakage on day 14. Choroidal neovascularization was identified by light microscopy in all the lesions that showed fluorescein staining and late leakage. The score of fluorescein staining was reduced in rats given 200 mg/kg per day or 600 mg/kg per day (P < 0.01) of tranilast. The thickness of the laser-induced lesions was reduced in a dose-dependent manner by tranilast, a significant difference was observed with 600 mg/kg per day (P < 0.05). Oral indomethacin treatment did not reduce fluorescein staining on day 14. CONCLUSIONS: Tranilast inhibits the development of choroidal neovascularization in this experimental model.

Effects of inhaled KAA-276, a selective histamine H1 receptor antagonist, on antigen- and histamine-induced bronchoconstriction in animals.

The antiasthmatic profile of KAA-276 (1-[1-(4-fluorophenylmethyl)-1H-benzimidazole-2-yl]-5-[2-[4-(2- carboxethyl) phenyl]ethyl]-1,5-diazacyclooctane sulfate, CAS 167264-26-8), a newly synthesized histamine H1 receptor antagonist, given by inhalation as an aerosol was investigated and compared with the profiles obtained using other routes of administration. When given by inhalation, or by intravenous or oral routes, KAA-276 inhibited antigen-induced bronchoconstriction in rats with ID50 (a dose to inhibit the antigen-induced response by 50%) values of 0.054%, 1 mg/kg, and 51.2 mg/kg, respectively. KAA-276 prevented the histamine-induced wheal reaction in rats dose-dependently with ID50 values of 0.22% by inhalation, 0.18 mg/kg by the intravenous route, and 2.3 mg/kg by the oral route. To judge from these results, inhaled KAA-276, unlike intravenous or oral KAA-276, had no inhibitory effect on the histamine-induced wheal reaction at a dose (0.054%) that is effective against the antigen-induced airway asthmatic response. Inhaled KAA-276 suppressed antigen-induced bronchoconstriction in actively sensitized guinea pigs, and histamine-induced bronchoconstriction in monkeys. These results suggest that inhalation of KAA-276 would benefit patients with bronchial asthma without inducing unwanted systemic effects.

Pharmacological characterization of a novel long-acting histamine H1 receptor antagonist, KAA-276.

The pharmacological profile of a newly synthesized histamine H1 receptor antagonist, KAA-276 (1-[1-(4-fluorophenylmethyl)-1H-benzimidazol-2-yl]-5-12-[4-( 2-carboxyethyl)-phenyl]ethyl]-1,5-diazacyclooctane sulfate), was characterized. In a H1 receptor binding assay in vitro, KAA-276 inhibited [3H]mepyramine binding to guinea pig cerebellar membrane preparations with an IC50 of 0.66 nM. The inhibitory potency of KAA-276 was greater than that of terfenadine, but similar to that of astemizole and ketotifen. KAA-276 antagonized the histamine-induced constriction of ileum and trachea isolated from guinea pigs in a dose-dependent manner with a concomitant reduction in the maximum response. Furthermore, the inhibitory effect of KAA-276 on histamine induced contraction was potentiated depending on the duration of preincubation time and revealed an irreversible property. KAA-276 given orally, intraduodenally, and by inhalation significantly inhibited histamine-induced bronchoconstriction dose-dependently in guinea pigs. Inhalation of KAA-276 exhibited inhibitory activity with a rapid onset and long duration, while intraduodenal administration resulted in action with a slow onset. Therefore, KAA-276, an irreversible and selective histamine H1 receptor antagonist, was shown to be a useful drug for therapeutic strategies against bronchial asthma when administered by the aerosol route.

Tranilast inhibits the proliferation, chemotaxis and tube formation of human microvascular endothelial cells in vitro and angiogenesis in vivo.

