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Genetic structure and diversity of nine Japanese kokanee (landlocked) Oncorhynchus nerka stocks and anadromous O. nerka from the North Pacific and the Canadian Lake Cultus population were examined using microsatellite and mitochondrial DNA. Sequence analyses of the cytochrome b region of mtDNA for Japanese kokanee O. nerka stocks on Honshu and Hokkaido islands revealed that most Japanese stocks were monomorphic of one major haplotype, which was also dominant in the Lake Cultus population and anadromous O. nerka in the North Pacific. Assignment tests using microsatellite DNA revealed that there was no clear-cut population structure in Japanese kokanee O. nerka stocks.

Interrelationships between thyroid hormone and estrogen actions have been documented with regard to a variety of physiological functions. Both hormones stimulate transcription of target genes by binding to their nuclear receptors that interact with specific responsive elements (estrogen and thyroid hormone response elements, i.e ERE and TRE, respectively) in the regulatory regions of the gene. In vitro studies have suggested that interplay between the two hormones might be due to cross-talk at hormone responsive elements, with the respective hormone receptors and ligands able to interact, although physiological relevance has yet to be proved. We have proposed a simpler mechanism for thyroid hormone effects on estrogen responses via increase in estrogen receptor alpha (ERalpha) with resultant increase in progesterone receptors, prolactin production and tumor growth. A pituitary cell line, GH3, has been widely used to investigate the function of mammo-somatotropic cells, especially regarding regulation of GH and prolactin production. In the present study, an ERE-luc reporter was transfected into GH3 cells and the responses to endogenous ERalpha were examined. We demonstrated that: (1)l -3,5,3'-triiodothyronine (T3) induces mRNA expression of ERalpha; (2) T3 alone is able to induce ERE-luc activity and this is inhibited by OH-tamoxifen; (3) T3 synergistically acts on estradiol (E2)-induced ERE responses; and (4) ERE-luc activity is enchanted by co-transfection of an ERalpha expression vector. These results support the hypothesis that estrogen responses are potentiated by T3 through up-regulation of ERalpha levels.

Low-dose long-term erythromycin treatment has recently been reported to be very effective in patients with chronic respiratory infection and inflammation. This effect of erythromycin was thought to be not antibacterial but anti-inflammatory. However, the exact mechanism of the effect of erythromycin has not yet been clarified. The aims of this study were to investigate the effects of erythromycin on cytokine production and its mechanisms of actions in rat alveolar macrophages. Using rats with or without administration of erythromycin for 3 months, the production of the cytokines tumour necrosis factor-or (TNF-alpha), cytokine-induced neutrophil chemoattractant (CINC)-1 and CINC-2alpha by enzyme-linked immunosorbent assay and the expression of TNF-alpha and CINC-1 messenger ribonucleic acid (mRNA) by Northern blotting in rat alveolar macrophages were analysed. CINC-1 is the rat counterpart of human interleukin-8, and CINC-2alpha of human macrophage inflammatory peptide-2. Erythromycin reduced cytokine production and secretion when cytokines was induced by lipopolysaccharide treatment. Conversely, erythromycin slightly upregulated the expression of cytokine mRNA. These results suggest that erythromycin inhibits cytokine production and exhibits anti-inflammatory effects by means of a translational and/or posttranslational mechanism.

Using wood mice (Apodemus speciosus) captured in the wild in Niigata, we analysed the proportion of various endocrine cells in pancreatic islets for both immunohistochemical and microscopic characteristics. In both the dorsal and ventral portions of the pancreas, the centre of the pancreatic islets was occupied predominantly by insulin-positive (B) cells, surrounded by glucagon-positive (A), somatostatin-positive (D), and pancreatic-polypeptide-positive (PP) cells. Although the proportions of the various endocrine cells in pancreatic islets varied from one mouse to the next, in most animals B cells accounted for more than half of all endocrine cells. Dorsal and ventral portions of the pancreas differed in the proportions of various endocrine cells, specifically, in the A-to-PP cell ratio: the proportion of PP cells higher in the ventral portion. The same tendency is seen in humans, rats and mice. Microscopic examinations revealed morphologically distinct secretory granules in A, B and D cells. The morphology of these granules was similar that of secretory granules found in rats and mice.

The pathogenesis of posttransplant coronary artery disease, which is thought to be a major form of chronic rejection after cardiac transplantation, is not fully understood. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) on endothelial cells induces reduction of NO release and up-regulation of adhesion molecules, thereby contributing to the development of vascular atherosclerosis. Herein, we investigated the expression of LOX-1 mRNA in murine allografted hearts that develop diffuse coronary obstruction. Allogeneic (C57BL/6 to BALB/c) and syngeneic (C57BL/6 to C57BL/6) heterotopic cardiac transplants were removed the 10th posttransplant day. LOX-1 mRNA expression was measured by RT-PCR. The heartbeat of the allografts gradually weakened and was almost stopped on day 10, whereas syngeneic hearts continued to pulsate throughout the experiment. Histologically, allografts showed fibrous luminal narrowing of the coronary arteries with severe mononuclear cell infiltration. In contrast, the vascular architecture of syngeneic grafts was almost normal. Marked increase in LOX-1 mRNA expression was observed only in allografts. The results indicate that alloimmune responses induce up-regulation of LOX-1 mRNA in transplanted hearts. Increased LOX-1 may be involved in the progression of obstructive vascular changes.

