Technical note: Monitoring grazing bites and walking activity with pedometers.

Three types of pedometers installed on loosely fitted neck collars were investigated to determine the accuracy with which the devices measured the number of grazing bites performed by cows. The pedometer memory stored the summed number of bites over 1-h intervals for up to 10d, and the battery had a life of more than 3 mo. The values recorded by the pedometers were linearly related to the number of bites measured by visual observation and were unaffected by rumination. The correlation coefficients between the pedometer values and the number of bites were all >0.9. Circadian and day-to-day variations in the number of grazing bites were obtained by all 3 pedometers. The regression coefficients differed among the pedometers. The pedometers also responded to the occurrence of walking steps. The values recorded by the pedometers were linearly related to the observed number of walking steps. The correlation coefficients between the pedometer values and the number of steps were all >0.9. Although the number of walking steps affected the number of grazing bites, the number of bites greatly exceeded the number of walking steps. One type of pedometer, equipped with a 2-dimensional accelerometer, was used to analyze both grazing and walking behaviors. The back-forth and right-left movements of the pedometer had similar values during walking, whereas the values of the back-forth movements were greater during grazing. I conclude that pedometers can be used to measure the number of grazing bites but that this technique requires calibration to relate the pedometer values to the number of grazing bites. Observations of the back-forth and right-left movements of pedometers on neck collars will aid in distinguishing the grazing and walking behaviors of cows.

Rapid and validated fluorometric HPLC method for determination of gabapentin in human plasma and urine for clinical application.

WHAT IS KNOWN AND OBJECTIVE: A rapid derivatization and validated HPLC method for gabapentin in human plasma and urine is needed for clinical use. The objective of this study was to establish a rapid and validated analytical method for the determination of gabapentin in human plasma and urine using isocratic fluorometric HPLC for clinical application. METHODS: This analytical method is based on precolumn fluorescent derivatization using 4-fluoro-7-nitro-benzofurazan. The derivatization was coupled to fast HPLC separation using a 2·3 µm-particle size ODS column (100 × 4·6 mm i.d.). RESULTS AND DISCUSSION: The derivatization of gabapentin was optimized and HPLC separation was achieved over an ODS column with a run time of 3·5 min. Calibration curves in human plasma and urine were linear over the concentration ranges of 0·05-10 and 10-1000 µg/mL, respectively. Intra- and inter-assay precision and accuracy values of plasma were within 8·0% and 101-109% and within 8·3% and 94-108%, respectively. Those of urine were within 8·5% and 97-106% and within 9·5% and 97-105%, respectively. This validated method was applied to a pharmacokinetic study in healthy subjects. Interindividual variations in plasma disposition and urinary excretion of gabapentin were observed. WHAT IS NEW AND CONCLUSION: A rapid and validated isocratic fluorometric HPLC method for the determination of gabapentin in human plasma and urine for clinical application has been established. This method can be utilized to evaluate the pharmacokinetic disposition of gabapentin in humans.

Characterization of aortic aneurysms in cardiovascular disease patients harboring Porphyromonas gingivalis.

OBJECTIVE: Porphyromonas gingivalis was recently shown to cause intimal hyperplasia in a mouse model by a novel cholesterol-independent mechanism, suggesting to be a pathogen-specific feature of cardiovascular diseases. The aim of this study was to characterize the clinical and histopathological features of aortic aneurysms in cardiovascular disease patients harboring oral P. gingivalis. SUBJECT AND METHODS: Aortic aneurysm specimens were collected from 76 Japanese patients who underwent surgery, of whom dental plaque specimens were also collected from 31 patients. Bacterial DNA was extracted from each specimen to detect P. gingivalis by polymerase chain reaction. Histopathological analyses of the aortic aneurysm specimens, including immunohistochemical staining for embryonic myosin heavy chain isoform (SMemb) and S100 calcium-binding protein A9 (S100A9), were also performed. RESULTS: The number of aneurysms occurring in the distal aorta was significantly higher in subjects positive for P. gingivalis in dental plaque compared with those who were negative. The expressions of S100A9 and SMemb were also significantly greater in the subjects positive for P. gingivalis in dental plaque. On the other hand, there were no significant differences in adipocellular accumulation between the groups. CONCLUSIONS: These results suggest that aortic aneurysms in patients harboring oral P. gingivalis have greater expression of S100A9 and proliferative smooth muscle cells, which was different from the present patients without oral P. gingivalis.

