DNA vaccination or genetic immunization is a rapidly developing technology that offers new approaches for the prevention and therapy of disease.Regarding the inoculation method of DNA vaccine, we recommend the gene gun delivery system, which is a highly reliable method compared to intramuscular inoculation.DNA vaccines could have potential advantages over other types of vaccines in that these vaccines can induce strong cellular immune responses, cytotoxic T lymphocytes and type 1 helper T cells, without resorting to live organisms or complicated protein formulation.The cellular immune responses are especially required for the protection against infections with intracellular pathogens such as viruses and Mycobacterium tuberculosis and protection against cancers, suggesting that they seem to be suitable targets of DNA vaccines.We describe here that their application to bacterial infections requires optimization of codon usage in the DNA vaccines to the host animal to improve translational efficiencies of the bacteria genes.DNA vaccines for a variety of pathogens and cancers have now entered phase I/II human clinical trials.
The action of fendiline on cardiac electrical activity was investigated in guinea pig papillary muscle by monitoring frequency− and time−dependent changes in membrane potential, action potential(AP)configuration and conduction velocity.Isolated guinea pig papillary muscles driven at 0.1 to 3 Hz showed a concentration−dependent reduction of + Vmax, overshoot, and AP duration at −20mV(APD20)in the presence of fendiline(1−320μM), reflecting inhibition of Na+ and L−type Ca2+ channels, respectively.No significant change in resting potential and AP duration at 90% repolarization(APD90)were observed.Inhibition of + Vmax and APD20 was more prominent at higher frequency of stimulation(2 Hz)than at lower ones(0.2 Hz), demonstrating frequency−dependent block of Na+ and Ca2+ channels including an open channel block.A good relationship between changes in + Vmax and APD20 suggested some commonality in the mechanism of inhibition of Na+ and Ca2+ channels by fendiline.Time−dependence of effects of fendiline, observed in presence of bolus dose(200μM), showed an earlier onset of inhibition of + Vmax and APD20, particularly at higher frequencies.Missed beats and conduction block also appeared earlier in preparations driven at higher frequency.These findings suggest a frequency−dependent(and open channel)block of Na+ and Ca2+ channels by fendiline, leading to inhibition of fast and slow conduction in addition to its reported inactivated Ca2+ channel block.
The present study examined whether rifampin attenuated glutathione(GSH)depletion by acetaminophen reactive metabolites generated in the in vitro P450 enzyme system prepared from mouse liver and the possible mechanism involved in this effect.The results showed that GSH concentration was decreased concentration−dependently by acetaminophen in the in vitro P450 enzyme system.Rifampin significantly attenuated acetaminophen−mediated GSH depletion in a concentration−dependent manner.The concentration−response curve for GSH depletion of acetaminophen was shifted to the right in a parallel fashion in the presence of rifampin at the concentration of 3.2×10-5 M, which appeared to result from the competitive binding of rifampin to acetaminophen metabolites.Cytochrome c was markedly reduced by acetaminophen metabolites in this enzyme system, and GSH concentration−dependently increased the cytochrome c reduction by acetaminophen metabolites.These findings suggested that cytochrome c was reduced by the GSH conjugate of acetaminophen metabolites rather than by acetaminophen−derived superoxide anion(O2·-)and other unbound free radicals.Rifampin was shown to possess an effect similar to that of GSH.It is concluded that the decrease in GSH depletion by rifampin is most likely attributable to the binding of rifampin to the acetaminophen toxic species, and the increase in cytochrome c reduction by rifampin is attributable to the conjugate formed between rifampin and acetaminophen metabolites.
The effects of the new pulmonary surfactant secretagogue YM−40461, 1−(2−dimethylaminoethyl)−1−(3, 4, 5−trimethoxyphenyl)urea, on tracheal mucociliary transport(MCT)were assessed using guinea pigs with acute bronchitis.Acute bronchitis was induced by SO2 gas exposure(400 ppm for 3 h).MCT velocity was measured by means of the dye gelatin technique.YM−40461 at doses of 1−10 mg/kg, p.o.induced recovery of MCT function, with an ED50 value of 2.4 mg/kg.Maximal recovery(78.0±12.5%)was observed 2 gh in the animals treated with 10 mg/kg of Ym−40461.Ambroxol and bromhexine showed less effect on the MCT dysfunction than YM−40461.An artificial surfactant(Surfacten) also aided recovery.YM−40461 at a dose of 10 mg/kg, p.o.significantly improved surfactant production without affecting mucus secretion.These results show that YM−40461 ameliorates MCT dysfunction caused by SO2 exposure by activation of pulmonary surfactant secretion.
