Identification of a Lineage D Betacoronavirus in Cave Nectar Bats (Eonycteris spelaea) in Singapore and an Overview of Lineage D Reservoir Ecology in SE Asian Bats.

Coronaviruses are a diverse group of viruses that infect mammals and birds. Bats are reservoirs for several different coronaviruses in the Alphacoronavirus and Betacoronavirus genera. They also appear to be the natural reservoir for the ancestral viruses that generated the severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus outbreaks. Here, we detected coronavirus sequences in next-generation sequence data created from Eonycteris spelaea faeces and urine. We also screened by PCR urine samples, faecal samples and rectal swabs collected from six species of bats in Singapore between 2011 and 2014, all of which were negative. The phylogenetic analysis indicates this novel strain is most closely related to lineage D Betacoronaviruses detected in a diverse range of bat species. This is the second time that coronaviruses have been detected in cave nectar bats, but the first coronavirus sequence data generated from this species. Bat species from which this group of coronaviruses has been detected are widely distributed across SE Asia, South Asia and Southern China. They overlap geographically, often share roosting sites and have been witnessed to forage on the same plant. The addition of sequence data from this group of viruses will allow us to better understand coronavirus evolution and host specificity.

An Open-Label Trial of Yokukansan on Sleep Disturbance in Alzheimer's Disease and Other Dementia.

BACKGROUND: An effective hypnotic drug with a low risk of adverse reactions is required for Alzheimer's disease (AD) patients, because the therapeutic interventions to improve sleep quality may help alleviate some symptoms of AD including cognitive function. OBJECTIVE: The aim of this study was to investigate the efficacy and safety of Yokukansan in sleep disturbances in patients with AD and other dementia. DESIGN: An open-label trial. SETTING: Two sites consist of university and hospital in Japan. PARTICIPANTS: Thirteen patients (7 men and 6 women, average age = 76.0 ± 7.2 (mean ± SD) years old) including 12 AD and 1 frontotemporal dementia. INTERVENTION: Treatment with Yokukansan (5-7.5 g/day) was given for 8 weeks. MEASUREMENTS: The primary outcome measure was the Sleep Disorder Inventory (SDI) based on the Neuropsychiatric Inventory, an instrument developed by the Alzheimer's Disease Cooperative Study. Secondary outcome measures included the objective actigraphic evaluations, Neuropsychiatric Inventory-Questionnaire (NPI-Q), MINI-Mental State Examination (MMSE). These assessments were evaluated at baseline, and weeks 4 and 8. RESULTS: After 4 and 8 weeks treatment with Yokukansan, significant improvements were observed in the SDI total score, caregivers' distress score, and NPI-Q total score. In actigraph data, wake after sleep onset (WASO) time (min), was significantly improved. The MMSE score did not change during the treatment. No serious adverse events were caused by YKS. CONCLUSION: The present results suggest that Yokukansan is safe and beneficial in the treatment of sleep disturbances and that it can possibly reduce the burden of care of demented patients.

Isotype-specific changes in the amount of beta-tubulin RNA in synchronized tobacco BY2 cells.

The 3'-ends of the beta-tubulin cDNA were amplified from tobacco BY2 polyA+ RNA. According to the differences in the predicted amino acid sequence at the extreme C-terminal, they were grouped into three different isotypes, NTB1 in which "EEGDYYEEDEEDLNEA", NTB2 in which "EEEYYEDEEEA QED" and NTB3 in which "DECEYEEEEEYDHEGN" follows the conservative "YQQYQDATAD" sequence. Using unique 3'-untranslated regions as probes, changes in the RNA levels of each beta-tubulin isotype were determined by dot-blot hybridization. The levels exhibited characteristic rhythms in the cell cycle. NTB1 RNA was highest in S phase in comparison to NTB2 RNA level which was highest in late G2. On the other hand, NTB3 RNA level was highest in early G2.

Upregulation of prostacyclin synthesis-related gene expression by shear stress in vascular endothelial cells.