Molecular analysis of aortic intimal hyperplasia caused by Porphyromonas gingivalis infection in mice with endothelial damage.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis infection is thought to be a significant etiological factor in the development of cardiovascular diseases. However, scant definitive evidence has been presented concerning the pathological molecular mechanisms of these disorders. In the present study, we performed a molecular analysis of the developmental mechanisms of aortic intimal hyperplasia induced by P. gingivalis. MATERIAL AND METHODS: The effects of P. gingivalis-induced bacteremia on intimal hyperplasia were evaluated using a mouse model of aortic hyperplasia created by photochemical-induced endothelial cell injury. Alterations of gene expression profiles in injured blood vessels of the mice were extensively analyzed using DNA microarray assays to identify the key molecules involved in P. gingivalis-induced hyperplasia. In addition, human aneurismal specimens from patients with or without P. gingivalis infection were analyzed histochemically. RESULTS: Intravenous administration of P. gingivalis dramatically induced intimal hyperplasia in the mouse model. Concomitantly, S100 calcium-binding protein A9 (S100A9) and embryonic isoform of myosin heavy chain (SMemb), a proliferative phenotypic marker of smooth muscle cells, were significantly overexpressed on the surfaces of smooth muscle cells present in the injured blood vessels. Similarly, increased expressions of S100A9 and SMemb proteins were observed in aneurismal specimens obtained from P. gingivalis-infected patients. CONCLUSION: We found that bacteremia induced by P. gingivalis leads to intimal hyperplasia associated with overexpressions of S100A9 and SMemb. Our results strongly suggest that oral-hematogenous spreading of P. gingivalis is a causative event in the development of aortic hyperplasia in periodontitis patients.

Technical note: Estimation of feed intake while grazing using a wireless system requiring no halter.

A simple, compact bite counter was used to record dairy cow jaw movements. This information was used to estimate feed intake. The device is composed of a pendulum, a microcontroller, and a transmitter attached to a collar. The microcontroller memory can store the number of bites over 10-min intervals for up to 3 mo and has a battery life of more than 1 yr. The number of bites measured by personal observation, and the values reported by the counter were compared for 5 multiparous, nonlactating Holstein cows. The correlation was linear with an R(2) value of 0.9, unaffected by rumination, and little affected by walking. The collar system avoided the problems often experienced with counters attached to halters. The utility of the bite counter recordings in estimating intake was tested using 8 multiparous lactating cows. The slopes of the regression lines relating the number of bites to feed intake were dependent on the level of available pasture mass (120 or 190 g standing dry matter/m(2)). The feed intake could be estimated by applying linear regression to the number of bite counts versus pasture disappearance. In both cases the R(2) values of the regression lines were >0.7. Although the counter recorded jaw movements during grazing when the head was down, it did not record rumination or mastication when the head was raised because the counter/collar did not contact the jaw in this position. The bite counters were easy to attach to the cows using the collar and could be used effectively by farmers and researchers.

Cutaneous pharmacokinetics of topically applied maxacalcitol ointment and lotion.

UNLABELLED: Maxacalcitol (22-oxacalcitriol), a vitamin D3 analogue, is widely used for the treatment of psoriasis in Japan. The effects of topically applied dermatologic preparations have routinely been assessed by their pharmacodynamic profiles and their concentrations in the skin correlate well with these profiles. OBJECTIVES: Recently, a maxacalcitol lotion formulation (M514102) was developed for the treatment of psoriatic lesions on the face and scalp. To predict the clinical efficacy of the lotion, we investigated the cutaneous bioavailability of topically applied lotion and compared this with that of its ointment formulation in healthy subjects. METHODS: In the first study, 12 subjects were divided into two groups of 6 each and were treated with the ointment or lotion. Six drug application sites were randomly selected on the left volar forearm. After 0, 2, 4, 6, 8 and 10 h, the formulations were gently removed and tape stripping was performed. Maxacalcitol was extracted from the tape strips and quantified by liquid chromatographic tandem mass spectrometry. In the second study, four drug application sites were randomly selected on the left volar forearm in the 12 subjects. The ointment was applied and spread over two sites and the lotion was applied in the same manner over the remaining two sites. After 8 h, the preparations were gently removed and followed by tape stripping. RESULTS: The average concentrations of maxacalcitol in the stratum corneum (SC) at 2, 4, 6, 8 and 10 h after application were 6.9 +/- 3.3, 12.8 +/- 6.2, 11.8 +/- 4.6, 13.1 +/- 5.2 and 12.3 +/- 3.1 microg/g for the ointment and 3.1 +/- 1.0, 9.1 +/- 3.1, 13.9 +/- 3.4, 13.1 +/- 4.1 and 15.5 +/- 3.1 microg/g for the lotion, respectively. A steady state was observed at approximately 4 and 6 h after application of the ointment and lotion, respectively. In the second study, there was no significant difference between the average of the SC concentrations of the ointment and lotion at 8 h. CONCLUSIONS: In conclusion, we observed that assessment of cutaneous bioavailability by using tape stripping was reproducible. Accordingly, the cutaneous bioavailability of the lotion was comparable to that of its ointment. Hence, treatment with the lotion is expected to be as effective as that with the ointment.