The present study observed the effects of an activation of neuropeptide Y(NPY)Y1 receptors on adrenergic and purinergic components of double−peaked vasoconstrictor responses to periarterial nerve stimulation in the isolated, perfused canine splenic arteries.The results showed that 3−30 nM Leu31 Pro34 neuropeptide Y(LP−NPY)produced a dose−dependent potentiation of double−peaked vasoconstrictor responses to trains of 30−s pulses at 1, 4 or 10 Hz of stimulation.The potentiation of LP−NPY of the nerve−stimulated vasoconstrictions were completely inhibited by subsequent blockade of α1−adrenoceptors or Y1 receptors with 0.1μM prazosin or with 1μM BIBP 3226((R)−N2−(diphenylacetyl)−N−[(4−hydroxyphenyl)methyl]−argininamide), respectively.The remaining responses in the presence of LP−NPY and prazosin were abolished by P2X receptor desensitization with 1μM α, β−methylene ATP.Moreover, 30 nM LP−NPY failed to modify the vasoconstrictor responses to nerve stimulation after treatment with prazosin.A subsequent administration of α, β−methylene ATP completely suppressed the remaining responses after prazosin and LP−NPY.The vasoconstrictions induced by 0.003−1 nmol noradrenaline and 0.003−1μmol ATP were slightly, but not significantly enhanced by 30 nM LP−NPY.The observations indicated that activation of postjunctional NPY Y1 receptors may have an important role in the modulation of adrenergic rather than purinergic transmission of the sympathetic co−transmission.
The effects of calcium polycarbophil(CP), a water−absorbing polymer, on bowel movement were examined in comparison with known laxatives and anti−diarrheal agents in dogs, a species that resembles humans for stool output.CP increased stool frequency, fecal water content and fecal weight in a dose−dependent manner, but did not induce diarrhea.Sennoside and carboxymethylcellulose sodium(CMC−Na)increased fecal water content and induced diarrhea at lower doses than that which enhanced stool frequency.Trimebutine decreased stool frequency, fecal weight and fecal water content, resulting in inhibition rather than stimulation of defecation.In sennoside−induced diarrhea, loperamide and CP improved stool consistency and this was accompanied by reduced fecal moisture and frequency of diarrhea.In contrast, CMC−Na aggravated stool consistency with increased fecal water content and frequency of diarrhea, and trimebutine had little noticeable effect apart from reducing fecal weight.Our results show that CP has both laxative and anti−diarrheal effects in dogs and differed from conventional laxatives and anti−diarrheal agents.CP may be a suitable agent for treatment of idiopathic constipation, secretory diarrhea and irritable bowel syndrome with alternating constipation and diarrhea and with either predominating in terms of less side effects such as diarrhea or constipation.
We previously reported that intrahepatic flow disturbance can be detected by vital staining of the liver with a fluorescent dye(Masuda et al., Biochem Pharmacol, 53, 1779−1787(1997)).To evaluate further use of this method, a detailed study was performed.The isolated perfused rat liver was vitally stained with rhodamine 6G(R−6G)and perfusion−fixed, and cross and horizontal sections were examined by fluorescence microscopy.In the control liver, R−6G staining was localized to periportal hepatocytes and was distributed evenly throughout the liver, indicating a homogeneous perfusion.Finer examination of the thick sections and reconstruction of a three−dimensional flow pattern revealed intricate vascular networks together with the sinusoids in different portions of the liver.In a flow−redistribution model, i.e., under hepatic nerve stimulation, the extensive flow redistribution to the deeper portion of the liver was found to occur via short branches sprouted from large portal veins, with minimal perfusion of the liver margin.Thus, visualization of hepatic microvasculature enables anatomical analysis of flow disturbance.The method is indirect but simple and may help detect intrahepatic flow disturbance that could be evoked by various factors.