Prostacyclin (prostaglandin I2, PGI2) has a variety of functions, including inhibition of smooth muscle cell proliferation, vasodilation, and antiplatelet aggregation. PGI2 production in endothelial cells has been reported to increase biphasically after shear loading, but the underlying mechanism is not well understood. To clarify the mechanism for the second phase of PGI2 upregulation, we examined the gene expression of the enzymes involved in PGI2 production in human umbilical vein endothelial cells (HUVECs) after shear-stress (24 dyne/cm2) loading. The production of 6-keto-PGF1alpha, a stable metabolite of PGI2, increased time-dependently under shear stress. The arachidonic acid liberation from membrane phospholipids in HUVECs after 12 hours of shear loading was increased significantly compared with the static condition. No change was observed for cytosolic phospholipase A2 expression, as detected by reverse transcription-polymerase chain reaction and Western blotting. Cyclooxygenase (COX)-1 mRNA increased after 1 hour of shear loading, and the increase lasted for 12 hours, the longest time tested, whereas COX-2 mRNA increased after 1 hour of shear loading and peaked at 6 hours. An increase of COX-1 expression was detected at 12 hours of shear loading by Western blotting. No expression of COX-2 was detected in the static control, but induced expression was observed at 6 hours after shear loading. PGI2 synthase was also found to be upregulated. These results suggest that the elevated PGI2 production by shear stress is mediated by increased arachidonic acid release and a combination of increased expression of COXs and PGI2 synthase.

Shear stress down-regulates gene transcription and production of adrenomedullin in human aortic endothelial cells.

Vascular endothelial cells are potent modulators of vascular tone in response to shear stress. Levels of vasoactive peptides such as adrenomedullin (AM), endothelin-1 (ET-1), C-type natriuretic peptide (CNP), and nitric oxide (NO) are affected by fluid shear stress. AM, a potent vasodilator and suppressor of smooth muscle cell proliferation, contains the shear stress responsive element (SSRE) "GAGACC" in its promoter region. To examine the role of AM in the shear stress response, cultured human aortic endothelial cells (HAoECs) were exposed to fluid shear stresses of 12 and 24 dynes/cm2 in a cone-plate shear stress loading apparatus for various time periods, and the levels of AM gene expression and peptide secretion from HAoECs were measured by Northern blotting analysis and radioimmunoassay (RIA), respectively. Both AM gene transcription and AM peptide levels were down-regulated by fluid shear stress in a time- and magnitude-dependent manner. Our results demonstrate that the normal level of arterial shear stress down-regulates AM expression in HAoECs, suggesting that AM participates in the modulation of vascular tone by fluid shear stress.

Expression of platelet-activating factor receptor transcript-2 is induced by shear stress in HUVEC.

Platelet-activating factor (PAF) is an important lipid involved in inflammation reaction and circulation regulation. The receptor for human PAF is synthesized from two spliced transcripts of the same gene. Our observation in the present study shows that HUVEC express transcript-1 only in the static condition. Shear stress induces the expression of transcript-2 in these cells but not transcript-1, resulting in increased PAF receptor expression as measured by FACS analysis. These results suggest that shear stress may increase the susceptibility of endothelial cells to PAF by inducing transcript-2 expression.

P2X1 purinoceptor in human platelets. Molecular cloning and functional characterization after heterologous expression.

ADP is an important physiological platelet agonist. The molecular identity of the ADP receptor(s) in human platelets, however, is still unclear. Although P2T purinoceptor was believed to be the ligand-gated cation channel for ADP in human platelets, recent patch clamp studies now suggest it is P2X1 type. In the present study, we have cloned a cDNA encoding a P2X1 purinoceptor from human platelets using degenerate reverse transcription and polymerase chain reaction. Northern blotting with a P2X1-specific probe revealed a band of 1.8 kilobases in human platelets as well as in several megakaryoblastic cell lines. 1321N1 human astrocytoma cells expressing the cloned P2X1 cDNA exhibited both ATP- and ADP-stimulated Ca2+ influx that could be blocked by the purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid and suramin. Additionally, a polyclonal antibody raised against glutathione-S-transferase-P2X1 fusion peptide reacted with a 70-kDa band on Western blot of human platelets. It is therefore concluded that functional P2X1 purinoceptors are present in human platelets.

Effect of ointment bases on topical and transdermal delivery of salicylic acid in rats: evaluation by skin microdialysis.

