Different roles of nitric oxide synthase-1 and -2 between herpetic and postherpetic allodynia in mice.

We investigated using the mice role of nitric oxide synthase (NOS) in the spinal dorsal horn in herpetic and postherpetic pain, especially allodynia, which was induced by transdermal inoculation of the hind paw with herpes simplex virus type-1 (HSV-1). The virus inoculation induced NOS2 expression in the lumbar dorsal horn of mice with herpetic allodynia, but not postherpetic allodynia. There were no substantial alternations in the expression level of NOS1 at the herpetic and postherpetic stages. Herpetic allodynia was significantly inhibited by i.p. administration of the selective NOS2 inhibitor S-methylisothiourea, but not the selective NOS1 inhibitor 7-nitroindazole. NOS2 expression was observed around HSV-1 antigen-immunoreactive cells. On the other hand, postherpetic allodynia was significantly inhibited by i.p. administration of 7-nitroindazole, but not S-methylisothiourea. The activity of reduced nicotinamide adenine dinucleotide phosphate diaphorase, an index of NOS1 activity, significantly increased in the laminae I and II of the lumbar dorsal horn of mice with postherpetic allodynia, but not mice without postherpetic allodynia. The expression level of NOS1 mRNA in the dorsal root ganglia was similar between mice with and without postherpetic allodynia. The results suggest that herpetic and postherpetic allodynia is mediated by nitric oxide in the dorsal horn and that NOS2 and NOS1 are responsible for herpetic and postherpetic allodynia, respectively. It may be worth testing the effects of NOS2 and NOS1 inhibitors on herpetic pain and postherpetic neuralgia in human subjects, respectively.

A phylogenetic view on species radiation in Apodemus inferred from variation of nuclear and mitochondrial genes.

Species of field mice (genus Apodemus) are the most common rodents inhabiting woodlands and forests of the Palaearctic region. We examined the cytochrome b (cyt b) gene in mitochondrial DNA (1140 bp) and the interphotoreceptor retinoid binding protein (IRBP) gene in nuclear DNA (1152 bp) in nine species of Apodemus. Based on the genetic variation, the nine species were grouped into four lineages: (1) Agrarius group (A. agrarius, A. peninsulae, A. semotus, and A. speciosus), (2) Argenteus group (A. argenteus), (3) Gurkha group (A. gurkha), and (4) Sylvaticus group (A. alpicola, A. flavicollis, and A. sylvaticus). It was shown that these four lineages diverged within a short period of evolutionary time, suggestive of a radiation event. Soon after the radiation, the Agrarius group was likely to have differentiated again into the species lineages simultaneously. In contrast, the European clade, the Sylvaticus group, radiated rather recently. The relative ratio of the extent of sequence divergence among the four main lineages to that among the members of the subfamily Murinae (including Mus and Rattus) was calculated to be 72.4% in the cyt b gene with transversional substitutions, and 58.5% in the IRBP gene with all substitutions, using the Kimura two-parameter method. The value for the three European lineages was 27.6% in the cyt b gene and 12.3% in the IRBP gene. These results may have a correlation with the notion that deciduous broadleaf forests remained in Central East Asia through the late Tertiary to the present, while those in Europe to a large extent had disappeared by the Pliocene.

Heparin and heparan sulfate enhancement of the inhibitory activity of plasminogen activator inhibitor type 1 toward urokinase type plasminogen activator.

To study effects of glycosaminoglycan on the interaction between two chain urokinase type plasminogen activator (tcu-PA) (EC 3.4.21.31) and plasminogen activator inhibitor type 1 (PAI-1) the second order rate constant (k1) between high molecular weight tcu-PA and active recombinant prokaryotic PAI-1 (rpPAI-1) was determined employing a continuous method using chromogenic substrate S-2444 either in the presence or absence of various kinds of glycosaminoglycans. k1 was (5.9 +/- 1.6).10(6)/mol per s in the absence of effector molecule, and following addition of heparin (1.0 U/ml) k1 was enhanced to (3.22 +/- 0.73).10(7). A significant enhancement of k1 was also obtained by heparan sulfate (1.87 +/- 0.25).10(7). Dermatan sulfate or chondroitin sulfate did not show a significant effect on k1 although a slight decrease was obtained by mono-dextran sulfate (4.2 +/- 1.2).10(6). The intrinsic fluorescence of rpPAI-1 was shown to be slightly increased following addition of heparin (1.49 +/- 0.22%, n = 6), suggesting that heparin may enhance the inhibitory activity of PAI-1 toward tcu-PA both by a template mechanism and by a modification of PAI-1 structure.