Eicosapentaenoic acid (EPA) is an unsaturated fatty acid contained in fish oils. In order to clarify the mechanism of its anti-inflammatory effects on the lung, we studied arachidonic acid (AA) metabolism by in vitro exposure of rat alveolar macrophages to EPA. EPA was found to inhibit the endogenous production of leukotriene B4 (LTB4) from AA. At a low concentration of EPA, generation of LTB5 was increased, while at a high concentration it was decreased. These results suggest that at a lower concentration EPA may be competitive with AA as a substrate, and that at a higher concentration it may directly inhibit AA metabolism via inhibition of 5-lipoxygenase or phospholipase A2.

Understanding the origin and radiation of modern Asian hornbills and the influential ecological roles they play as seed dispersal agents within Asian rainforests should help reveal the evolution of these roles. We constructed a dated phylogeny of hornbills using mitochondrial DNA sequences of the cytochrome b gene and discovered that all clades leading to frugivorous hornbills originated in the mid-Eocene ∼48 Ma. This 'explosive' radiation coincided with a remarkable floral invasion of Asian rainforests from the Indian microcontinent. Analysis of phylogenetic data, in conjunction with palaeontological events, suggests that the invasion of distinctive flora comprised two waves, one during the mid-Eocene, when India was offshore of the Sunda Shelf, and the other late Eocene, when India collided with the Asian mainland. We propose that frugivorous vertebrates, such as hornbills, were present during the first wave and assisted rapid colonization of the Asian flora.

We examined the effect of hyperoxia on arachidonic acid (AA) metabolism in bovine carotid artery endothelial cells (CAEC) and pulmonary artery endothelial cells (PAEC). Confluent monolayers were exposed to hyperoxic gases (95% O 2 or 60% O 2 ) from 12–72 h. Control cells were incubated under normoxic condition (air-5% CO 2 ). After exposure of the cells to normoxic or hyperoxic conditions, prostaglandin (PG) synthesis activity was analyzed in cell homogenates using thin layer chromatography; release of 6-keto-PGF 1 α, a stable metabolite of PGI 2 , into the culture medium was measured using a radioimmunoassay. The major metabolites formed from exogenously supplied 14 C-AA were 6-keto-PGF 1 α and a small amount of PGE 2 . Hyperoxia (95% O 2 ) decreased the synthesis of these cyclooxygenase products beginning at 24 h; moderate hyperoxia (60% O 2 ) had no such effect. There was no significant difference between CAEC and PAEC with respect to the depletion effect of hyperoxia. After 72 h of exposure to 95% O 2 , endothelial injury was observed in CAEC but not in PAEC. We conclude that hyperoxia decreases cyclooxygenase activity in endothelial cells, and that this decrease is dependent on the severity of the hyperoxia. In addition, CAEC are more susceptible to hyperoxia-induced injury than PAEC. The depletion of cyclooxygenase activity and the resultant effect on PGI 2 and PGE 2 production may be a factor in the development of hyperoxia-induced endothelial injury.

We conducted comprehensive analyses on intron positions in the Mus musculus genome by comparing genomic sequences in the GenBank database and cDNA sequences in the mouse cDNA library recently developed by Riken Genomic Sciences Center. Our results confirm that introns have a tendency to be located toward the 5′ end of the gene. The same type of analysis was conducted in the coding region of seven eukaryotes (Saccharomyces cerevisiae, Plasmodium falciparum, Caenorhabditis elegans, Drosophila melanogaster, M. musculus, Homo sapiens, Arabidopsis thaliana). Introns in genes with a single intron have a locational bias toward the 5′ end in all species except A. thaliana. We also measured the distance from the start codon to the position of the intron, and found that single introns prefer the location immediately after the start codon in S. cerevisiae and P. falciparum. We discuss three possible explanations for these findings: (1) they are the consequence of intron loss by reverse-transcriptase; (2) they are necessary to accommodate the function; and (3) they are concerned with the mechanism of pre-mRNA splicing.

It is often difficult to identify sexes of many fish species by conventional cytological method because of the lack of heteromorphic sex chromosomes. Isolation of sex-specific molecular markers is thus important for sexing and for understanding sex chromosome evolution in these species. We have identified genetic sexes by PCR-based male-specificity of a growth hormone pseudogene (GH-psi) in masu and Biwa salmon, two subspecies of the Oncorhynchus masou complex, and their hybrid Honmasu. PCRs with primers designed from sequences of chinook salmon GH genes amplified GH-I and GH-II fragments in both sexes, but a third GH-psi fragment was detected in predominant proportion of males and very few phenotypic females. The consistency of phenotypic sex with genetic sex identified by GH-psi for masu salmon, Biwa salmon and Honmasu was 93.1, 96.7 and 94%, respectively. The remaining individuals showed inconsistency or deviation from sex-specificity: a few phenotypic males lacked the GH-psi, whereas a few phenotypic females possessed the GH-psi. Sequence of the putative GH-psi fragment from such females was identical to that from genetic males, and shared about 95% homology with the corresponding GH-psi fragment from chinook salmon. This result confirmed that these females were really GH-psi-bearing individuals. PCR analyses with primers designed from masu salmon GH-psi gave identical results, indicating that the absence of GH-psi in a few males was not resulted from primer mismatching. These GH-psi-bearing females and GH-psi-absent males were more likely to originate from spontaneous sex reversion than from crossing-over between GH-psi and the sex determination gene/region.