Microdialysis has been used to determine the concentration of salicylic acid in skin tissue and plasma periodically for 4 h to evaluate the effect of ointment bases on topical and transdermal delivery of salicylic acid. The ointment bases examined were solbase (water-soluble), poloid and white petrolatum (oleaginous), hydrophilic poloid (water in oil (w/o) type emulsion lacking water) and absorptive ointment (w/o-type emulsion containing water). The ointments (0.1 g) containing 25 micromol salicylic acid were applied for 2 h to the surface of rat skin (1 cm2) with (intact) or without the stratum corneum. For intact skin, the extent of topical delivery from different ointments, evaluated by the area under the concentration-time curve (AUC) of salicylic acid in the skin tissue (AUCskin), increased in the order solbase << white petrolatum, poloid, hydrophilic poloid << absorptive ointment. The ratio of AUCskin (topical delivery) to the AUC of salicylic acid in plasma (AUCplasma, transdermal delivery) varied remarkably among the different bases, the greatest ratio being observed for absorptive ointment. When the ointments were applied to skin surface without stratum corneum, AUCskin for solbase was much higher (about 45 times that for intact skin), whereas only a small (two-fold) increase was observed for poloid and hydrophilic poloid and the increase was negligible for white petrolatum and absorptive ointment. For skin without the stratum corneum, the ratio AUCskin/AUCplasma for the different ointments was comparable, although the magnitudes of AUCskin and AUCplasma still varied substantially. The variance of AUC values arises as a result of the different rates of release of salicylic acid from the bases. These results indicate that: the topical and transdermal delivery of salicylic acid in intact skin varies substantially among different ointment bases, and the greatest topical delivery is observed for absorptive ointment; use of absorptive ointment increases the retention of salicylic acid in the stratum corneum; and the stratum corneum functions strongly as a penetration barrier for solbase, moderately for poloid and hydrophilic poloid, and less for absorptive ointment and white petrolatum.

Possible involvement of m-calpain in vascular smooth muscle cell proliferation.

Vascular smooth muscle cell (VSMC) proliferation still remains a poorly understood process, although it is believed to play a critical role in pathological states, including atherosclerosis and hypertension. Several reports have suggested that proteases may be directly involved in this process; however, it was still unclear which protease is responsible for VSMC proliferation. In this study, by use of a cell-permeable calpain inhibitor (calpeptin; benzyloxycarbonyl-Leu-nLeu-H), its analogue (benzyloxycarbonyl-Leu-Met-H), the cell-impermeable serine protease inhibitor leupeptin, and antisense oligonucleotide against m-calpain to inhibit proliferation of primarily cultured human VSMCs, we investigated whether calcium-activated neutral protease (calpain) is involved in VSMC proliferation. Calpeptin and its analogue, more specific for m-calpain, equally inhibited the proliferation of VSMCs in a dose-related manner, whereas a more limited antiproliferative effect was observed in leupeptin-treated VSMCs. Antisense oligonucleotide against m-calpain, but not scrambled antisense, dose-dependently inhibited m-calpain expression and proliferation of VSMCs. Maximal inhibition was an approximately 50% reduction of cell number and m-calpain antigen observed at 50 micromol/L of antisense oligonucleotide. Calpeptin or antisense oligonucleotide against m-calpain increased the expression of the endogenous calpain substrate pp125FAK (focal adhesion kinase), whereas the expression of the endogenous calpain inhibitor calpastatin was not affected. These results suggest that the proliferation of VSMCs requires protease activity, some of which is due to m-calpain.

Cerebellar control of the cardiovascular responses during postural changes in conscious rabbits.

In conscious control rabbits, tilting the head 30 degrees up from a position 30 degrees down induced initially an inhibition in the renal sympathetic nerve activity (RSNA), however this inhibition immediately released and became a transient increase. Following these responses in RSNA, blood pressure (BP) initially decreased but recovered to the control level within 3-5 s. After bilateral destruction of the lateral nodulus-uvula in the cerebellum, in contrast, the same tilting of the head caused an immediate large increase in RSNA without early inhibition, which was sustained at a high level. BP increased transiently, but then decreased and remained at a level lower than the control. These results indicate that the timing and duration of this transient increase in RSNA during tilting the head up are controlled by the lateral nodulus-uvula and may be important in the rapid adaptation of blood pressure. In addition, this suggests that the lateral nodulus-uvula may play an important role in the cardiovascular control under conditions of consciousness during changes in head position and body posture.

Two short peptide sequences repeated three times in carrot beta-tubulin.

There are two repeated peptides, [S,T,Y]QQ[Y,M] and MFR[R,X][V,K], which appear three times each in the region corresponding to the third exon of carrot beta-tubulins. The former peptide is flanked on both sides by a pair of identically charged groups and the latter includes two or three basic amino acids in it. The sequences are generally conserved well among the eukaryotes except for fungi, in which one of the peptides, SQQY, does not exist.

Expanded polytetrafluoroethylene (ePTFE) vascular graft loses its thrombogenicity six months after implantation.