Stromelysin-1 (MMP-3) is critical for intracranial bleeding after t-PA treatment of stroke in mice.

BACKGROUND: Tissue-type plasminogen activator (t-PA) is approved for treatment of ischemic stroke patients, but it may increase the risk of intracranial bleeding (ICB). Matrix metalloproteinases (MMPs), which can be activated through the plasminogen/plasmin system, may contribute to ICB after ischemic stroke. OBJECTIVES: To explore the contribution of plasminogen, MMP-3 and MMP-9 to ICB associated with t-PA treatment after ischemic stroke. METHODS: Using a thrombotic middle cerebral artery occlusion (MCA-O) model, ICB was studied in mice with genetic deficiencies of plasminogen (Plg(-/-)), stromelysin-1 (MMP-3(-/-)), or gelatinase B (MMP-9(-/-)) and their corresponding wild-type (WT) littermates. The induction of MMP-3 and MMP-9 was also studied in C57BL/6 WT mice. RESULTS: ICB induced by t-PA (10 mg kg(-1)) was significantly less than WT in Plg(-/-) (P < 0.05) and MMP-3(-/-) (P < 0.05) but not in MMP-9(-/-) mice. Furthermore, administration of the broad-spectrum MMP inhibitor GM6001 after t-PA treatment reduced ICB significantly (P < 0.05) in MMP-3(+/+) mice, but had no effect on MMP-3(-/-) mice. MMP-3 expression was significantly enhanced at the ischemic hemisphere; with placebo treatment, it was expressed only in neurons, whereas it was up-regulated in endothelial cells with t-PA treatment. Although MMP-9 expression was also significantly enhanced at the ischemic brain, the amount and the distribution were comparable in mice with and without t-PA treatment. CONCLUSIONS: Our data with gene-deficient mice thus suggest that plasminogen and MMP-3 are relatively more important than MMP-9 for the increased ICB induced by t-PA treatment of ischemic stroke.

Effect of MDR1 C3435T polymorphism on cure rates of Helicobacter pylori infection by triple therapy with lansoprazole, amoxicillin and clarithromycin in relation to CYP 2C19 genotypes and 23S rRNA genotypes of H. pylori.

BACKGROUND: Polymorphism in MDR1 is associated with variation in the plasma level of a proton pump inhibitor. AIM: To investigate whether MDR1 polymorphism is associated with eradication rates of Helicobacter pylori by a triple therapy with lansoprazole, amoxicillin and clarithromycin in relation to CYP2C19 genotype status and bacterial susceptibility to clarithromycin. METHODS: A total of 313 patients infected with H. pylori completed the treatment with lansoprazole 30 mg b.d., clarithromycin 200 mg b.d. and amoxicillin 750 mg b.d. for 1 week. MDR1 C3435T polymorphism and CYP2C19 genotypes of patients and sensitivity of H. pylori to clarithromycin were determined. RESULTS: Logistic regression analysis revealed that the MDR1 polymorphism as well as CYP2C19 genotypes of patients and clarithromycin-resistance of H. pylori were significantly associated with successful eradication. Eradication rates for H. pylori were 82% (83/101: 95% CI = 73-89), 81% (112/139: CI = 73-87), and 67% (44/73: CI = 48-72) in patients with the MDR1 3435 C/C, C/T and T/T genotype, respectively (P = 0.001). CONCLUSIONS: Polymorphism of MDR1 is one of the determinants of successful eradication of H. pylori by the triple therapy with lansoprazole, amoxicillin and clarithromycin, together with CYP2C19 genotype and bacterial susceptibility to clarithromycin.