1. First developed as an antiallergic drug, tranilast inhibits chemical mediator release from mast cells. In the present study, we examine the effects of tranilast on angiogenesis in vitro and in vivo and discuss the application of tranilast for angiogenic diseases. 2. Tranilast inhibited significantly the proliferation (IC50: 136 microM, 95% confidence limits: 134-137 microM) and vascular endothelium growth factor (VEGF)-induced chemotaxis (IC50: 135 microM, 95% confidence limits: 124-147 microM) of human dermal microvascular endothelial cells (HDMECs) at concentrations greater than 25 micrograms ml-1. No toxicity to HDMECs measuring by LDH release and no inhibitory effects on metalloproteinase (MMP)-2 and MMP-9 activity were observed even at 100 micrograms ml-1 (306 microM). 3. Tube formation of HDMECs cultured on the matrigel as an in vitro angiogenesis model was inhibited by tranilast in a concentration-dependent manner. The IC50 value and 95% confidence limits were 175 microM and 151-204 microM, respectively. 4. In vivo angiogenesis was induced in mice by the subcutaneous injection of matrigel containing 30 ng ml-1 VEGF and 64 micrograms ml-1 heparin. Tranilast was administered orally twice a day for 3 days. Tranilast dose-dependently suppressed angiogenesis in the matrigel and a significant change was observed at a dose of 300 mg kg-1. 5. These results indicate that tranilast is an angiogenesis inhibitor which may be beneficial for the improvement of angiogenic diseases such as proliferative diabetic retinopathy, age-related macular degeneration, tumour invasion and rheumatoid arthritis.

O-methylasparvenone, a nitrogen-free serotonin antagonist.

O-Methylasparvenone (1) and asparvenone (2) were isolated from an Aspergillus parvulus Smith broth in a microbial screening for 5-HT2C ligands and found to be 5-HT2C antagonists. They represent the first nitrogen-free serotonin ligands. The absolute configuration of 1 was determined to be S by X-ray analysis of the corresponding Mosher-ester. A short and efficient synthesis of rac-1 was developed. This protocol was applied to the synthesis of derivatives of 1 and a structure-affinity relationship was established.

Neutralization of HIV type 1 infectivity by serum antibodies from a subset of autoimmune patients with mixed connective tissue disease.

Mixed connective tissue disease (MCTD) is a rheumatic disorder with clinical similarities to HIV-1 infection, and with characteristic autoimmune anti-RNP antibodies specific for the U1 snRNP splicing complex. Anti-RNP antibodies cross-react with the HIV-1 surface, owing to multiple homologies between the gp120/41 envelope complex and the 70K protein of U1 snRNP. A key epitope of 70K, its RNA-binding site, is homologous to a dominant B and T cell epitope in the third variable loop (V3) of gp120. In this study, we tested the ability of anti-RNP sera to inhibit HIV-1 infectivity in vitro. Of nine sera tested, five were 70-99% effective in neutralizing one or more HIV-1 strains. One serum was > 99% effective in neutralizing HIV-1MN, and 86 and 77% effective against the primary isolates HIV-1(CO) and HIV-1(JR-FL), respectively, an efficacy equal to that of a pool of broadly neutralizing antibodies from HIV-1-infected subjects (HIVIG). The mean neutralizing titer of anti-RNP sera against HIV-1(JR-FL) was 3.9-fold higher than that of HIVIG. Neutralizing potency was associated with high reactivity to gp120 by ELISA, and with the presence of serum rheumatoid factor, known to enhance antibody neutralization of other viruses. The current findings provide further evidence that individuals unexposed to HIV-1 may develop immunologic resistance by alternative mechanisms, possibly including molecular mimicry, or exposure to as yet unidentified retroviruses. Thus MCTD, which involves both B and T cell reactivity to self-epitopes homologous to HIV-1, may elucidate new strategies for generating protective immunity to this virus.

Cross-reactivity between autoimmune anti-U1 snRNP antibodies and neutralizing epitopes of HIV-1 gp120/41.