We determined the effect of l−cis diltiazem, the enantiomer of diltiazem(d−cis isoform), on the energy metabolism of isolated guinea pig hearts during ischemia−reperfusion.We used 31P−NMR to measure the high−energy phosphate content and intracellular pH(pHi)during global ischemia for 30 min followed by reperfusion for 30 min.Before ischemia, the left ventricular developed pressure(LVDP)was reduced less by 10 μM l−cis diltiazem than by 3 μM diltiazem or 500 nM nifedipine.However, 10 μM l−cis diltiazem preserved the intracellular ATP content during ischemia and reperfusion, reduced the end−diastolic pressure increase during ischemia and reperfusion, and restored LVDP after reperfusion.Nifedipine at 50 nM, which reduced the LVDP more than 10 μM l−cis diltiazem, showed no cardioprotective effect.Ten micromolar l−cis diltiazem and 3 μM diltiazem, but neither 50 nor 500 nM nifedipine, reduced the pHi decrease that occurred 25 or 30 min after the onset of ischemia.Therefore, l−cis diltiazem has a cardioprotective effect on ischemic and reperfused myocardium and is less cardiodepressive than diltiazem and nifedipine.The effect of l−cis diltiazem during ischemia and reperfusion involves energy preservation, which is probably independent of its Ca2+−channel blocking action.
In order to demonstrate the localization of an ethacrynic acid−sensitive Cl- pump in the rat kidney, immunohistochemical analysis was performed using an anti−Cl- pump antibody raised against rat brain Cl- pump protein with confocal laser scanning microscopy.The antibodies against Na+, K+−ATPase, aquaporin 2 and a type B intercalated cell marker, 43−kDa protein, were also used for comparison.Anti−Cl- pump antibody recognized a 51−kDa renal protein of the same size as that in the brain on Western blots.Cl-−pump−like immunoreactivity was observed on the basolateral membranes of 42±3% of cortical collecting duct(CCD)cells and of 38±1% of outer medullar collecting duct(OMCD)cells.Such immunoreactivity in CCD was sometimes co−localized with Na+, K+−ATPase, but in OMCD, the Cl- pump−like immunoreactivity co−existed with neither Na+, K+−ATPase, aquaporin 2 nor the type B intercalated cell marker 43−kDa protein.Thus, the Cl- pump was demonstrated to be localized on the basolateral membranes of type A intercalated cells of cortical and medullary collecting ducts in the rat kidney.
The effects of Ginkgolide B(BN52021)on in vitro activation responses of human peripheral blood mononuclear cells(PBMC)from asthmatic patients was measured using 2−channel flow cytometric analysis of activation−associated cell surface antigens or ELISA assays for cytokines known to be expressed by PBMC during T1 or T2 immunological activation.BN52021 is an anti−inflammatory extract of Ginkgo biloba and has been used therapeutically.It is a known inhibititor of platelet activating factor(PAF), which is important in the pathogenesis of asthma, and may synergise with cyclosporin A(CyA)to inhibit pathogenic immune activation in asthmatics.We compared the inhibitory effects of BN52021 and CyA(1μM each)on activation of PBMC of asthmatic patients stimulated by phorbol myristate acetate and calcium inophore.Inhibition of production of the cytokines IL−4 and IL−5 by BN52021 was insignificant compared to CyA.However, BN52021 significantly reversed the increase in activation−associated CD45RA expression, with a trend towards decreased expression of HLA−DR.Lymphocyte activation markers were not significantly altered by CyA.Since they appear to have differing effects on activated cells, the anti−inflammatory effects of CyA and BN52021 in atopic asthma is potentially additive.The present approach may be useful for preliminary evaluation of novel therapeutic modalities for asthma treatment.
The effect of TEI−6363(5−[E−4−N, N−dimethylaminophenylmethylene]−4−hydroxy−2−[1−methyl imidazole−2−ilthio]−4−[4−phenylbutyl]−2−cyclopentenone), a chemically synthesized prostaglandin A1 derivative, on cell proliferation and osteoblastic differentiation was investigated concurrently.ROS17/2.8 cells(a rat osteosarcoma−derived cell line)were treated with TEI−6363 at two concentrations, 10-7 and 10-6M, and viable cells were counted to assess cytotoxic effects and determine the growth curve.After 96 h of treatment, there was no evidence of any effect of TEI−6363 on cell viability at either concentration.However, a clear inhibitory effect on cell proliferation was observed after treatment with 10-6M TEI−6363 for 24 h or longer.A pulsetreatment experiment showed that TEI−6363 induced the inhibition of proliferating ROS17/2.8 cells 24 h after addition.The inhibition of proliferation was associated with G1−arrest demonstrated by flow cytometric analysis, and incorporation of [3H]thymidine by ROS17/2.8 cells was decreased.Osteoblastic differentiation(assessed on the basis of increased alkaline phosphatase activity and collagen synthesis)was induced by TEI−6363 treatment at 10-6M following G1−arrest and inhibition of cell proliferation.These results suggest that TEI−6363 arrested the cell cycle of ROS17/2.8 cells at the G1 phase and induced osteoblastic differentiation.These results did not appear to be dependent on a marked cytotoxic effect.
