Amelioration of neurovascular deficits in diabetic rats by a novel aldose reductase inhibitor, GP-1447: minor contribution of nitric oxide.

The effects of a novel potent aldose reductase inhibitor, GP-1447 [3-[(4,5,7-trifluorobenzothiazol-2-yl)methyl]-5-methylphenylace tic acid] on the sciatic nerve blood flow in streptozotocin-induced diabetic rats were examined. Blood flow was analyzed in terms of mass, i.e. the volume of blood in tissue, and of velocity, i.e. the velocity of the blood flow. In diabetic rats, a 63% decrease in blood flow due to a decrease in velocity was observed. The blood mass in the same animals fluctuated, thereby increasing its range of values. Treatment with GP-1447 at a dose of 30 mg/kg per day for 4 consecutive weeks following a 3-week period without treatment ameliorated the reduced blood flow by 51%, and was accompanied by a recovery of velocity. The increase in the range of blood mass values was reversed by treatment with GP-1447. The restoration of the range of blood mass values, but not that of the blood flow, by GP-1447 was blocked by treatment with the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine. Motor nerve conduction velocity (MCV) changes in parallel with blood flow values, while it is inversely proportionate to alterations in the range of blood mass values. It is suggested that the observed beneficial effect of GP-1447 on blood flow is involved in the restoration of decreased MCV in diabetes. It would appear that GP-1447-induced amelioration of neurovascular defects is not mediated solely by the improvement of the NO system.

A karyotype study on the Drosophila robusta species-group (Diptera: Drosophilidae).

The karyotypes of the Drosophila robusta species-group, especially its species-subgroup okadai, were studied by using brain cell-Giemsa staining method. Drosophila gani possessed a diploid number of 12 chromosomes, comprising 4 pairs of metacentric, 2 pairs of acrocentric chromosomes, and both D. okadai and D. neokadai did 2n = 12, with 3 pairs of metacentrics, 2 pairs of acrocentrics and 1 pair of microchromosomes. The okadai subgroup characteristically retained a middle-sized acrocentric X chromosome, which implies that this subgroup may occupy an ancestral position for the robusta group.

Molecular phylogeny of twelve Asian species of epilachnine ladybird beetles (Coleoptera, Coccinellidae) with notes on the direction of host shifts.

We determined the nucleotide sequences of a part of the mitochondrial cytochrome c oxidase I gene (1,000 bp) for twelve species of Asian phytophagous ladybird beetles belonging to the genus Epilachna, and constructed molecular phylogenetic trees for ten "Henosepilachna" species, using two "Epilachna" species as outgroups. Based on the suggested phylogenetic trees, we discussed taxonomic issues and the direction of host shift in these epilachnines.

Two Cryptic Species of the Phytophagous Ladybird Beetle Epilachna vigintioctopunctata (Coleoptera: Coccinellidae) Detected by Analyses of Mitochondrial DNA and Karyotypes, and Crossing Experiments.

Analyses of a part of mitochondrial cytochrome c oxidase I gene sequences (645 bp) for seventeen individuals of Epilachna vigintioctopunctata (Fabricius) from eight localities in east and southeast Asia revealed that the populations are divided into two genetically distinct groups (Chiba, Tokyo, Naha, Iriomote, Bangkok vs. Kuala Lumpur, Padang, Bogor). The number of nucleotide substitutions between sequences of different groups was 57-60, while that between sequences within each group was 1-8. Karyotypes of the two groups were also distinctly different. Crossing experiments showed that there exist strong postmating barriers between the two groups: eggs obtained from between-group crossings usually did not hatch, whereas more than 90% of eggs from within-group crossings hatched. It is concluded that E. vigintioctopunctata, a notorious pest of solanaceous crops in Asia and Australia, is composed of at least two reproductively isolated biological species that probably occupy different geographic ranges.

An enzymatic assay for myo-inositol in tissue samples.

An enzymatic assay for myo-inositol (MI) was modified. The method is based on the oxidation of MI by NAD(+)-dependent MI dehydrogenase, coupled to reoxidation of NADH by iodonitrotetrazolium chloride and diaphorase. The resultant formazan is measured spectrophotometrically. In order to remove interference by glucose, preliminary phosphorylation of glucose by hexokinase was employed before the above reaction. The assay is quantitative for MI in amounts ranging from 1 to 20 nmol. This method gives a negligible blank, even in the measurement of rat serum. The reduced MI content in the sciatic nerve and lens of streptozotocin-induced diabetic rats recovered in a dose-dependent manner by treatment with a novel potent aldose reductase inhibitor, GP-1447 ¿3-[(4,5, 7-trifluorobenzothiazol-2-yl)methyl]-5-methylphenylacetic acid¿.