The significant enhancement of fibrinolysis by calcium ion in a cell free system: the shortening of euglobulin clot lysis time by calcium ion.

We investigated the roles of calcium ion (Ca2+) on euglobulin clot lysis time (ECLT) and found that the physiological concentration of Ca2+ significantly (4-5 times) shortened ECLT. The shortening was observed at the concentration of Ca2+ higher than 1.5-2.0 mM. Other divalent cations such as Mg2+, Zn2+ or Mn2+ didn't change ECLT. Anti-tPA antibody or plasminogen activator inhibitor-1 prolonged ECLT in the absence of Ca2+, whereas they had no effect on ECLT shortened by Ca2+. C1 inactivator also had no effect. When barium absorbed plasma was employed, the shortening of ECLT by Ca2+ wasn't observed, whereas it was recovered by the readdition of barium absorbed fraction. When factor X deficient plasma was employed, the shortening of ECLT by Ca2+ was also not observed. Thus, Ca2+ enhances fibrinolysis in a cell free system by a novel pathway in which the presence of factor X is prerequisite.

Electric-foot-shock induced the suppression of fibrinolytic activity in rats.

Electric-foot-shock was given to rats to initiate constant mental stress and its effect on fibrinolytic activity was analyzed. After the termination of electric-foot-shock which was given for an hour, euglobulin clot lysis time in the stressed group significantly prolonged than those in the control group. tissue plasminogen activator activity was also significantly lower in the stressed group. These effects lasted at least for an hour and returned to the control values 24 hours after the stress. Whole blood serotonin levels, which mainly show serotonin contents in platelets, were higher in the stressed group. A negative correlation between whole blood serotonin and tPA activity in the stressed group was obtained. These results suggest that prolonged mental stress impairs fibrinolysis by decreasing tPA activity with a concomitant increase of serotonin contents in platelets.

Foot shock-induced changes in blood and brain serotonin and related substances in rats.

The effects of electric foot shock on peripheral and central serotonergic systems in rats have been studied. We have focused on the time course alterations with particular attention being paid to changes in 5-HT, 5-HIAA, tryptophan concentrations and 5-HIAA/5-HT ratios in blood and various parts of the brain, observed within 1 h following stress application. Blood and brain (7 regions) samples were taken immediately after electric foot shock, 30 min, 1 and 24 h later. In the blood stress induced a rise in tryptophan level as well as rises in 5-HT, 5-HIAA levels and 5-HIAA/5-HT ratio within 1 h following stressful treatment. Tryptophan concentration was found to be increased in every part of the brain within 1 h after electric foot shock application. In striatum it remained higher even after 24 h. 5-HT level showed a significant rise only in medulla, while hypothalamus was the sole region where a fall in 5-HT was found. In other parts of the brain 5-HT level remained unaffected by stress. 5-HIAA content increased in almost every brain area studied except cerebellum and striatum. 5-HIAA/5-HT ratios shared the same pattern of changes. Briefly, foot shock altered 5-HT turnover in various brain regions, in particular within the first hour following stress application, whereas delayed response to stress was rarely observed. Increased brain tryptophan level seems to be necessary to cope with the enhanced 5-HT metabolism caused by stress, reflecting as a rise in 5-HIAA concentration and 5-HIAA/5-HT ratio.

Medroxyprogesterone acetate (mpa) increases pai-1 secretion from huvec and elevates the plasma-levels of pai-I in-vivo.

Plasma levels of tissue type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) in patients administered with medroxyprogesterone acetate (MPA) (800 mg/day) or tamoxifen (20 mg/day) were assayed. Active free PAI-1 levels of patients given MPA were significantly higher than those either given tamoxifen or post adjuvant therapy, t-PA antigen levels were not different among the three groups. In in vitro study, treatment of cultured human umbilical vein endothelial cells (HUVEC) with MPA (10(-6)-10(-8) M) decreased PAI-1 levels significantly with a trend to decrease in t-PA levels.

The potential role of platelet PAl-1 in t-PA mediated clot lysis of platelet rich plasma.