Although expanded polytetrafluoroethylene (ePTFE) has been believed to be an inert material for vascular prosthesis, it shows less tendency of graft maturation by means of endothelialization. The aim of this study is to evaluate long-term alteration in thrombogenicity of ePTFE grafts after implantation. Serial levels of thrombin-antithrombin III complex (TAT), D-dimer, C-reactive protein (CRP), and prothrombin time (PT) were examined in 77 patients following ePTFE Y-graft implantation for up to five years. TAT showed biphasic elevation after implantation; TAT increased from 16.4+/-8.6 ng/ml to 27.4+/-10.5 ng/ml at one day, decreased to 18.5+/-4.5 ng/ml at one week, and increased again to 25.3+/-8.5 ng/ml at two weeks. Elevated TAT gradually decreased after the second peak to reach a lower level than that before surgery after six months. There was no significant difference in TAT level after six months due to the difference in diagnosis or anti-thrombotic therapy. We suggest that ePTFE grafts lose their thrombogenicity six months after implantation, after which anti-thrombotic therapy might be unnecessary.

[The prognostic factors in astrocytic tumors: analysis by the Kaplan-Meier method and the Weibull log-linear model].

As the prognosis of astrocytic tumors depends on various factors, identifying prognostic factors should be useful for developing strategies to cope with them. Between 1975 and 1994, more than 200 patients with astrocytic tumors were treated in Kagoshima University. Of these patients, 149 (grade I: 17, grade II: 42, grade III: 41, grade IV: 42, unproven: 7) have been followed up. Records of these patients were retrospectively reviewed for age at the time of initial symptoms, gender, histological grade (WHO), extent of tumor resection, radiation therapy, and administration of anticancer agents. We used the Kaplan-Meier method and the Weibull log-linear model to analyze the relation between survival time and these prognostic factors. Survival time was counted from onset of symptoms, and age of initial treatment was used as a covariant. The mean age of males at the initial diagnosis was 40.8 years (n = 77), and that of females was 39 years (n = 72). Using the Kaplan-Meier method, the mean survival time of the 149 patients was 101 months (males; 72.7 months, females; 134.5 months). Mean survival time of grade II was 144.3 months, that of grade III was 95.2 months, and grade IV (glioblastoma) was 15.9 months. Histological grades and mean ages of the groups showed a positive correlation. Among grades II, III and IV, the Kaplan-Meier survival curves were significantly different (p < 0.0001) according to the log-rank test. By the extent of surgical resection (subtotal or greater resection, partial resection, and less than partial resection), the mean survival time showed a significant difference (p < 0.05) on the log-rank test. However, we could not detect a significant difference in survival time between the group that received chemotherapy and the group which did not. The Weibull log-linear analysis indicated that gender, age, histological grade (WHO), extent of surgery, and dose of radiation therapy were prognostic factors. Covariants of grades II, III, and IV made survival time 0.314, 0.179, and 0.069 times as long as that of grade I. The survival time after "partial resection" became 1.415 times as long as the survival time after "less than partial resection". The covariant of "greater than subtotal resection" showed a prolonged survival time of 2.916 compared with that of "less than partial resection". As for age at treatment, the older the patient was, the shorter the survival time. The rate was 0.986 for each year of age. Irradiation of one Gy increased survival time by 1.015 times. Chemoimmunotherapy (dose of ACNU and interferon beta) could not be confirmed as an effective covariant.

Hemostasis activation during sclerotherapy of lower extremity varices.

The influence of compression sclerotherapy upon hemostasis activation was investigated in 41 consecutive patients with lower extremity varices by serial measurement of thrombin-antithrombin III complexes (TAT), D-dimer, fibrinogen and C-reactive protein (CRP). Blood sampling was carried out before operation and on the 7th and 28th post-operative day in patients randomly assigned to either the control group (n = 18), in which high ligation of sapheno-femoral junction and local excision of varices were performed, or the sclerotherapy group (n = 23) in which the comparable surgical intervention and compression sclerotherapy using hypertonic saline were performed simultaneously. In both groups, the TAT, D-dimer and fibrinogen concentrations at day 7 were significantly elevated compared to the value before operation while CRP showed no significant change during the observation period. In the sclerotherapy group, higher incidence of superficial thrombosis was observed and the TAT concentration at day 7 was significantly higher than that in the control group (p < 0.01), and the TAT at day 28 was still significantly elevated compared to the pre-operative level (p < 0.05). However, no relationship between TAT and D-dimer concentrations and the extent of superficial thrombosis was observed. We conclude that compression sclerotherapy for lower extremity varices causes latent activation of coagulation system and can be a risk factor for venous thromboembolism.