[Study on genetic polymorphism of isozyme in natural populations of Drosophila virilis].

Using techniques of polyacrylamide slab electrophoresis and agarose electrophoresis, we have detected genetic variation at 6 loci which coding for enzymes in 4 local samples from natural population of Drosophila virilis. We found 50% of the loci detected are polymorphic, depending on the criterion of polymorphism used. An individual is heterozygotes on the average at 27.13% of its loci. The amount of genetic variation fluctuates widely from locus to locus. At Est-alpha, Est-beta, Amy, most of the individuals are heterozygotes. At the other extreme , Mdh, aGpdh, Acph, few individuals are heterozygotes. For Mdh, we have measured the thermostability at 53 degrees C. No more genetic variation was found. We have measured the amount of genetic differentiation between different local populations. The result showed that there is no relationship between geographical distance and genetic distance. The results are discussed in the light of the continuing controversy over selection and natural theories of genetic variation. We think that both selection and stochastic processes must operate simultaneously in most systems.

[Transfer of skin conductance responses through stimulus equivalence classes].

This study investigated the transfer of classically conditioned skin conductance responses through stimulus equivalence classes. Fourteen subjects were taught 4 stimulus relations, A1-->B1, A1-->C1, A2-->B2, and A2-->C2 through matching-to-sample training. In subsequent matching-to-sample tests, 13 subjects demonstrated the formation of two stimulus equivalence classes, "A1, B1, and C1" and "A2, B2, and C2." They then received differential conditioning, in which Stimulus B1 was followed by electric shocks, while B2 was not followed by the shocks. Subsequent transfer tests revealed that skin conductance responses for Stimulus C1 were greater than those for C2, suggesting the transfer of conditioned responses through the stimulus equivalence classes.

Mitochondrial DNA polymorphisms in the two subspecies of Drosophila sulfurigaster: relationship between geographic structure of population and nucleotide diversity.

Recent empirical and theoretical studies on mitochondrial DNA (mtDNA) variation in higher animals have suggested that the extent of mtDNA polymorphism is largely affected by spatial population subdivision. To examine this we studied mtDNA polymorphism in two subspecies of Drosophila sulfurigaster: D. s. albostrigata and D. s. bilimbata. Drosophila sulfurigaster albostrigata is mainly distributed on the mainland of Southeast Asia. In contrast, D. s. bilimbata forms discontinuous populations on many islands scattered in the Pacific Ocean. Because of the difference in their distribution patterns, the two subspecies are thought to be different in the extent of spatial population subdivision. mtDNA was isolated from greater than 50 isofemale strains for each subspecies and were analyzed by eight restriction endonucleases. Nucleotide diversity within a population was higher in D. s. albostrigata than in D. s. bilimbata. However, haplotype diversity was 1.6 times greater in D. s. bilimbata (0.85) than in D. s. albostrigata (0.53). The large difference in overall heterogeneity was attributed to the difference in interpopulational nucleotide diversity. For the two subspecies the proportion of interpopulational gene diversity in a subdivided population was calculated to be 0.54 in D. s. bilimbata and 0.40 in D. s. albostrigata. These observations indicate that spatial population subdivision is a major factor in determining mtDNA polymorphism in these subspecies. The extent of mtDNA divergence between the subspecies was very high. The average nucleotide divergence between them was 7.6%, which is almost the interspecific level reported for other Drosophila species. The cause of the high degree of mtDNA divergence is discussed.

The origins of the Japanese mainland population of Drosophila albomicans.

Drosophila albomicans is a species widely distributed but mainly in Southeast Asia. In its traditional distribution, there are substantial genetic differentiations among three geographic areas, Southeast Asian continent, Taiwan and Nansei islands. In the last decade, however, this species has invaded the Japanese mainland and is now spreading its distribution area to western Japan. In this study, variations of chromosomal arrangements, allozymes and sex ratio in F2 hybrids with D. nasuta were examined to identify the origins of the newly colonizing population. The results strongly suggest that the origins of Japanese mainland population can be found in Taiwan.

Phylogenetic position of the subgenus Lordiphosa of the genus Drosophila (Diptera: Drosophilidae) inferred from alcohol dehydrogenase (Adh) gene sequences.