AFM characterization of single strand-specific endonuclease activity on linear DNA.

The specificity of nucleases for nicked and un-nicked double-stranded DNA has been characterized using atomic force microscopy (AFM). We have found that AFM has advantages over the usual macroscopic analyses, such as sucrose gradient centrifugation or electrophoresis, in characterizing nuclease digestion. In particular, short DNA fragments resulting from non-specific digestion were detected and, thus, the true length distribution of digested DNA was revealed. A simple numerical method is proposed to estimate the number of nicked sites per DNA molecule based on AFM images.

Analysis of carbon source-regulated gene expression by the upstream region of the Candida tropicalis malate synthase gene in Saccharomyces cerevisiae.

We investigated the regulation of expression of a gene encoding malate synthase (MS) of an n-alkane-utilizable yeast Candida tropicalis in the yeast Saccharomyces cerevisiae, where its expression is highly induced by acetate. By comparing levels of gene expression in cells grown on glucose, acetate, lactate, and oleic acid, we found that the increase in gene expression was due to a glucose repression-derepression mechanism. In order to obtain information concerning the regulation of the gene expression, a fusion gene which consists of the 5'-upstream region of MS-2 (UPR-MS-2) and the lacZ gene (encoding Escherichia coli beta-galactosidase), was introduced into S. cerevisiae, and beta-galactosidase activities were measured with cells grown on glucose or acetate. Deletion analysis of UPR-MS-2 revealed that the region between -777 and -448 (against the translation initiation codon) enhanced the level of gene expression in both glucose- and acetate-grown cells. In this region, sequences which resemble binding sites of Rap1p/Grf1p/Tufp, a global transcription activator, were found at seven locations and one was found for another pleiotropic activator Abf1p. The result also suggested the presence of multiple upstream repression sequences (URSs), which function specifically in glucose-grown cells, in the region between -368 and -126. In the repressing region, there were three tandem C(A/T)CTCCC sequences and also a putative binding site of Mig1p, a transcriptional repressor which mediates glucose repression of several other genes. When MIG1 gene of S. cerevisiae was disrupted, the expression of the UPR-MS-2-lacZ gene in glucose-grown cells increased approx. 10-fold. Furthermore, the effect of deletion of a putative Mig1p binding site was abolished in the MIG1-disrupted strain, suggesting Mig1p binds to this site and brings about glucose repression. When the SNF1 gene was disrupted, the high level gene expression observed in acetate-grown cells bearing UPR-MS-2 was abolished. This indicated that derepression of UPR-MS-2 -mediated gene expression was dependent on Snf1p, as is the case of genes encoding isocitrate lyase and gluconeogenic enzymes in S. cerevisiae.

Evidence for existence of polyol pathway in cultured rat mesangial cells.

The accumulation of polyols has been previously found in renal glomeruli isolated from streptozocin-induced diabetic (STZ-D) rats, although the intraglomerular polyol pathway has not been exactly localized. Because we have previously observed mesangial cell dysfunction in STZ-D rats, we examined whether the polyol pathway exists in mesangial cells as a possible candidate of the cause of cellular dysfunction. The activities of two polyol pathway enzymes, aldose reductase and sorbitol dehydrogenase, were clearly detected in the crude homogenate of cultured mesangial cells at higher levels than those of whole glomeruli when DL-glyceraldehyde or D-fructose was used as substrate. When cells were incubated in medium containing 55 mM glucose or galactose, a large amount of sorbitol or galactitol was accumulated intracellularly. The accumulation of polyols was effectively blocked by an aldose reductase inhibitor, ICI 128436. These results suggest that the polyol pathway exists in mesangial cells of rat glomeruli and may play a role in the development of mesangial cell dysfunction found in STZ-D rats.

Identification and characterization of aldose reductase in cultured rat mesangial cells.