Ethanol is a social drug and has been generally known to be a CNS depressant.A large fluctuation of blood alcohol concentration(BAC)is well−known to occur due to main factors such as the genetic polymorphism of the main alcohol metabolizing enzymes and the effect of blood.Few studies have substantially discussed the relationship between impaired CNS activities and BAC.In this study, focusing on the correlation of BAC, we investigated the acute effects of alcohol intake on cognitive performance in humans by objective evaluation methods consisting of the attention−demanding cognitive tasks.Tasks were administered to ten healthy male volunteers before and after ingesting established amounts of alcohol.With increased BAC, we observed prolongation of reaction time performances and lowering of a coordination performance.From the results, we concluded that cognitive performance deteriorates with an increase of BAC.Additionally, the BAC threshold that causes significant impairment of cognitive performance was estimated to be approximately 50 mg/dl(ca.10 mM).
To investigate the cellular sources of arachidonate metabolites during asthma, in vitro productions of thromboxane(TX)B2, prostaglandin(PG)D2, leukotriene(LT)B4 and cysteinyl(Cys)LTs from guinea pig eosinophils and macrophages simultaneously stimulated with the Ca ionophore A23187 were investigated.Eosinophils produced high levels of LTB4 and TXB2 and relatively low levels of CysLTs and PGD2.Although macrophages released abundant TXB2 and a little PGD2, 5−lipoxygenase metabolites were below detectable levels.In conclusion, eosinophils produced both cyclooxygenase and 5−lipoxygenase metabolites, while arachidonate metabolism in the macrophages was almost completely inclined toward the cyclooxygenase pathway.
We examined cross−sensitization of cocaine and synthetic local anesthetics to their seizure susceptibility after repeated administration.Seizure susceptibility of procaine and lidocaine increased after the end of two days of treatment with a subconvulsive dose of cocaine.Acute treatment with nomifensine but not GBR12935, a specific inhibitor of the dopamine transporter, facilitated lidocaine−induced convulsion.Furthermore, daily treatment with nomifensine for two days enhanced lidocaine−induced convulsion.These results suggest the possible involvement of the brain noradrenergic system in the changes in seizure susceptibility after repeated administration of some local anesthetics.
The effect of zaldaride, a calmodulin inhibitor, on 16, 16−dimethyl prostaglandin E2(dmPGE2)−induced intestinal ion secretion was investigated in rats.Zaldaride inhibited the dmPGE2−induced increase in water content in the colon, but not that in the small intestine.In the colonic mucosa, zaldaride attenuated the dmPGE2−induced short−circuit current;however, it did not affect the forskolin or dibutyryl cAMP−induced effect.These results suggest that zaldaride inhibits dmPGE2−induced intestinal ion secretion by reducing the activity of Ca2+/calmodulin−dependent adenylate cyclase linked to a receptor, and the colon may be an important site in the action of zaldaride.
To assess the genotoxicity of seven dental antiseptics, the ability of these agents to induce unscheduled DNA synthesis(UDS)was examined using Syrian hamster embryo(SHE)cells.Treatment of SHE cells with phenol or formaldehyde induced UDS in a concentration−dependent manner as determined by direct scintillation counting of [3H]thymidine incorporated into DNA during repair synthesis.Guaiacol or m−cresol induced UDS only in the presence of exogenous metabolic activation.p−Phenolsulfonic acid, sodium hypochlorite and p−chlorophenol failed to induce UDS in the presence or absence of exogenous metabolic activation.Our results suggest that phenol, guaiacol, m−cresol and formaldehyde are genotoxic to mammalian cells.
Pharmacological blockade of Ca2+−independent phospholipase A2(PLA2)is reported to disintegrate hippocampal synaptic plasticity, which is thought to be the cellular mechanism underlying learning and memory.Therefore, we investigated the effect of the Ca2+−independent PLA2 inhibitor bromoenol lactone(BEL)on spontaneous alteration behaviors of mice.When 3 nmol BEL was intracerebroventricularly injected 30 min prior to the test, the mice showed a poor alternation ratio, compared with control animals.The data suggest that Ca2+−independent PLA2 activity is required for spatial memory.