We analyzed the phylogenetic relationship between the species of Lordiphosa and other Drosophilidae using alcohol dehydrogenase (Adh) gene sequences. The phylogenetic trees consistently show that the four species Drosophila kurokawai, D. collinella, D. stackelbergi, and D. clarofinis, which include three species groups of Lordiphosa, form a monophyletic clade. This clade is placed as a sister group to the willistoni and saltans groups of Sophophora. On the other hand, three species of Lordiphosa, D. tenuicauda, D. pseudotenuicauda, and D. acutissima, all of which belong to the tenuicauda group, are not shown to be related to the major Lordiphosa lineage. In the phylogenetic trees, these species are included into the clade comprised of Drosophila and Hirtodrosophila, although it remains uncertain whether the tenuicauda group is a monophyletic group or not. These results indicate that Lordiphosa is polyphyletic and that most of the members of the subgenus have a close relationship to the neotropical groups of Sophophora. The above conclusion is compatible with the hypothesis of Okada (Mushi [1963] 37:79-100) and Lastovka and Máca (Acta Ent Bohemoslov [1978] 75:404-420) that Lordiphosa is most closely related to Sophophora; in contrast, our results contradict the hypothesis of Grimaldi (Bull Am Mus Nat Hist [1990] 197:1-139) that Lordiphosa is a sister group to the genus Scaptomyza.

Evolution of the noncoding regions in Drosophila mitochondrial DNA: two intergenic regions.

The sequences of the mitochondrial DNA (mtDNA) segment containing the two intergenic regions were determined for six species belonging to the Drosophila immigrans species group and compared to the corresponding segments of Drosophila species which had been studied previously. We found remarkable differences in the evolutionary rates of the two intergenic regions. The Intergenic I region, which lies between the tRNA(gln) and the tRNA(ile) genes, was found to be highly conserved in terms of both size (30 ntp) and nucleotide sequence among the species studied. In contrast, the sequences of the Intergenic II region, which lies between the tRNA(f-met) and the tRNA(ile) genes, showed considerable variation. The size of the Intergenic II region ranged from 0 to 88 ntp, and accurate alignment was possible only among sequences from geographical strains or very closely related species in the nasuta species subgroup. The observed differences in conservation of the two mtDNA intergenic regions are discussed in light of functional constraints on mtDNA sequences.

A simplified method for reading hemagglutinations on a flat-bottom microtitration plate in the mouse H-2 assay.

Kaliss' method for determining hemagglutination titer of mouse alloantiserum was modified to a simpler form. It became possible to read the hemagglutination directly on a flat-bottom microtitration plate using an inverted type microscope. Sensitivity of the modified method was almost the same as that of the original one. This method seems to be useful especially for the larger scale assay of H-2 specificities.

Analysis of mitochondrial DNA in two species of the bipectinata species complex of Drosophila.

Drosophila bipectinata and D. malerkotliana are members of the bipectinata species complex of the ananassae subgroup in the melanogaster species group of Drosophila. The mtDNA from 18 isofemale strains of these species was analyzed, using 13 restriction endonucleases. Altogether, eight mtDNA haplotypes were detected, of which the haplotype 1B was shared by the two species. Restriction cleavage map of mtDNA of these species was constructed. The net nucleotide substitutions per site calculated between these species was found to be 0.0002. This value appeared to be relatively much lower than expected at the interspecific level, even lower than that reported between two subspecies of Drosophila despite both being good species. This extreme closeness of their mtDNAs is discussed in the light of recent findings.

Possible mechanisms of sudden death and hemoconcentration induced by endothelin-1 and big endothelin-1 in mice.

We investigated the profiles of sudden death and hemoconcentration induced by endothelin-1 (ET-1) and big endothelin-1 (big ET-1) in mice using various compounds as pharmacological tools. In ET-1-induced sudden death (5 nmol/kg, i.v.), pretreatment with the Ca(2+)-channel blockers, diltiazem, nifedipine or verapamil at a dose of 2 mg/kg, i.v. significantly inhibited the mortality and prolonged the latency to death. These Ca(2+)-channel blockers, however, failed to inhibit the rise in hematocrit (Ht), namely hemoconcentration, induced by ET-1 (2.5 nmol/kg). A beta-adrenoceptor agonist, isoproterenol (1 mg/kg) tended to prolong the latency, whereas, a beta-adrenoceptor blocker, propranolol (2 mg/kg), and an alpha- and beta-adrenoceptor blocker, labetalol (5 mg/kg), aggravated the sudden death. Esculetin (10 mg/kg) and fenbufen (10 mg/kg), which are enzyme inhibitors in the arachidonate cascade, prevented only the hemoconcentration. Anti-arrhythmic drugs, lidocaine (1 mg/kg) and disopyramide (20 mg/kg) did not improve any parameters. Big ET-1 also caused sudden death (20 and 25 nmol/kg, i.v.) and hemoconcentration (10 nmol/kg, i.v.). Of several proteinase inhibitors, only a metalloproteinase inhibitor, phosphoramidon (2 mg/kg i.v.), prevented the sudden death and the hemoconcentration induced by big ET-1 but not by ET-1. Ca(2+)-channel blockers exerted their protective effects only when a lower dose of big ET-1 was employed. These results indicate that the sudden death caused by both peptide is mainly due to myocardial ischemia and respiratory disorder, and that hemoconcentration caused by them is due to their vasoconstrictor action but to their effects on the vascular permeability via secondary endogenous factors.