Although the enhanced activity of the polyol pathway has been detected in diabetic glomeruli, the intraglomerular localization of this pathway has not yet been well defined. In this study, we attempted to identify aldose reductase, a key enzyme of the polyol pathway, in cultured rat mesangial cells and to characterize the properties of this enzyme using enzymological and immunological methods. When the aldose reductase (DL-glyceraldehyde-reducing) activity was analyzed in mesangial cell extract, the Lineweaver-Burk plot showed concave downward curvature, and the Michaelis constant was 0.83 mM DL-glyceraldehyde, and this activity was noncompetitively inhibited by an aldose reductase inhibitor, ICI-128,436. The enzyme activity was enhanced by the addition of sulfate ion and partially suppressed by barbital. The enzyme cross-reacted with the antisera against rat lens and testis aldose reductases on Ouchterlony plate, and migrated to the region of molecular weight of about 36,500 Da on Western blotting. The presence of aldose reductase mRNA was also confirmed by Northern analysis using cDNA for rat aldose reductase, 10Q. From these results, it was concluded that the aldose reductase may exist in rat glomerular mesangial cells and may play a role in the development of diabetic glomerulopathy, though the coexistence of aldehyde reductase(s) may not be fully ruled out.

Cooperativity and intermediate structures of single-stranded DNA binding-assisted RecA-single-stranded DNA complex formation studied by atomic force microscopy.

The formation of a complex between RecA protein and single-stranded (ss) DNA was studied systematically by atomic force microscopy (AFM) by varying incubation time and the molecular ratio of RecA protein to single-stranded DNA binding (SSB) protein. New intermediate structures, such as small circular, tangled, and protruded structures in the absence of SSB and sharply turned structures in the presence of SSB, were clearly identified at the early stage of complex formation. These structures have probably resulted from competitive binding of RecA and SSB to DNA. After long incubation, only fully covered RecA-ssDNA and totally RecA-free SSB-ssDNA complexes were present regardless of RecA concentrations. Together with intermediate structures which consisted of only two parts, that is, ssDNA covered by SSB and by RecA proteins, the observation suggested strong neighbor cooperative binding of RecA to ssDNA assisted by SSB.

In vivo assessment of the cutaneous bioavailability of topically applied maxacalcitol.

Maxacalcitol (22-oxacalcitriol), a vitamin D3 analogue, is widely used for the treatment of psoriasis. The effects of topical dermatologic drugs have been assessed by their pharmacodynamic activities, and concentrations in the skin correlate with these activities. In this study, we assessed the cutaneous bioavailability of topically applied maxacalcitol ointment in vivo by tape stripping. Six drug application sites were randomly assigned on the left volar forearm of six healthy men. Fifty milligrams of maxacalcitol ointment (25 microg/g) was applied to each site. After 0 (15 s), 0.5, 1, 2, 4, or 6 h, the ointment was gently removed, and tape stripping was performed. Maxacalcitol was extracted from the tape strips and quantified by liquid chromatographic tandem mass spectrometry. Average concentrations of maxacalcitol in the stratum corneum (SC) were 2.61, 4.37, 6.23, 9.37, and 9.46 microg/g at 0.5, 1, 2, 4, and 6 h, respectively, after drug application. The steady state was attained approx. 4 h after drug application. The cutaneous bioavailability of topical maxacalcitol ointment can be assessed by the tape-stripping method. This approach will probably be useful in the assessment of the bioequivalence of topical dermatologic products and as a parameter for pharmacokinetic/pharmacodynamic studies.

Community-Based Intervention for Prevention of Dementia in Japan.

Population aging is accelerating, with prolonged life expectancy and a decrease in birth rate. As age is a significant risk factor for dementia, we are confronted with an ever-increasing prevalence of mild cognitive impairment (MCI)/dementia. Thus, the Japanese National Center for Geriatrics and Gerontology launched a project to promote community-based research, including the development of an effective screening system for high-risk groups and intervention for dementia prevention. This review introduces the project, the Obu Study of Health Promotion for the Elderly, with the following strategic triad: 1) Identification of the target population by population screening; we regarded patients with MCI as the target population, and developed a screening test battery to identify MCI in a population screening setting. 2) Scientific evaluation of community-based intervention; we developed an interventional method combining exercise and cognitive training ("cognicise"). In practical settings, "cognicise" is programmed into multicomponent exercise intervention, which was reported to have benefits of cognitive improvement and reduction of brain atrophy based on randomized controlled trials. 3) Standardization of the methods of population screening and community-based intervention for evidence-based policy making and widespread implementation. Dementia prevention, or at least delaying the onset of dementia and/or stopping/slowing the progression of dementia, should benefit the whole society as well as individuals. It is our continuing challenge to improve the screening system and community-based intervention for dementia prevention through accumulation of evidence.