We report extensive amino acid sequence homology between HIV-1 gp120/41, and > 33% of a U1 RNA-associated splicing protein, 70K. The latter is a target of autoimmune anti-RNP antibodies in mixed connective tissue disease (MCTD). The homologies, involving dominant epitopes of 70K and neutralizing epitopes of gp120/41, are the basis for mutual antibody cross-reactivity. A key finding is that the epitope GRAFVTIG in the V3 loop of gp120 (strain IIIB) is homologous to the functionally essential U1 RNA-binding site of 70K. ELISA data reveal a mean reactivity of anti-RNP antibodies to V3 IIIB that is as high as that of HIV sera. V3 MN, containing the framework sequence G-AF-T, also cross-reacts with anti-RNP antibodies, as do hydrophilic epitopes in gp41 homologous to the COOH end of 70K. Further, there is strong cross-reactivity between HIV sera and 70K in Western blots. In contrast, antibodies from a related autoimmune disorder, Sjögren's syndrome (SS), are neither V3 nor gp41 selective. We conclude that the substantial cross-reactivities reported here are due to conserved, antigenically dominant B cell epitopes having homologous counterparts in 70K and gp120/41. Because antibody production in both MCTD and HIV-1 infection is T cell dependent, the results imply that common T cell clones are also activated in these two disease paradigms. Further exploration of the mechanisms that activate these clones, and that control their divergent fates in MCTD and AIDS, may provide new insights into immune dysregulation in HIV infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of a thromboxane A2 synthetase inhibitor (OKY-046.HCl) on airway hyperresponsiveness in guinea pigs.

We studied the effect of (E)-3-[p-(1H-imidazol-1-ylmethyl)phenyl]-2-propenoic acid hydrochloride monohydrate (OKY-046.HCl), a specific thromboxane (TX) A2 synthetase inhibitor, on airway hyperresponsiveness of guinea pigs. OKY-046.HCl (30-100 mg/kg, intraduodenally (i.d.) or orally (p.o.)) suppressed dose dependently the airway hyperresponsiveness to acetylcholine (ACh) induced by formyl-methionyl-leucyl-phenylalanine (FMLP), platelet activating factor (PAF) and repetitive antigen. OKY-046.HCl (100 mg/kg) also inhibited the increase in TXB2 in bronchoalveolar lavage fluid (BALF) induced by FMLP, PAF and antigen. Aspirin 10 or 30 mg/kg i.d. or p.o.) suppressed the airway hyperresponsiveness induced by FMLP and PAF but not by antigen. Azelastine (10 mg/kg i.d.) was ineffective on PAF- and antigen-induced airway hyperresponsiveness. TXA2 mimetic drugs caused airway hyperresponsiveness that was not inhibited by OKY-046.HCl (30 mg/kg i.v.). Furthermore, OKY-046.HCl showed no effect on propranolol- and physostigmine-induced airway hyperresponsiveness which did not accompany TXB2 generation in BALF. The number of eosinophils in BALF increased after FMLP exposure, an effect which was not inhibited by OKY-046.HCl. These results suggest that OKY-046.HCl inhibits airway hyperresponsiveness by suppressing TXA2 generation. We suggest that OKY-046.HCl will be a new antiasthmatic drug.

Inhibitory action of OKY-O46.HCl, a specific TXA2 synthetase inhibitor, on platelet activating factor (PAF)-induced airway hyperresponsiveness of guinea pigs: role of TXA2 in development of PAF-induced nonspecific airway hyperresponsiveness.