To improve the in vivo potency of the potent GPIIb/IIIa antagonist 2-[(3S)-4-[(2S)-2-(4-amidinobenzoylamino)-3-(4-methoxyphenyl)propanoyl]-3-(2-methoxy-2-oxoethyl)-2-oxopiperazinyljacetic acid (4), the amidino group was converted to an oxadiazole ring, thiadiazole ring or substituted amidoxime group. These groups were expected to be metabolized to an amidino group in vivo. The compounds synthesized were evaluated for their potency to inhibit the ex vivo adenosine 5'-diphosphate (ADP)-induced aggregation of guinea pig platelets. Among the compounds examined, the methoxycarbonyloxyamidine 8a exhibited the most potent ex vivo inhibitory activity with a fast onset and prolonged duration of action after oral administration.

A novel underwater antenna (which we named an 'aquaerial') for telemetering the biological signals from freely swimming fish in freshwater natural environments is presented. It is designed for receiving a 90-100 MHz carrier wave and consists of plural unit receiving antennas (UAs). The plural UAs are placed underwater to cover the area where the target fish carrying the transmitter is swimming. The UAs are equally spaced and have a directional coupling amplifier to supply the signals received to the coaxial cable. The optimal length of the UA was found to be 16.5 cm (a half wavelength in water) and optimal spacing was 2 m (one wavelength along coaxial cable) when 95 MHz was used as the carrier frequency. Using this 'aquaerial', long-term monitoring of EEG signals from the olfactory bulb of the rainbow trout (Oncorhynchus mykiss) swimming freely in natural environments was achieved.

We evaluated the effectiveness of the Helios gene gun system, a recently developed, commercially available gene gun device. Following skin transfection with beta-galactosidase or interleukin-12 cDNA using the gene gun, beta-galactosidase expression was detected exclusively in the epidermal cell layer, and transgene expression of IL-12 cDNA was maximal 2 days post-transfection and remained detectable for at least 5 additional days. Furthermore, particle-mediated delivery of IL-12 cDNA into epidermal cells overlying an intradermal tumor resulted in a significant suppression of tumor growth of Lewis lung carcinoma. Appreciable levels of IFN-gamma production were readily detected at the skin transfection site, and were induced from splenocytes and lymph node cells in the IL-12 treated mice. These results show that in vivo delivery of IL-12 cDNA into skin by the Helios gene gun device can have a useful routine application for cancer therapy research.

To elucidate the role of major chemotactic factors, cytokine-induced neutrophil chemoattractant (CINC), leukotriene B4 (LTB4) and C5a in lipopolysaccharide (LPS)-induced acute lung injury in rat, we employed three reagents: anti-CINC-1 antibody, an LTB4 receptor antagonist (ONO-4057) and an anti-complementary agent (K-76COONa). Rats were divided into five groups: (1)control group; (2) LPS group, which received intratracheal instillation of LPS (100 microg/kg); (3) Anti-CINC group, which received intratracheal coinstillation of LPS with anti-CINC-1 antibody (1 mg/kg); (4) LTB4-Ra group, which received intravenous ONO-4057 (10 mg/kg) prior to intratracheal LPS; (5) Anti-C5a group, which received intravenous K-76COONa (100 mg/kg) prior to intratracheal LPS. The number of neutrophils in bronchoalveolar lavage (BAL) fluids 6 h after LPS instillation was significantly reduced in the Anti-CINC group, however, no reduction was found in either the LTB4-Ra group or Anti-C5a group. The levels of CINC-1, CINC-2alpha and CINC-3 in BAL fluids were significantly higher in the LPS group than in the saline-instilled control group. In vitro, the production of CINC-1 and CINC-3 from LPS-stimulated macrophages was significantly elevated compared to unstimulated macrophages 6 h later. The increase in CINC-2alpha production was markedly less than that of CINC-1 or CINC-3. These results indicate that CINCs, especially CINC-1 and CINC-3 play an important role in the recruitment of neutrophils to the lung in LPS-induced acute lung injury.

The reductive metabolism in vivo of a flavoring additive, trans-4-phenyl-3-buten-2-one (PBO; trans-methyl styryl ketone) was investigated in rats and dogs. In both species, the double bond-reduced product, 4-phenyl-2-butanone (PBA), was detected by HPLC as the predominant species in blood after i.v. administration of PBO. PBA detected in rat blood was identified by comparison to the authentic sample. In contrast, the carbonyl-reduced product, trans-4-phenyl-3-buten-2-ol (PBOL) was also detected as a minor metabolite of PBO in both species. The area under the curve of PBOL in rat blood was only 3% of that of PBA. PBO was mutagenic in the Ames test using Salmonella typhimurium TA 100 when S-9 mix was added, but PBA and PBOL were not. It appears that PBO is mainly metabolized to PBA in vivo in rats and dogs as a detoxification pathway.

The roles of prostaglandins and catecholamines in the hypotensive hemodynamic change during cardiopulmonary support with a venoarterial bypass (VAB) were investigated in a series of chronic animal experiments of gradually reduced pulmonary arterial blood flow (PAF). The VAB system consisted of a pulsatile ventricular assist device, an artificial lung, and the right atrium uptake and descending aorta return cannulae in four adult goats weighing 49-51 kg. The PAF was adjusted to 50, 10, and 0% of the total systemic blood flow. Indomethacin, an inhibitor of prostaglandin production; phentolamine, an alpha-antagonist of catecholamine; and noradrenaline, an agonist of catecholamine were administered at each PAF condition. The mean aortic pressure (mAoP) and the systemic vascular resistance decreased in proportion to the decrease in PAF. Indomethacin increased the mAoP at all PAF conditions, indicating a relationship between prostaglandins and hypotension. Phentolamine decreased the mAoP at all PAF conditions, indicating a normal response of catecholamine receptors. However, noradrenaline increased the mAoP at 50 and 10% PAF, but did not appreciably increase the value at 0% PAF, indicating complete response of catecholamine receptors to endogenous catecholamines at 0% PAF only. In conclusion, prostaglandins play a substantial role in hypotension during VAB, and catecholamines may subsequently increase in compensation for extreme hypotension.