Combination of a free radical scavenger and heparin reduces cerebral hemorrhage after heparin treatment in a rabbit middle cerebral artery occlusion model.

BACKGROUND AND PURPOSE: We sought to investigate the effects of EPC-K1, a free radical scavenger, on reducing heparin-produced cerebral hemorrhage in a rabbit model of middle cerebral artery (MCA) photothrombosis and to investigate whether the combination of EPC-K1 and heparin enhances neuroprotection from cerebral ischemic damage. METHODS: In the heparin-alone group (n=8), heparin was administered intravenously for 24 hours, starting from 3 hours after MCA occlusion. In the EPC-K1-alone group (n=8), EPC-K1 was administered as a bolus injection (10 mg/kg) twice at 3 and 6 hours after MCA occlusion. In the combination group (n=8), EPC-K1 and heparin both were administered as in the single-drug procedures. In the vehicle group (n=10), saline were infused for 24 hours. RESULTS: Heparin prolonged activated partial thromboplastin time by approximately 3 times that of control animals. In the heparin-treated animals, the hemorrhage size was significantly increased (P<0.0001) and neurological symptoms were significantly worse (P<0.01) than in control animals at 48 hours. The combination of EPC-K1 and heparin dramatically reduced heparin-produced cerebral hemorrhage (P<0.0001), with a significant reduction in infarct volume (reduction by 63.2% and 57.2% of heparin-alone and control animals, respectively, P<0.0001) and a significant improvement in neurological symptoms (P<0.01 versus heparin-alone and control animals, respectively). CONCLUSIONS: These data indicate that free radical formation may play a key role in intracerebral hemorrhage exacerbated by heparin treatment and that the combination of a free radical scavenger and heparin augmented neuroprotection from acute brain ischemia. The results of the present study may suggest a potential clinical approach for the treatment of acute stroke.

ME3277, a GPIIb/IIIa antagonist reduces cerebral infarction without enhancing intracranial hemorrhage in photothrombotic occlusion of rabbit middle cerebral artery.

GPIIb/IIIa antagonists are expected to have a beneficial effect on acute cerebral infarction, however, the occurrence of intracranial hemorrhage has not been as widely investigated. A rabbit focal thrombotic occlusion model of the middle cerebral artery was established by creating a photochemical reaction between green light and Rose Bengal. Hemorrhagic transformation was common in the area of cerebral infarction. Using this model, the effect of a GPIIb/IIIa antagonist, ME3277 (low dose, (L); 0.15 mg/kg + 0.125 mg/kg x h, middle dose, (M); 0.3 mg/kg + 0.25 mg/kg x h and high dose, (H); 0.6 mg/kg + 0.5 mg/kg x h), aspirin (20 mg/kg) and sodium ozagrel (thromboxane A2 synthase inhibitor, 1 mg/kg + 2 mg/ kg x h) were evaluated. Drugs were intravenously administrated 30 minutes after the photochemical reaction for 24 hours. Aspirin inhibited the ex vivo platelet aggregation induced by arachidonic acid and collagen but not by adenosine diphosphate (ADP), while sodium ozagrel only inhibited the arachidonic acid-induced aggregation. ME3277 dose-dependently inhibited the platelet aggregation induced by all the inducers (approximately 60% in L, 80% in M, and 90% in H). At 24 hours of middle cerebral artery (MCA) occlusion, infarct volume was significantly reduced by aspirin and each dose of ME3277. These agents improved neurologic deficits, with ME3277 being more potent than aspirin. Sodium ozagrel did not alter the infarct volume nor neurologic deficits. No drug was found to worsen hemorrhage volume despite increasing bleeding time (2-3 fold) in the skin. In this model, the occluded artery was spontaneously recanalized and re-thrombosed frequently. One mechanism by which antiplatelet agents reduced infarct volume was inhibition of rethrombosis of the MCA. These results suggest that treatment with a GPIIb/IIIa antagonist is a useful intervention for acute cerebral infarction prolonging dose bleeding time to 3 times the basal value.