We studied a role of TXA2 in the development of PAF-induced nonspecific airway hyperresponsiveness in guinea pigs using a TXA2 synthetase inhibitor (OKY-O46.HCl) and a stable TXA2 mimetic agent (STA2). Inhalation of PAF (1 microgram/ml) and STA2 (1 or 10 ng/ml) increased the airway response to acetylcholine (ACh), histamine, leukotriene D4 and electrical vagal stimulation. Intraduodenal administration (i.d.) of OKY-O46.HCl (100 mg/kg) inhibited PAF-induced airway hyperresponsiveness. However, OKY-046.HCl (30 mg/kg, i.v.) did not suppress STA2-induced airway hyperresponsiveness. Neither hexamethonium (1 mg/kg, i.v.) nor hemicholinium-3 (10 mg/kg, i.v.) prevented the increase in the airway response to ACh after inhalation of PAF and STA2. In the presence of atropine (0.5 mg/kg, i.p.), PAF-induced airway hyperresponsiveness to histamine did not change. OKY-046.HCl (100 mg/kg, i.d.) inhibited the increase in ACh (10(-8) M)-induced 45Ca uptake into the lung tissue from PAF-inhalated guinea pigs. Inhalation of STA2 increased the number (Bmax) of muscarinic and H1-histaminergic receptors in the lung tissue from guinea pigs, but no changes were found on beta-adrenoceptors. These results suggest that TXA2 should act on the smooth muscle cells or alter functions of muscarinic and H1-histaminergic receptors, except beta-adrenoceptors, and then increase the membrane permeability to extracellular Ca2+. We also assume that OKY-046.HCl can inhibit PAF-induced nonspecific airway hyperresponsiveness by suppressing the generation of TXA2.

[Anti-allergic effects of (E)-3-[p-(1H-imidazol-1-ylmethyl)phenyl]-2-propenoic acid (OKY-046), a specific thromboxane (TX) A2 synthetase inhibitor: effect of OKY-046 on II-IV type allergic reactions].

We studied the effects of OKY-046 on types II, III and IV allergic reactions, as classified by Coombs and Gell. In Type II, OKY-046 at 30-100 mg/kg intraduodenally (i.d.) and at 1-30 mg/kg intravenously (i.v.) inhibited the bronchoconstriction in a dose-dependent manner after Forssman antigen injection. Aspirin (3 mg/kg, i.v.) also suppressed it. OKY-046 (30-100 mg/kg, i.d.) suppressed the increase of TXB2 level in the plasma in a dose-dependent manner. However, there was no effect of OKY-046 and aspirin on the decrease in complement activity (CH50), platelets and leukocytes. Additionally, OKY-046 (300 mg/kg, p.o.) prolonged the survival time following Forssman antigen injection. However, the immune hemolysis reaction was not prevented by OKY-046 (10(-6)-10(-3) M). FUT-175 protected against the Forssman shock at 1 mg/kg, i.v. and the in vitro immune hemolysis reaction at 10(-5) M. In Type III, OKY-046 (300 mg/kg, p.o.) significantly suppressed the direct passive Arthus reaction and immune complex nephritis in rats. There was no effect of OKY-046 on the delayed-type hypersensitive response to picryl chloride in mice. We think that OKY-046 should be a beneficial drug for the treatment of types II and III allergic reactions.

[Anti-allergic effects of (E)-3-[p-(1H-imidazol-1-lylmethyl) phenyl]-2-propenoic acid (OKY-046), a specific thromboxane (TX) A2 synthetase inhibitor: effects on type I allergic reactions].

We studied the effects of OKY-046 on type I allergic reactions. OKY-046 (100 mg/kg) given orally suppressed antigen-induced bronchoconstriction and TXB2 generation in broncho alveolar lavage fluid in rats passively sensitized with anti-DNP-As monoclonal IgE. At the dose of 30 mg/kg given intraduodenally, it also inhibited antigen-induced bronchoconstriction in guinea pigs passively sensitized with anti DNP-As serum and actively sensitized with ovalbumin. However, aspirin (30 mg/kg) didn't suppressed them significantly. Azelastine (10 mg/kg) inhibited bronchoconstriction in passively sensitized rats and actively sensitized guinea pigs. In 48 hour homologous PCA reactions of rats and mice, oral administration of OKY-046 (300 mg/kg) and tranilast (100 mg/kg) suppressed the extravasated dye in the skin. OKY-046 decreased histamine release from passively sensitized rat peritoneal exudate cells. There was no effect of OKY-046 on SRS-A and leukotriene release from actively sensitized guinea pig lungs and passively sensitized rats. In conclusion, we think that OKY-046 should be an useful asthmatic drug or anti-allergic drug by oral administration.