The relationship between chiral inversion and stereoselective amino acid conjugation of a new nonsteroidal anti-inflammatory agent, R, S-2-[4-(3-methyl-2-thienyl)phenyl]propionic acid (R,S-MTPPA) was investigated in rats and dogs. Only the S-enantiomer was found in plasma after oral administration of S-MTPPA to both species. In contrast, the R- and S-enantiomers were both detected after the dosing of R-MTPPA. In rats, the area under the curve of S-MTPPA in plasma was only 9% of that of R-MTPPA when R-MTPPA was dosed, whereas in dogs it was 2.5 times larger than that of the R-enantiomer. After administration of R-MTPPA, both enantiomers appeared in the urine. In rats, a small amount of S-enantiomer was found in the urine, whereas in the case of dogs the amount of the S-enantiomer was larger than that of the R-enantiomer. It appears that R-MTPPA is chirally inverted to S-MTPPA in both rats and dogs, although the extent of chiral inversion is greater in dogs than in rats. In dogs, the taurine conjugate was detected in plasma, urine and feces as a major metabolite after oral administration of either R- or S-MTPPA. In this case, only S-MTPPA-taurine was detected in the urine after the administration of S-MTPPA, and it was also the main component after administration of R-MTPPA.

The results of this study show the quinone-dependent reduction of tertiary amine N-oxides to the corresponding tertiary amines by rat liver preparations. The reduction of imipramine N-oxide to imipramine mediated by liver mitochondria, microsomes, and cytosol proceeded in the presence of both NAD(P)H and menadione under anaerobic conditions. When menadione was replaced with 1, 4-naphthoquinone or 9,10-anthraquinone, similar results were obtained in the cytosolic reduction. The quinone-dependent reducing activity in liver cytosol was inhibited by dicumarol and carbon monoxide. This result suggested that the activity is caused by DT-diaphorase, a cytosolic quinone reductase, and hemoproteins in liver cytosol. In fact, catalase and hemoglobin showed the ability to reduce imipramine N-oxide when supplemented with DT-diaphorase. The hemoproteins also exhibited the N-oxide reductase activity with reduced menadione, menadiol. The N-oxide reductase activity of the hemoproteins was also exhibited with 1,4-dihydroxynaphthalene, 1,4,9, 10-tetrahydroxyanthracene, or 1,4-dihydroxy-9,10-anthraquinone. Furthermore, hematin revealed a significant N-oxide-reducing activity in the presence of menadiol. The reduction appears to proceed in two steps. The first step is reduction of menadione to menadiol by a quinone reductase with NADPH or NADH. The second step is nonenzymatic reduction of tertiary amine N-oxides to tertiary amines by menadiol, catalyzed by the heme group of hemoproteins. Cyclobenzaprine N-oxide and brucine N-oxide were also transformed similarly to the corresponding amine by the quinone-dependent reducing system.

The full length porcine granulocyte/macrophage colony stimulating factor (GM-CSF) cDNA, including secretion signal peptide coding region was recloned into baculovirus transfer vector pAcYM1. The vector was then transfected with Autographica californica nuclear polyhedrosis virus (AcNPV) DNA into SF21AE cells and the recombinant virus AcPGM was recovered. Recombinant porcine GM-CSF (rpGM-CSF) was obtained from the serum-free culture medium of Tn5 cells infected with the AcPGM virus, and was shown to be a glycosylated 21 kDa protein as confirmed by tunicamycin treatment and [3H]-glucosamine uptake. The biological activities of rpGM-CSF in AcPGM-infected cell culture supernatants were demonstrated by porcine bone marrow cell proliferation and haematopoietic cell colony formation assays. The use of rpGM-CSF enabled us to culture porcine monocytes/macrophage and dendritic-like cells, derived from either porcine bone marrow or peripheral blood, for up to 4 months.

We investigated the effects of tamoxifen on the growth of 7,12-dimethylbenz(a)anthracene induced rat mammary tumors, the activity of thymidylate synthetase and thymidine kinase (key enzymes involved in de novo and salvage pathways for pyrimidine nucleotide synthesis), and also their gene expression. The effects on immunohistochemistry using bromodeoxyuridine in the tumors and bone mineral density of the femur in rats were also studied. Chronic administration of tamoxifen markedly reduced the expression of thymidylate synthetase mRNA, followed by a reduction in enzyme activity and S-phase cells in the mammary tumors, and significantly enhanced the bone mineral density. Tamoxifen not only attenuated bone loss in aging but also enhanced bone volume in mammary tumor-bearing rats in which tumor growth was suppressed via both the de novo and salvage pathways for pyrimidine nucleotide synthesis.

The localization of two salmon-type gonadotropin-releasing hormone (sGnRH) precursors, pro-sGnRH-I (short type) and pro-sGnRH-II (long type), was investigated by using in situ hybridization techniques in the brain of the landlocked sockeye salmon, Oncorhynchus nerka. We used 30-mer oligonucleotide probes complementary to pro-sGnRH-I and pro-sGnRH-II cDNA. No significant differences were observed in the localization of sGnRH neurons expressing pro-sGnRH-I and pro-sGnRH-II mRNAs; both were expressed in the olfactory nerve, the olfactory bulbs, the regions between the olfactory bulb and telencephalon, the ventral telencephalon, the preoptic area, and the hypothalamus. Almost all sGnRH neurons examined co-expressed both precursors. The expression of two sGnRH precursors in the same neuron and the wide distribution of such neurons in the brain suggest that there are no functional differences between the two precursors.