Direct measurement of superoxide-dependent chemiluminescence from rat skin following UV-dependent fluoroquinolone-induced dermatitis.

Fluoroquinolones are widely used for the treatment of bacterial infection. However, some members of the group cause UV-dependent dermatitis. Therefore, animal models are required to screen and predict if a derivative is likely to cause photodermatitis. Under anaesthesia, the hair on the dorsal side of rats was shaved, then a fluoroquinolone derivative, Y-26611 or ofloxacin was administered subcutaneously (SC) followed by irradiation of a 3 cm diameter circular area of the skin with UV-B(ultraviolet-B) for 45 min. Peak photodermatitis was observed 24 h after irradiation in Y-2611-treated skin. Leucocyte infiltration into the skin, mostly polymorphonuclear leucocytes, was verified by histological techniques. O2.- radical generated by activated leucocytes was detected directly from the skin by SC application of a sensitive O2.- -dependent luminescence reagent, MCLA. O2.- -dependent photon emission (chemiluminescence) from the skin was amplified by phorbol myristate acetate and was markedly suppressed by superoxide dismutase. This is the first report of direct detection of chemiluminescence from the skin of a living animal.

TRA-418, a novel compound having both thromboxane A(2) receptor antagonistic and prostaglandin I(2) receptor agonistic activities: its antiplatelet effects in human and animal platelets.

TRA-418 is a novel compound that has been found in our screening for compounds having both thromboxane A2 (TP) receptor antagonistic and prostaglandin I2 (IP) receptor agonistic activities. In the binding assays, TRA-418 showed a 10-fold higher affinity to TP-receptors than IP-receptors. TRA-418 inhibited platelet aggregation induced by the TP-receptor agonist, U-46619 and by arachidonic acid at concentrations lower than those required for inhibition of ADP-induced aggregations. Furthermore, TRA-418 inhibited not only platelet aggregation induced by ADP alone, but also that induced by ADP in the presence of the TP-receptor antagonist, SQ-29548. When the IC50 values of TRA-418 for platelet aggregation were estimated in platelet preparations from monkeys, dogs, cats, and rats using ADP and arachidonic acid as the platelet stimulating agents, it was found that the values estimated in monkey platelets were quite similar to those estimated in human platelets. In ex vivo platelet aggregation in monkeys, TRA-418 exhibited significant inhibitory effects on arachidonic acid-induced aggregation in platelet preparations from monkeys treated at 3 micro g kg min-1 or higher doses, where neither a significant decrease in blood pressure nor a significant increase in heart rate was observed. These results are consistent with the fact that TRA-418 has a relatively potent TP-receptor antagonistic activity together with a relatively weak IP-receptor agonistic activity.

Milrinone, a phosphodiesterase inhibitor, suppresses intimal thickening after photochemically induced endothelial injury in the mouse femoral artery.

The effect of milrinone, a phosphodiesterase (PDE) inhibitor, on intimal thickening after endothelial denudation was investigated. Intimal thickening was induced in the femoral arteries of mice by a photochemical reaction between rose bengal and transluminal green light which caused endothelial injury followed by platelet adhesion, aggregation, and formation of an occlusive thrombus in the irradiated segment of the mouse femoral artery. In this model, intimal thickening occurred following spontaneous thrombolysis. The intima/media ratio at 21 days after irradiation was 0.556+/-0.104 in the untreated group. Oral administration of milrinone (0.3-3.0 mg/kg) for 3-21 days suppressed intimal thickening by up to 56% in a dose- and time-dependent manner. In an in vivo experiment using bromodeoxyuridine incorporation, milrinone suppressed cell proliferation at 1.0 mg/kg p.o. On the other hand, the minimum doses of milrinone for suppression of ex vivo platelet aggregation induced by collagen (0.8 microg/ml) or ADP (0.5 microM) were 3.0 and 10.0 mg/kg, respectively. These results indicate that milrinone may not suppress intimal thickening by inhibiting platelet function but by preventing vascular smooth muscle cell (VSMC) proliferation, probably through a mechanism mediated via 3', 5'-adenosine cyclic monophosphate (cAMP).