[Inhibitory effects of OKY-046.HCl, a selective thromboxane (TX) A2 synthetase inhibitor, on platelet activating factor (PAF)-induced airway hyperresponsiveness in guinea pigs].

We studied the inhibitory effects of OKY-046.HCl on PAF-induced airway hyperresponsiveness (AHR) in guinea pigs. 1) Inhalation of PAF (1 or 10 micrograms/ml) caused AHR to acetylcholine (ACh) aerosol and increased TXB2 generation in broncho-alveolar lavage fluid (BALF) at 30 min and 60 min, but the AHR and the TXB2 generation disappeared at 2 hr. OKY-046.HCl (100 mg/kg, intraduodenally) inhibited the AHR, which was accompanied by its inhibition of the TXB2 generation. However, no changes of 6-keto-PGF1 alpha in BALF were found. 2) There were no changes in the number of leukocytes; the activities of alkaline phosphatase, N-acetyl-beta-D-glucosaminidase, and lactate dehydrogenase; and the LTC4/D4/E4 in BALF. 3) In bronchus-lung preparations, PAF (1 microgram/min) also caused the AHR and increased TXB2 generation. OKY-046.HCl (100 micrograms/min) inhibited the AHR and TXB2 generation. 4) PAF (1 microgram/ml) evoked TXB2 generation in BALF from normal guinea pigs. OKY-046.HCl (10(-4)M) inhibited its increase. 5) Stable TXA2 (STA2, 1 ng/ml) inhalation also caused AHR to ACh at 30 min. STA2 (0.45 ng/min) also caused the AHR in bronchus-lung preparations. These results suggest that OKY-046.HCl can inhibit PAF-induced AHR by suppressing the generation of TXA2. We also supposed that TXA2 is released from lung parenchyma, airway epithelium and cell components in BALF.

Beneficial effect of OKY-046, a selective thromboxane A2 synthetase inhibitor, on experimental cerebral vasospasm.

Effects of OKY-046, a selective inhibitor of thromboxane (TX)A2 synthetase and a platelet aggregation inhibitor, on in vitro and in vivo models of cerebral vasospasm were studied. The contraction of the isolated rabbit basilar artery by an exposure to 1.0 ml of whole rabbit blood plus 0.05 or 0.1 units/ml of thrombin was diminished by the treatment with 10(-4) M of OKY-046 and/or 10(-6) M of cinanserin. When the whole blood of rabbits treated intravenously with 1 mg/kg/min of OKY-046 was used, the contraction of the basilar artery was decreased to about half of the control contraction. Angiographically recognized cerebral vasospasm in vivo, by a transorbital injection of 5.0 to 7.0 ml of autologous arterial blood into the cisterna magna of dogs, was suppressed by 0.05 and 0.5 microgram of OKY-046. Moreover, the decrease in the regional cerebral blood flow in autologous blood infused-dogs was inhibited by 0.5 microgram of OKY-046. The increase in TXB2 in the cerebrospinal fluid of dogs was significantly inhibited, and the level of 6-keto-PGF1 alpha was slightly increased by the treatment of OKY-046. The ratio of 6-keto-PGF1 alpha/TXB2 was increased from 1.5 to 5.2 in OKY-046-treated dogs. No effect on the basal tone and response to vasoactive agonists such as norepinephrine, KCl and PGE1 was observed in the isolated spiral thoracic aorta of guinea pigs or rabbits. Taken together with our previous findings, we conclude that the inhibition of cerebral vasospasm in the in vitro and in vivo models by the treatment of OKY-046 might be due to an inhibition of platelet aggregation, an inhibition of TXA2 generation and an increase in the ratio of PGl2/TXA2.

Determination of collagen band positions on polyacrylamide gel without staining.

Protein band positions on a slab polyacrylamide gel were determined without staining, by blotting the bands onto a nitrocellulose membrane which was then stained to visualize the electrophoretic pattern. The unfixed, unstained bands were cut from the gel according to the copy pattern placed underneath the slab gel. Collagen alpha 1 and alpha 2 chains were separately extracted from the gel using this procedure.