Novel and potent aldose reductase inhibitors: 4-benzyl- and 4-(benzothiazol-2-ylmethyl)-3,4-dihydro-3-oxo-2H-1,4-benzothiazine-2-ac etic acid derivatives.

A number of 1,4-benzothiazine-2-acetic acid derivatives (1, 2 and 3) and their bioisosteres (15b, 16, 18 and 20b) were synthesized and evaluated in vitro for the ability to inhibit aldose reductase (AR) in porcine lens. The compounds which exhibited potent activity in vitro were also assayed in vivo for inhibitory activity against sorbitol accumulation in the erythrocytes, sciatic nerve and lens of streptozotocin-diabetic rats. The 4-(substituted benzothiazol-2-ylmethyl)-1,4-benzothiazine-2-acetic acid derivatives (2 and 3) showed more potent AR inhibitory activity than did the 4-(4-bromo-2-fluorobenzyl)-1,4-benzothiazine-2-acetic acid derivatives (1). 4-(4,5,7-Tri-fluorobenzothiazol-2-ylmethyl)-3,4-dihydro-3- oxo-2H-1,4-benzothiazine-2-acetic acid (2q, SPR-210) showed not only a potent AR-inhibitory activity in vitro (IC50 9.5 x 10(-9) M) but also a significant reduction in sorbitol accumulation in rat sciatic nerve (ID50 0.1 mg/kg) and lens (ID50 9.8 mg/kg). Optical resolution of the racemic SPR-210 was achieved by means of a diastereomer salt method using (-)-brucine. The biological activities of both enantiomers, (+)- and (-)-SPR-210, were comparable to that of the racemate.

Possible involvement of different mechanisms in sudden death induced by endothelin-1 and big endothelin-1.

The effects of diltiazem and phosphoramidon on sudden death induced by endothelin (ET)-1 and by big ET-1 were compared in rodents. Diltiazem (2 mg/kg, i.v.) remarkably diminished the lethal toxicity of ET-1 with a reduction in the extent of the rise in plasma immunoreactive ET-1-like activity (IR-ET-1), tissue IR-ET-1 accumulation in the heart and the rise in plasma potassium concentration. In big ET-1-induced lethality, diltiazem only slightly prolonged the latency and did not reduce the mortality. Although diltiazem moderately inhibited the rise in plasma IR-ET-1 and potassium concentration in these mice, it did not affect the accumulation of IR-ET-1 in the heart, lung or kidney. Phosphoramidon (2 mg/kg, i.v.) decreased the lethality of big ET-1 with the decrement in elevation of IR-ET-1 in the heart, lung and plasma as well as with the decrease in plasma potassium concentration, but it failed to improve any parameters in ET-1-induced lethality. In anesthetized rats, ET-1 (5 nmol/kg, i.v.) elevated ST-segment of electromyocardiograms, and diltiazem (2 mg/kg, i.v.) significantly reversed this change. Big ET-1 (25 nmol/kg, i.v.) also induced the ST-segment elevation, which was significantly inhibited by phosphoramidon but not by diltiazem. These findings suggest that accumulation of ET-1 in the heart, which may lead to lethal cardiac ischemia, is an important factor in the lethality of ET-1, while additional factors (such as hemoconcentration and bronchoconstriction) may be involved in big ET-1-induced lethality.

Frequency distribution of histocompatibility-2 antigenic specificities in the Japanese wild mouse genetically remote from the European subspecies.