The potential role of platelets in platelet rich plasma clot lysis induced by tissue plasminogen activator (t-PA) was investigated. At the various concentrations of both single chain t-PA (sct-PA) and two chain t-PA (tct-PA) (1.5nM, 3nM, and 6nM), we compared the t-PA mediated lysis time of platelet rich plasma clot (PRP-clot) with that of platelet poor plasma clot (PPP-clot). At the concentrations ranged from 1.5 to 6 nM of both types of t-PA, the clot lysis time of PRP-clot was longer than that of PPP-clot. This elongation was more significant in the tct-PA induced clot lysis than that in the sct-PA induced clot lysis. At the concentration of 3nM of tct-PA, the lysis time of PRP-clot was longer by a factor of 30% in comparison with that of PPP-clot. When the release and the aggregation of platelets were blocked by prostaglandin E1 (PGE1) and theophylline in this experiment, the lysis time of PRP-clot was essentially the same as that of PPP-clot. We then measured the antigen levels of total PAI-1 and t-PA-PAI-1 complex in the lyzed solutions of PRP-clot and PPP-clot to analyse the possible effect of plasminogen activator inhibitor-1 (PAI-1) present in platelets. Most of PAI-1 in a PPP-clot lyzed sample existed as t-PA-PAI-1 complex. In the lyzed solution of PRP-clot, however, the antigen levels of both total PAI-1 and t-PA-PAI-1 complex were significantly higher than those in PPP-clot, and larger amounts of PAI-1 existed as free PAI-1 which possesses activity. These data suggest that at least certain amounts of PAI-1 in platelets exist as an active form and inhibits t-PA activity resulting in the prolongation of the clot lysis time. Activation of platelets, therefore, seems to play an important role in the platelet rich plasma clot lysis induced by t-PA.

Impaired fibrinolytic activity induced by ingestion of butter: effect of increased plasma lipids on the fibrinolytic activity.

To investigate the effects of the increased plasma lipid level on fibrinolysis, we measured the levels of fibrinolytic components in serially obtained plasma samples from healthy volunteers after the intake of different amounts of butter. Plasma triglyceride level increased significantly after butter intake compared to the control group. Eight hours after the intake of 100g of butter, plasminogen activator inhibitor 1 (PAI-1) level in plasma was significantly higher and euglobulin clot lysis time was significantly prolonged compared to those of the control group. There was no effect on plasma tissue plasminogen activator level. These results suggest that the temporary increase in plasma triglyceride level induced high PAI-1 level, resulting in impaired fibrinolytic activity. The effect of temporary hyperlipidemia on platelet function was also analyzed and revealed that the response of platelets to ADP and collagen was lower in the butter intake group compared to those of the control.

Impaired fibrinolysis in hypertension and obesity due to high plasminogen activator inhibitor-1 level in plasma.

In order to elucidate the influence of the risk factors of coronary heart disease on the fibrinolytic activity, relationships between blood pressure, body mass index (BMI), plasma lipoprotein (a) (Lp(a)) level and the plasma levels of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) were analyzed in the subjects with mild hypertension. Systolic blood pressure showed a positive correlation with total PAI-1 and free PAI-1. Diastolic blood pressure showed no correlation with these proteins involved in the fibrinolytic system. BMI had a positive correlation with total PAI-1, free PAI-1 and euglobulin clot lysis time (ECLT). Plasma Lp(a) level showed correlation with neither blood pressure nor fibrinolytic parameters, but it showed weak negative correlation with body mass index (BMI). These results suggest that high blood pressure and obesity tend to increase free PAI-1 which reduces fibrinolytic activity. Lp(a), however, seems not to influence directly the fibrinolytic system but may work to decrease fibrinolytic activity only in conjunction with other risk factors. The effects of daily drinking of alcohol and smoking on the fibrinolytic system were also investigated in the present study and we obtained the results that habitual drinking increased plasma levels of both tPA and PAI-1 whereas smoking did not affect fibrinolytic activity. These results suggest that risk factors for coronary heart disease such as hypertension and obesity are closely related to the impaired fibrinolysis.

Serotonergic measures in blood and brain and their correlations in rats treated with tranylcypromine, a monoamine oxidase inhibitor.