Shear stress induces expression of CNP gene in human endothelial cells.

To elucidate the effect of blood flow on gene transcription of C-type natriuretic peptide (CNP), human umbilical vein endothelial cells were exposed to shear stress in a cone-plate viscometer. Expression of CNP mRNA, evaluated by reverse transcription-polymerase chain reaction, was markedly increased by exposure to shear stress of 24 dyne/cm2 at 3 h. The CNP mRNA level was maintained until 12 h. Thus, the present study demonstrated for the first time that shear stress induces expression of CNP gene in human endothelial cells.

An infection-resistant PTFE vascular graft; spiral coiling of the graft with ofloxacin-bonded PTFE thread.

OBJECTIVE: To develop an infection-resistant polytetrafluoroethylene (PTFE) vascular graft for potential clinical use in grafting in sites of bacterial contamination and in replacement of the infected grafts. SETTING: Experimental study in rabbits. MATERIALS AND METHODS: An antibiotic ofloxacin (OFLX) was bonded to a sheet of PTFE by impregnation, which was cut and twisted into fine threads. The in-vitro antibacterial activity of OFLX-PTFE thread was determined by measuring the zone of growth inhibition against Escherichia coli. The thread was spirally coiled around a ridged outerwall PTFE to make the OFLX-PTFE graft. OFLX-PTFE graft or control graft was interposed in the inferior vena cava (IVC) of rabbits and the entire graft was covered with fibrin containing a fixed number of E. coli. Three or 7 days after the grafting, the grafts with perigraft tissue were harvested and subjected to bacteriological studies. RESULTS: In spite of early phase rapid elution of OFLX, a significant antibacterial activity was retained for more than 2 weeks. The antibacterial activity of OFLX-PTFE threads implanted in the subcutaneous space of rabbits decreased to 48% after 24 h and to approximately 1% after a week. The swab culture of all the control grafts was positive, while only one of 13 PTFE-OFLX grafts was positive. The number of viable bacteria in the perigraft tissue of OFLX-PTFE grafts was remarkably low in comparison with that of control grafts. Thus, the OFLX-PTFE grafts exhibited a marked in-vivo antibacterial activity. CONCLUSION: By a unique method, it was possible to furnish PTFE graft with an excellent infection-resistant property, without affecting the original biological behaviour.

[Ventilatory insufficiency due to upper cervical injury treated by diaphragm pacing: a case report].

A 33-year-old man suffered chronic ventilatory insufficiency with tetraplegia after an operation for atlantoaxial dislocation. He was alert and his tetraplegia gradually improved. However, continuous mechanical ventilation was necessary for him. Thirteen months after the operation, a diaphragm pacer (Avery Laboratory Inc.) was implanted on the right phrenic nerve in the cervical region. Nineteen days after the implantation of the right side pacer, a left side diaphragm pacer was also implanted. Diaphragm pacing was started two weeks after the second implantation. The pacing period was gradually prolonged and continuous pacing for 9 hours by the right side pacer and three hours by the left side pacer was able to be obtained. One year after implantation, twelve continuous hours of diaphragm pacing became possible. During diaphragm pacing, blood gas analysis was satisfactory and the patient could move sitting on a wheel chair, watch television and write letters using a word processor. We were unable to achieve total ventilatory support for him using these diaphragm pacers. We thought that the main cause of our partial failure originated from the procedure used in implanting the electrode onto the phrenic nerve. Left side pacing needs higher amplitude than that used on the right side to obtain sufficient tidal volume. The patient refused our continuing the left side pacing because of pain around the anterior chest and shoulder. Another problem to be watched is diaphragm fatigue. However, diaphragm pacing has been continued for six years and it has been useful in improving his quality of life.(ABSTRACT TRUNCATED AT 250 WORDS)

Skin microdialysis: detection of in vivo histamine release in cutaneous allergic reactions.

In vivo histamine release in cutaneous allergic reactions was chronologically examined using the skin microdialysis technique. Antigen challenge was made in ovalbumin-sensitized guinea pigs 1 h after the implantation of the microdialysis probe. A marked histamine release after intradermal injection of ovalbumin solution was observed in the early phase (up to 40 min). No isolated delayed histamine release was observed thereafter over 8 h. Also, in an atopic volunteer, a marked release of histamine after intradermal injection of mite extract was observed only in the early phase, although erythema and induration at the site of injection were continuously observed in the late phase (up to 4 h). These findings suggest that the skin microdialysis technique is a useful, simple technique for chronological determination of chemical mediators released in the allergic skin in vivo.