Fifty-eight Japanese wild mice, Mus musculus molossinus, collected from twenty-eight localities were surveyed for twelve H-2 antigens using the haemagglutination method. Significantly higher frequencies of H-2.3 and H-2.5 specifities and relatively lower frequencies of the other public specificities were observed. This was confirmed by examining four specificities, H-2.3, 5, 13 and 23, in 370 mice. Quantitative absorption of the alloantisera by erythrocytes and spleen lymphocytes of molossinus mouse revealed definite absorption of H-2.3, 5 and 8 antigens, though their antigenic strength was apparently weaker than the controls, B10 congenic mice. Comparative assay of the strength of H-2.5 antigen in either homozygous and heterozygous conditions distinctly eliminated the possibility of gene-dose effect for the reduced strength of the public antigens in molossinus mouse. To explain this, divergence time between molossinus and domesticus was computed based on the allelic frequency data already reported on ten loci in both subspecies. It is roughly 1.5 x 10(6) years, which could allow for the occurrence of considerable genetic changes in many public antigens, except those conserved through selection.

Substituted 1,8-naphthyridin-2(1H)-ones as selective phosphodiesterase IV inhibitors.

New substituted 1,8-naphthyridin-2(1H)-ones have been found to exhibit highly selective phosphodiesterase (PDE) IV inhibition. These compounds inhibited polymorphonuclear leukocyte activation induced by N-formylmethionyl-leucyl-phenylalanine and caused relaxation of isolated guinea pig trachea precontracted by histamine or leukotriene D4. In anesthetized guinea pigs, presensitized with the antigen, these compounds also alleviated airway constriction induced by the antigen. Since these compounds differ in their chemical structure compared with theophylline and other PDE IV inhibitors so far reported and some of them have been shown to be well tolerated in acute toxicity studies, they will provide a new tool for investigating the possible relationship between PDE IV inhibition and anti-asthmatic activity.

Effects of a novel potent aldose reductase inhibitor, GP-1447, on aldose reductase activity in vitro and on diabetic neuropathy and cataract formation in rats.

GP-1447 {3-[(4,5,7-trifluorobenzothiazol-2-yl) methyl]-5-methylphenylacetic acid}, a novel aldose reductase (AR) inhibitor, exhibited highly potent and specific inhibition of AR activity from human placenta, human muscle, porcine and rat lens with IC50 values ranging from 3 to 10 nM. Lineweaver-Burk plots revealed non-competitive inhibition between DL-glyceraldehyde or beta-NADPH and inhibition of AR by GP-1447. In contrast to epalrestat, AR activity inhibited by GP-1447 did not recover after dialysis for 24 hr. Administration of GP-1447 to streptozotocin (STZ)-induced diabetic rats for 5 days beginning 1 week after STZ injection effectively inhibited the accumulation of sorbitol in the sciatic nerve, lens and retina with ED50 values of 0.25, 1.6 and 2.9 mg/kg/day, respectively. The motor nerve conduction velocity (MCV) in STZ-induced diabetic rats was significantly decreased 4 weeks after the induction of diabetes. Treatment with GP-1447 for the following 2 weeks dose-dependently restored the decreased MCV with an ED50 value of 0.28 mg/kg/day. Administration of GP-1447 (3 and 15 mg/kg/day for 12 weeks beginning 3 days after STZ injection) completely prevented cataract formation and was accompanied by marked inhibition of sorbitol accumulation in the lens. Furthermore, partial reversibility of cataract formation and morphological changes of the lens was observed in diabetic rats treated with 15 mg/kg/day of GP-1447 for 5 weeks beginning 8 weeks after the induction of diabetes. From these results, GP-1447 would be expected to exert potent ameliorating effects on some diabetic complications. Potent inhibition of cataract formation will be one of the characteristics of this compound.

Inhibitory activities of metal chelators on endothelin-converting enzyme. II. In vivo studies.

The effects of metal chelators on endothelin (ET)-converting enzyme (ECE) activity in vivo were examined. Three compounds, (2,3-dimercapto-1-propanol (DMP), toluene-3,4-dithiol (TDT) and 8-mercaptoquinoline (8-MQ)), which inhibited ECE in in vitro studies, exhibited inhibitory activity towards big ET-1-induced sudden death in mice, while EDTA did not. Similar results were obtained in big ET-1-induced hypertension. Big ET-1-induced hemoconcentration was inhibited by pretreatment with 8-MQ or EDTA but not with DMP or TDT. The elevation of immunoreactive ET-1 (IR-ET-1) in plasma after administration of big ET-1 was inhibited by pretreatment with the three compounds but not by EDTA. On the other hand, no chelator inhibited the elevation of IR-ET-1 in lung tissue after injection of big ET-1. Taking into consideration the in vitro results, more selective chelating activity of the compounds towards Zn2+ rather than Ca2+ and Mg2+ may contribute to the inhibition of big ET-1-induced responses in vivo. The ET-1 formation involved in big ET-1-induced hemoconcentration may have different physiological characteristics from that involved in big ET-1-induced sudden death or hypertension.