Tranylcypromine, a monoamine oxidase inhibitor, was administered to male Wistar rats in order to investigate its effects on blood and brain serotonin related substances after 1, 4, and 24 h following injection and possible relations between serotonergic measures in central nervous system and periphery. The dose of the drug tested was responsible for an increase in blood serotonin with a simultaneous fall in its metabolite 5-hydroxyindoleacetic acid (5-HIAA) compared to either pretreatment or control values. These changes were the most marked after 4 and 24 h following tranylcypromine injection. Almost all brain areas studied (cerebellum, medulla, hypothalamus, striatum, midbrain, hippocampus, and cortex) were to be affected by monoamine oxidase inhibitor treatment. They exhibited a rise in serotonin content starting from 1 h after drug administration and lasted in many parts of the brain up to 24 h, which was accompanied by a parallel fall in 5-HIAA level. All these changes were significant when compared to baseline and control values. Alterations in blood serotonin correlated positively with changes in brain serotonin and negatively with brain 5-HIAA, while the opposite pattern of correlations was found regarding blood 5-HIAA and the content of serotonin and 5-HIAA in various brain areas studied. This pattern of correlations speaks in favor of an existence of mutual relations between blood and brain serotonin related substances. Our results suggest that blood serotonin and 5-HIAA may serve as an index of monoamine oxidase inhibitor action on the central serotonergic system.

Correlation between serotonergic measures in cerebrospinal fluid and blood of subhuman primate.

The relationship between the concentration of serotonin (5-hydroxytryptamine; 5-HT), its precursor; tryptophan (Trp) and the main metabolite 5-hydroxyindoleacetic acid (5-HIAA) in cerebrospinal fluid (CSF) and blood of monkey have been studied. 5-HT, Trp and 5-HIAA underwent circadian changes in both CSF and blood. Significant correlations were found between 5-HT, 5-HIAA and Trp in CSF and blood. The significance of these findings and their relationship to the use of peripheral serotonergic system as a functional model of the central nervous system are discussed.

Plasminogen activator system in human breast cancer.

We measured antigen levels of 2 kinds of plasminogen activator, tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (UK), as well as those of their primary inhibitors, type-I plasminogen activator inhibitor (PAI-1) and type-2 plasminogen activator inhibitor (PAI-2), in tissue extracts from benign and malignant breast tumors. Tumor tissue samples from 40 fibroadenomas and 40 breast cancers were examined. t-PA antigen levels were the same in the 2 groups. Malignant tumors contained higher levels of UK antigen than did benign tumors. In the case of breast cancer, UK antigen levels of tumors with axillary lymph-node involvement were significantly higher than those of tumors without lymph-node involvement. PAI-1 and PAI-2 antigen levels of breast-cancer tissue samples were higher than those of fibroadenoma samples. PAI-1 antigen levels of carcinomas with lymph-node involvement were also significantly higher than those of carcinomas without node involvement. PAI-2 antigen levels, on the contrary, were higher in carcinomas without node involvement. UK, PAI-1 and PAI-2 antigen levels are potentially excellent independent factors for prediction of the metastatic potential of breast cancers.

Relationship between serotonergic measures in periphery and the brain of mouse.

Circadian rhythm and the relationship between the concentration of serotonin (5HT) and related substances (5-hydroxyindoleacetic acid; 5HIAA and tryptophan; Trp) in mouse brain, stomach and blood have been studied. All factors underwent circadian changes in the brain and blood. 5HT and 5HIAA levels in the stomach showed no circadian fluctuation. The concentrations of 5HT in the brain and blood did not correlate. Significant correlations were found between other serotonergic parameters analyzed in brain, stomach and blood. A significant negative correlation was observed between brain 5HIAA and blood 5HIAA. The concentration of tryptophan in the brain was correlated with the plasma total tryptophan level. There was fairly significant correlation (p less than 0.06) between brain serotonin and plasma tryptophan levels. The brain serotonin and tryptophan levels were strongly correlated (R = 0.410, p less than 0.03). Significant negative correlation was found between serotonin in the blood and serotonin in the stomach as well as between its level in the brain and in the stomach. The significance of these findings and their relationship to the use of peripheral serotonergic system as a model of neurons are discussed.

Alcohol-induced depression: involvement of serotonin.

We examined tryptophan and serotonin (5-hydroxytryptamine) levels in the blood after consumption of alcohol. Forty-five minutes after drinking, whole blood serotonin concentration was significantly reduced, whereas no changes were observed in tryptophan level. The diurnal rhythm of 5-HT in subjects who the day before had drunk alcohol was quite different from the control group, but very similar to that of patients with depression. The results strongly suggest that the mechanism of depression after alcohol drinking may be related to serotonin.