The disposition of S-2-[4-(3-methyl-2-thienyl)phenyl]propionic acid (CAS 155680-07-2, S-MTPPA, code: M-5011) was studied after oral administration to rats, dogs and monkeys using the 14C-labeled drug. After oral dosing, S-MTPPA was well absorbed from the gastrointestinal tract, to the extent of 97.7% in rats. The concentration of S-MTPPA in rat plasma reached a peak (Cmax: 13.07 micrograms/ml) at 15 min (tmax) after dosing and declined with a half-life (t1/2) of 2.5 h. The values of the parameters tmax, Cmax and t1/2 for dogs were 30 min, 26.2 micrograms/ml and 7.0 h, and those for monkeys were 15 min, 12.8 micrograms/ml and 3.0 h, respectively. The radioactivity was widely distributed in tissues and almost completely excreted in urine and feces within 48 h after oral administration to rats. The excretion of radioactivity in bile, urine and feces within 48 h after oral administration of 14C-S-MTPPA to bile duct-cannulated rats amounted to 75.0, 18.6 and 1.4% of the dose, respectively. The drug was metabolized mainly by oxidation of the thiophenyl moiety and by glucuronidation of the carboxyl group in rats and monkeys. The major urinary and fecal metabolite in dogs was identified as the taurine conjugate of MTPPA.

The patient, a 59-year-old woman, was admitted for further evaluation after a routine checkup in 1990 revealed an interstitial shadow chronic hypersensitivity pneumonia caused by her pigeons, was diagnosed. The patient later disposed of the pigeons and henhouse, where upon, she experienced dyspnea on exertion. In January 1995, the patient was admitted to our hospital with a progressive pulmonary interstitial shadow. Lung biopsy after thoracoscopic surgery revealed centrilobular fibrotic lesions, as well as, small honeycomb-like and granulomatous lesions. An antibody to the pigeon serum was detected in the patient's serum by the ouchterlony method. Chronic hypersensitivity pneumonia caused by pigeons is rare, and previously documented histological findings are few.

1. To elucidate the possible involvement of pertussis toxin (PTX)-sensitive G proteins in the post receptor mechanism of alpha 2-adrenoceptors and imidazoline receptors, we examined the effect of pretreatment of the central nervous system with PTX on the antidysrhythmic effect of dexmedetomidine, a selective alpha 2-adrenoceptor agonist, and rilmenidine, a selective I1-imidazoline receptor agonist on halothane-adrenaline dysrhythmias in rats. 2. Dexmedetomidine (0, 1.0, 2.0, 5.0 micrograms kg-1 min-1.i.v.) and rilmenidine (0, 1.0, 3.0, 10, 20 micrograms kg-1, i.v.) prevented the genesis of halothane-adrenaline dysrhythmias in a dose-dependent fashion. Both idazoxan (10, 20 micrograms kg-1, intracerebroventricularly (i.c.v.)), an alpha 2-adrenoceptor antagonist with high affinity for imidazoline receptors, and rauwolscine, (40 micrograms kg-1, i.c.v.), an alpha 2-adrenoceptor antagonist with low affinity for imidazoline receptors inhibited the action of dexmedetomidine (5.0 micrograms kg-1, min-1, i.v.), but the inhibitory potency of idazoxan was much greater than that of rauwolscine. While the pretreatment with PTX (0.1, 0.5, 1.0 micrograms kg-1, i.c.v.) did not change the dysrhythmogenecity of adrenaline, this treatment completely blocked the antidysrhythmic property of rilmenidine (20 micrograms kg-1, i.v.) as well as dexmedetomidine (5.0 micrograms kg-1 min-1, i.v.). 3. It is suggested that central I1-imidazoline receptors as well as alpha 2-adrenoceptors may be functionally coupled to PTX-sensitive G proteins.

Almost all respiratory diseases except benign lung tumors and lung dysplasia entail acute lung injury. The many clinical conditions associated with acute lung injury include aspiration pneumonia, bacterial pneumonia, and sepsis. The fundamental cause of acute lung injury is pulmonary vascular hyperpermeability. Pulmonary vascular hyperpermeability can be attenuated by nitric oxide and cyclic GMP, and potentiated by oxygen radicals and elastase released from neutrophils. Adhesion molecule inhibition could become an effective therapy against acute lung injury, because the adhesion molecules are very important in the pathogenesis of this condition. Adhesion molecules could also be useful markers of disease activity in various lung diseases. Neutrophil elastase inhibitors may become important as therapeutic agents against acute exacerbations of idiopathic interstitial pneumonia, because this pathological condition is a type of acute lung injury. Similarly, N-acetyl cysteine could also become a useful therapeutic agent against idiopathic interstitial pneumonia, because it is a precursor of glutathione, which is the major antioxidant in the fluid lining of the bronchial epithelium.