[Postoperative therapy of recurrent esophageal squamous cell carcinoma].

Some 218 resected cases and 43 autopsied cases after resection during the last 13 years were analyzed with regard to the mode of recurrence and survival. Several factors were investigated with the following results. 1) Extensive clearance of upper mediastinal lymph nodes during radical surgery may reduce the incidence of tumor recurrence. It is preferable to remove the cervical lymph node, however, after positive metastasis to cervical lymph node. 2) Beginning in 1981, interstitial brachytherapy of 192Ir seed assemblies using afterloading techniques was administered to two patients with esophageal carcinoma infiltrating the aorta and four patients infiltrating the trachea and main bronchus which were not completely resected in either case due to invasion of adjacent organs. No significant difference in life-prolongation was observed, but it may have been effective for prevention of localized recurrence in cases with aortic infiltration and with infiltration of the trachea and bronchus. 3) From 1984, eleven patients with recurrent squamous cell esophageal carcinoma were treated with combination chemotherapy sandwiched with irradiation (CDDP/5FU-RT-CDDP/5FU). Eight patients (73%) had complete or partial response, and six patients survived over one year after combination chemotherapy sandwiched with irradiation. Combination chemotherapy sandwiched with irradiation appears to be effective in the treatment of patients with recurrent squamous cell carcinoma of the esophagus. Toxic effects are moderate and manageable.

[The effects of eicosapentaenoic acid ethyl ester (EPA-E) on arterial thrombosis in rabbits and vascular lesions in rats].

The effects of highly purified ethyl ester of eicosapentaenoic acid (EPA-E) on ellagic acid-induced thrombus formation in the stenosed femoral artery in rabbits and on the progression of laurate-induced vascular lesions in rats were examined. (1) EPA-E in single administration significantly prevented the thrombus formation induced by ellagic acid in the stenosed femoral artery of rabbits. (2) EPA-E restored the hypercoagulation activity at 5 min and 3 hr after the ellagic acid injection. Platelet aggregability induced by arachidonic acid decreased at 5 min and 3 hr after the ellagic acid injection; EPA-E had little effect on this change. EPA-E showed almost no effect on fibrinolytic activity, blood viscosity and filterability of washed red blood cells after the ellagic acid injection. (3) Moreover, EPA-E in daily administrations showed a significant preventative effect against the laurate-induced vascular lesions in rats. The efficacy of EPA-E on arterial thrombosis and vascular lesions observed in this study suggests that EPA-E is a candidate for the treatment of peripheral circulatory disturbance.

[Absorption and distribution of rokitamycin in beagle dogs].

Tissue distribution of rokitamycin (TMS-19-Q) was studied after oral administration of 50 mg/kg to fasted Beagle dogs. Tissue concentrations of TMS-19-Q reached their peaks at 1-2 hours after the administration. The order of tissue concentrations of TMS-19-Q was liver greater than kidney greater than spleen greater than lung greater than mesenteric lymph node greater than heart greater than tonsil greater than serum greater than prostate gland greater than uterus greater than skin. At the peak time, drug concentrations in tonsil and lung were about 1.1 and 1.4 times higher, respectively, than that in serum.

[Studies on the metabolic fate of isepamicin sulfate (HAPA-B). II. Accumulation of HAPA-B after multiple administration in rats].

The accumulation of isepamicin sulfate (HAPA-B) in tissues and plasma was studied upon multiple intramuscular and intravenous administrations of 25 mg/kg of HAPA-B daily for 8 or 15 days to male rats. Shapes of plasma concentration curves in multiple intramuscular and intravenous administrations were very similar to that in a single administration. Drug concentrations in kidney at 24 hours after 8- and 15-multiple administrations through both routes were 3 to 4 and 4 to 5 times as high as that after a single administration. The concentration in kidney increased by multiple administrations for 8 days, but did not increase so highly there after till the 15-multiple administrations. On the other hand, the elimination of HAPA-B from kidney after multiple administrations was similar to that after a single administration. The accumulation of HAPA-B upon multiple administration was also observed in lung, heart, spleen and liver, but peak concentrations of the drug in these tissues were lower than that in kidney.