Reactive oxygen intermediates such as hydrogen peroxide play an important role in the pathophysiology of acute lung injury, not only as terminal effectors, but also as second messengers in signal transduction; we studied their role in adhesion molecule expression and cytokine production. N-acetylcysteine, an antioxidant, decreased the TNF alpha-induced expression of intercellular adhesion molecule-1 on cultured epithelial cells from human bronchi (BEAS-2A), and inhibited IL-8 production by those cells. In vivo, N-acetylcysteine attenuated the sequestration of polymorphonuclear neutrophils in rat lungs caused by intratracheal lipopolysaccharide. These findings suggest that adhesion molecule expression and cytokine production in the lung are mediated by the production of reactive oxygen intermediates. Because adhesion molecules and cytokines play a crucial role in the pathophysiology of neutrophil-mediated acute lung injury, the inhibition of adhesion molecule expression and cytokine production with anti-oxidants such as N-acetylcysteine may be a useful therapeutic strategy.

The hypothermic simple immersion technique has been widely used to preserve the donor heart for transplantation. However, there is still controversy as to which temperature provides the best protection against prolonged ischemia. The low molecular weight solutes within cells depress the freezing point to -0.6 degree C. In practice, however, most cells do not freeze internally unless they are cooled below -10 degrees C. We investigated the effects of subzero nonfreezing storage at -1 degree C on the preservation of myocardial metabolism and function.Isolated Wistar rat hearts were subjected to 6 hours of preservation with the intracellular type University of Wisconsin solution; the hearts of the subzero group were preserved at subzero nonfreezing (-1 degree C) temperature, and the hearts of the control group were at 4 degrees C. Recovery of cardiac function, myocardial adenine nucleotides content, and myocardial water content were evaluated after preservation.Subzero group resulted in significantly better aortic flow, cardiac output, and aortic systolic pressure than in the control group. Myocardial adenosine triphosphate, adenosine diphosphate, and total adenine nucleotides at end-storage were significantly better preserved in subzero group when compared with the control group. Myocardial water content at reperfusion significantly increased in the control group compared with the subzero group.Storage in the intracellular type solution at subzero nonfreezing (-1 degree C) temperature as compared with 4 degrees C appears to prolong myocardial preservation with respect to the enhancement of postischemic functional recovery, preservation of myocardial adenine nucleotides during ischemia, and prevention of myocardial edema at reperfusion.

Zonisamide (1,2-benzisoxazole-3-methanesulfonamide), an anticonvulsant agent, is primarily metabolized to 2-sulfamoylacetylphenol by reductive cleavage of the 1,2-benzisoxazole ring. Rabbit liver cytosol with an electron donor of aldehyde oxidase exhibited a significant zonisamide reductase activity that was sensitive to inhibition by menadione, an inhibitor of aldehyde oxidase. The result suggested that the cytosolic activity is caused by aldehyde oxidase, a cytosolic enzyme. In fact, rabbit and rat liver aldehyde oxidase had the ability to reduce zonisamide when supplemented with its electron donor. Apparent KM and Vmax values of aldehyde oxidase for zonisamide were 217 microM and 42 nmol/10 min/mg protein in the case of the rabbit liver enzyme, and 542 microM and 382 nmol/10 min/mg protein in the case of the rat liver enzyme, respectively. In rabbits, hamsters, mice, and guinea pigs, zonisamide reductase activity of the liver cytosols with 2-hydroxypyrimidine, an electron donor of aldehyde oxidase, was much higher than that of the liver microsomes with NADPH. In rats, zonisamide reductase activity was examined with liver microsomes and cytosols from seven strains. The 2-hydroxypyrimidine-dependent cytosolic activity exhibited marked strain differences, unlike the NADPH-dependent microsomal activity. 1,2-Benzisoxazole was also reduced to salicylaldehyde by rabbit liver cytosol and aldehyde oxidase in the presence of 2-hydroxypyrimidine. Stoichiometric studies showed that 2-sulfamoylacetylphenol was formed accompanying nearly equimolar ammonia from zonisamide.

We studied the effect of sensory nerve peptide substance P (SP) and neurokinin A (NKA) in isolated perfused guinea pig lungs. SP and NKA increased pulmonary arterial pressure, capillary pressure, pulmonary venous resistance, and lung weight, but they did not change pulmonary arterial resistance or pulmonary vascular permeability. The effects of SP on pulmonary vascular dynamics were greater than those of NKA. Ozagrel hydrochloride, a thromboxane synthase inhibitor, partially attenuated the effect of SP. This indicates that thromboxane contributes to SP-induced pulmonary vasoreactivity. However, ozagrel hydrochloride did not change the effects of NKA. FK224, an NK-1/NK-2 receptor antagonist, abolished both SP- and NKA-induced pulmonary vasoconstriction. This indicates that SP and NKA acted on the pulmonary vasculature through the NK-1 or NK-2 receptor, or both. Papaverine, a smooth muscle relaxant, abolished the effects of SP. The SP-induced increase in lung weight was caused by a rise in pulmonary hydrostatic pressure, especially that caused by pulmonary venoconstriction.

The present study demonstrated a significant variation of liver aldehyde oxidase activity in twelve strains of rats. The highest activity was found with Sea:SD rats and the lowest one with WKA/Sea rats. When assayed using benzaldehyde as a substrate, the difference in the activity between the two strains was 63.5-fold. Little significant differences in Km values of the enzyme were observed among several strains. On the other hand, Western blot analysis for the enzyme suggested that the strains possess different quantities of the enzyme, which are roughly parallel to aldehyde oxidase activity observed in the strains.

Liver aldehyde oxidase (EC 1.2.3.1) was capable of reducing N-arylacetohydroxamic acids, N-hydroxy-2-acetyl-aminofluorene, N-hydroxy-4-acetylaminobiphenyl and N-hydroxyphenacetin, to the corresponding amides in the presence of an electron donor of the enzyme under anaerobic conditions. When supplemented with an electron donor of the enzyme, a significant reduction of N-hydroxy-2-acetylaminofluorene occurred, which was sensitive to an inhibitor of the enzyme. These observations were made with cytosolic fractions prepared from the livers of rabbits, guinea pigs, rats and mice.

The questions of whether oxygen-derived free radicals are induced during preservation of lungs and, if so, how such radicals might relate to reperfusion injury were investigated by means of an isolated canine lung model. Lungs were obtained from 16 mongrel dogs and divided into groups 1 (n = 6), 2 (n = 5), and 3 (n = 5). The lungs of groups 1, 2, and 3 were flushed through the pulmonary artery with Euro-Collins solution alone, the solution with superoxide dismutase (120,000 U/L), and the solution with allopurinol (1 mmol/L), respectively, at 4 degrees C and then stored for 4 hours in the respective solutions at 4 degrees C with clamped bronchi. They were then reperfused for 2 hours by means of an isolated lung model. Lung lipid peroxidation was sequentially determined. The lung functional status was assessed by systolic pulmonary arterial pressure and end-inspiratory airway pressure. The lung edema was assessed by lung wet/dry weight ratio. Lipid peroxidation was induced after 1 hour of preservation and the first 30 minutes of the reperfusion in group 1 and only 2 hours of the reperfusion in group 2, whereas no induction was observed in group 3. Values for systolic pulmonary arterial pressure and end-inspiratory pressure in group 1 were significantly higher than those in group 3 (p0.05). The lung wet/dry weight ratio in group 1 was significantly higher than that in groups 2 and 3 (p0.05). The present results indicate that the administration of free radical scavengers in the preservation may effectively improve conditions for lung transplantation.

To clarify the relationship between cigarette smoking and regulators of vascular tone derived from endothelium, the authors studied the acute and subacute effects of nicotine on endothelin and eicosanoid production by bovine pulmonary artery endothelial cells (BPAEC). The acute effects of nicotine were assessed by measuring the release of endothelin, prostaglandin I2 (PGI2), and thromboxane A2 (TXA2) from BPAEC after their incubation with a medium containing various concentrations of nicotine (10(-9) M - 10(-3) M) for 24 h. The subacute effects were assessed after 1 week's culture of BPAEC in the presence of nicotine (10(-7) M, 10(-3) M). Acute exposure to a high concentration of nicotine (10(-5) M - 10(-3) M) led to a significant inhibition of endothelin production by BPAEC. Nicotine, 10(-3) M, inhibited PGI2 production, but led to release of lactate dehydrogenase. Subacute exposure to nicotine (10(-3) M) also inhibited the production of endothelin and PGI2 by BPEAC. A high concentration of nicotine reduced the production of endothelin and PGI2 by BPAEC, probably due to the cytotoxicity of nicotine. Concentrations of nicotine comparable to the plasma levels of nicotine found in smokers (10(-9) - 10(-6) M) did not affect the release of endothelin and PGI2 from BPAEC. The mechanism of nicotine-induced vasoconstriction may be independent of the release of endothelin or PG derivatives from endothelial cells.

Clinical observations indicate that in heart-lung grafting the heart is less frequently rejected than in heart grafting alone. In addition, our results of allograft experiments indicated that the graft survival period is longer in the former (11.8 +/- 0.8 days versus 6.6 +/- 0.5 days, p0.05), suggesting that the simultaneous grafting of the lung in heart-lung allografts suppressed the rejection of the grafted heart. We assessed the effect of simultaneous lung xenografting, splenectomy, and FK506 treatment on the survival of the xenografted heart in a concordant model. With Wistar rats as recipients and golden hamsters as donors, cardiac survival was compared between en bloc heart-lung and heart heterotopic xenografts. The cardiac survival of heart-lung and heart xenografts was not prolonged by FK506 treatment alone but was prolonged by splenectomy. Splenectomy plus FK506 (1.0 mg/kg/day) showed suppression of antibody production and a remarkable synergistic effect in prolongation (33.2 +/- 7.4 days versus 36.8 +/- 8.1 days) in heart-lung and heart xenografts. Simultaneous lung xenografting significantly shortened the survival period of the xenografted heart in splenectomy plus FK506 (0.5 mg/kg/day) recipients (4.8 +/- 0.8 days versus 9.0 +/- 3.5 days, p0.05), in contrast to the prolongation of the survival period of the grafted heart in heart-lung allografting.

The present study provides the first evidence for delta 13-reduction of 15-ketoprostaglandins (15-keto-PGs) by bovine ocular tissues. The 9,000xg supernatants of cornea, iris, ciliary body, retina, and RPE-choroid except lens exhibited delta 13-reductase activity toward 15-keto-PG E2 and F2 alpha in the presence of NADPH or NADH as an electron donor. Among the tissues tested, the highest activity was observed in ciliary body and iris, followed by RPE-choroid, retina, and cornea. The NADPH- and NADH-linked double bond reductase activities were inhibited by dicumarol, quercitrin, indomethacin and disulfiram, but not by potassium cyanide. NADPH-linked 15-keto-PG F2 alpha delta 13-reductase was purified from bovine iris-ciliary body cytosol by fractionation with ammonium sulfate and high performance liquid chromatography (HPLC) with TSK gel DEAE-5PW, and TSK gel Blue-5PW. The purified enzyme was homogenous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its molecular weight was estimated to be about 57,000 by electrophoresis, and about 55,000 by gel filtration HPLC with Superose 12.

We examined the role of lipopolysaccharide binding protein (LBP) in the airspace and the CD14 receptor on alveolar macrophages in TNF alpha production and neutrophil (PMN) sequestration in lungs induced by intratracheal injection of lipopolysaccharide (LPS). LPS alone (Salmonella minnesota wild-type; 20 ng) or LPS + LBP complex [LPS (20 ng) + rabbit LBP (500 ng); preincubated for 30 min at 37 degrees C] was injected intratracheally into isolated rabbit lungs perfused with lactate-Ringer-albumin solution. Human PMN (5 x 10(7)) were added to the perfusate after 2 hr perfusion. Samples of lung perfusate were collected every 30 min for 180 min, after which bronchoalveolar lavage (BAL) was also performed. TNF alpha concentration in the perfusate and BAL fluid were determined using a bioassay with L-929 fibroblasts. PMN accumulation in the lung was determined by myeloperoxidase assay of the lung homogenate. LPS alone did not significantly increase TNF alpha production or PMN accumulation in lungs, whereas LPS/LBP complex increased TNF alpha concentration in the perfusate and PMN accumulation. Intratracheal injection of anti-CD14 antibody (40 micrograms) with LPS/LBP complex prevented TNF alpha production and subsequent PMN sequestration. We conclude that LBP in the airspace enhances the effect of LPS on TNF alpha production via a CD14-dependent pathway, and this subsequently contributes to PMN sequestration in the lungs. Airspace accumulation of LBP secondary to increased vascular and epithelial permeability may play a critical role in the development of septic shock and lung injury by promoting TNF alpha production via a CD14-dependent mechanism.

Xenotransplantation may be considered a possible solution to the clinical donor-organ shortage. We studied the effects of the removal of preformed natural antibodies against donor tissue by double-filtration plasmapheresis (DFPP) and the administration of a platelet-activating factor antagonist on the prolongation of xenograft survival using the pig-to-dog model. The porcine hearts were perfused with the blood of mongrel dogs. Pairs of animals were divided into four groups: group I (n = 3) was given no pretreatment before perfusion; group II (n = 6) was pretreated by DFPP; group III (n = 3) was given a platelet-activating factor antagonist intravenously at a dose of 0.1 mg/kg 5 minutes before and after perfusion; group IV (n = 5) was pretreated by DFPP and then given a platelet-activating factor antagonist. The titer of natural antibodies was measured by lymphocytotoxicity and hemagglutination. Heart specimens were examined by immunohistochemical staining for deposits of dog antibodies. No immunosuppressive drugs were given in this study. The porcine hearts resumed beating immediately after perfusion in all groups. The mean duration of the graft beating was 17.0 +/- 2.0 minutes for group I, 215.2 +/- 22.6 minutes for group II (p0.001 compared with group I), 18.3 +/- 2.1 minutes for group III, and 317.0 +/- 26.5 minutes for group IV (p0.001 compared with group II). Immunoglobulin (Ig) M, IgG, complements (C3, C4), and fibrinogen were removed significantly from the perfusing blood, and natural antibody titer was scarcely detectable in groups II and IV after DFPP.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical mediators play minor roles in the acute lung injury caused by venous air embolism. The function of PAF in this condition was examined. Eight open chest mongrel dogs, mechanically ventilated, received air at an infusion rate of 0.1 ml/kg per min for 15 min. In addition, four dogs received air under continuous infusion of PAF at 10 ng/kg per min, the other four dogs under pretreatment with the PAF antagonist, CV-3988, at 10 mg/kg. In conclusion, PAF did not mediate pulmonary vasoconstriction but did modulate it and might possibly modulate cardiac deterioration in air embolism.

Sodium nifursyrenate [beta-(5-nitro-2-furyl)-p-carboxystyrene sodium salt, NSA-Na] is an antibacterial nitrofuran which has been widely used for prevention and treatment of bacterial infections in fish in Japan. When NSA-Na was anaerobically incubated with rabbit liver cytosol and 2-hydroxypyrimidine, 1-(p-carboxyphenyl)-5-cyano-3-oxo-1,4-pentadiene (cyano-pentadienone), 1-(p-carboxyphenyl)-5-cyano-3-oxo-1-pentanone (cyano-pentanone), and 1-(p-carboxyphenyl)-5-cyano-3-pentanone (cyano-pentanone) were isolated and identified as the metabolites of the nitrofuran. In addition, when cyano-pentenone and cyano-pentanone were aerobically incubated with the liver preparation and NADPH, 1-(p-carboxyphenyl)-5-cyano-3-hydroxy-1-pentene (cyano-pentenol) and 1-(p-carboxyphenyl)-5-cyano-3-pentanol (cyano-pentanol) were also isolated and identified as the metabolites of the nitrofuran in its further metabolism, respectively. The anaerobic incubation of NSA-Na with rat liver cytosol and 2-hydroxypyrimidine resulted in the formation of cyano-pentadienone and cyano-pentanone. In this case, however, cyano-pentenone was not detectable. On the other hand, when NSA-Na was anaerobically incubated with sea bream liver cytosol and NADPH, the formation of cyano-pentenone, cyano-pentanone, and cyanopentenol, but not cyano-pentadienone, was observed. Furthermore, cyano-pentanone was metabolized to cyano-pentanol by the fish liver preparation with NADPH under aerobic conditions. When NSA-Na was given orally to rabbits, cyano-pentanone, cyano-pentenol, cyano-pentanol, and beta-(acetamido-2-furyl)-p-carboxystyrene (acetamidofuran) were identified as the urinary metabolites of the nitrofuran.(ABSTRACT TRUNCATED AT 250 WORDS)