Biomedical subjects
Publications and source records attributed to M Niwa.
The nucleotide sequences of the promoter region for rat inducible nitric oxide synthase (iNOS) were elucidated. The sequences were highly homologous with the mouse iNOS gene. The regulation of the iNOS gene may be distinguished between rodent and mammalian.
1. Contribution of nitric oxide to the convulsive seizures induced by fluoroquinolones (FQs) coadministered with 4-biphenyl acetic acid (BPAA), the active metabolite of fenbufen, was assessed in mice. 2. Enoxacin + 4-biphenyl acetic acid caused clonic seizures in all treated mice, followed by tonic seizures and death. These events were associated with a significant increase in intracerebellar cyclic GMP. 3. Pretreatment with the nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine methylester (L-NAME), but not with D-NAME, significantly reduced the incidence of convulsions and lethality, as well as the increase in cyclic GMP. 4. Pretreatment with N-methyl-D-aspartic acid (NMDA)-receptor antagonist, MK-801, inhibited only the transition of clonic seizure to tonic seizure without affecting the incidence of clonic seizure and lethality. 5. These findings suggest that FQs + BPAA exert convulsions by activating NOS partly through the mediation of the NMDA receptor in the brain cells.
Seven days after an intracerebroventricular injection of 0.8 microgram kainic acid, a time of neural tissue-repair after damage, we applied our receptor autoradiographic method to examine changes in the endothelin receptors in kainic acid-induced neural lesions of the rat brain. There were belt-shaped areas with the de novo expressed [125I]endothelin-1 binding sites in the damaged hippocampus CA1, CA3, and CA4 subfields. We also noted a homogeneous zone with a low binding-density, the area sandwiched by the belt-shaped areas. In a "remote" area corresponding anatomically to the deep soma layer of the piriform cortex plus lateral parts of amygdaloid complex we noted a well-defined area with "punched hole-figure" of low density [125I]endothelin-1 binding sites. The lesion was surrounded by areas rich in binding sites. The de novo expressed [125I]endothelin-1 binding sites were characterized endothelin B receptor. Microglia were present in the area with "punched hole-figure" and in the hippocampus pyramidal cell layer with neuronal death. In contrast to microglia, astrocytes were rich with hypertrophia in kainic acid-induced neural lesions anatomically corresponding to areas with the de novo endothelin B receptor. Taken together with the present observations of microscopic evidence of cellular distribution, we suggest that the de novo expressed endothelin B receptor was carried by astrocytes aggregating in neural lesions. In light of our findings, the possibility that astrocytes can be activated by the endothelin B receptor in response to neural tissue repair after damage to neurons would have to be considered.
A set of the title compounds having different substituents (R1, R2) on their phenyl groups was synthesized to find sigma receptor binding affinity. Among the compounds, 2b (R1 = R2 = Cl) has the most potent sigma 1-binding activity, while 2a (R1 = R2 = H, SA4503) was most selective to sigma 1 over sigma 2 receptor. The crystal structures of 2a and 2b were shown, by X-ray crystallography, to be similar except for the one torsional angle of their propylene parts. Quantitative structure-activity relationship study suggested the affinity of the compounds to the sigma 1 receptor was dependent on the electronic feature, Swain-Lupton's R or Sz that was derived by molecular orbital method, of R1 and R2.
A new approach was developed to embody the alpha-helical protein structure having an arbitrary combination and arrangement of helices by the successive ligation of a haloacetyl peptide segment with a cysteinyl peptide. A four-helix-bundle protein was efficiently constructed by the repetitive ligation of alpha-helical peptide segments. The use of HPLC-purified unprotected peptide segments facilitated the purification of the intermediates to afford the highly homogeneous desired protein. The use of the bromoacetyl moiety and the chloroacetyl moiety for the ligation was judged to make no difference in practice. A trial of introducing an additional intramolecular disulfide cross-link was also examined. The resulting protein showed high stability in the chaotropic and thermal denaturation and in enzymatic degradation.
1. The receptor autoradiographic method done on the rat lower brain stem and cerebellum plus 125I-endothelin-1, BQ-123, an antagonist for the endothelin ETA receptor, and sarafotoxin S6c, an agonist for the ETB receptor, revealed minute amounts of the ETA receptor coexisting with the ETB receptor in the caudal solitary tract nucleus of the rat lower brain stem. 2. The ETB receptor is present predominantly in other parts of the lower brain stem. 3. Knowledge of the heterogeneous distribution of the central endothelin receptor subtypes aids in understanding the neurophysiology of endothelins.
1. The present study investigated the inhibitory effect of losartan, a type 1 angiotensin II (AT1) antagonist, and of combined treatment with losartan and GR144053, a fibrinogen receptor (GPIIb/IIIa) antagonist, on neointima formation subsequent to vascular injury in the hamster carotid artery. Vascular injury was achieved by a roughened-tip 2F catheter and the neointimal area was measured up to 2 weeks inducing the injury. 2. Compared to non-treated hamsters (intimal area (IA/internal elastic laminal area (IELA) ratio = 60.3 +/- 5.9%, n = 12), losartan dissolved in drinking water (1, 3 and 10 mg kg-1 per day, n = 8 each) reduced neointimal area dose-dependently, a significant decrease (IA/IELA = 39.7 +/- 5.6%) being attained with the highest dose when it was administered from 1 day before injury. However, neointima formation was not prevented even with the highest dose of losartan when the administration was started after injury. 3. When the administration of GR144053 (1.0 mg kg-1 per hour) via an implanted osmotic pump was started 30 min before the injury and continued for the next 2 weeks, no suppression of neointima formation was observed, although platelet aggregation evoked ex vivo by adenosine diphosphate (ADP) at the end of treatment period was efficiently inhibited. 4. In separate experiments in which 5-bromo-2-deoxy-Uridine (BrdU) was used to test smooth muscle cell (SMC) proliferation 1 and 7 days after injury, the ratio of SMC proliferation in the injured area was only slightly decreased by losartan when its administration was started after the injury, despite the marked reduction of SMC proliferation when treatment was started before the injury. Treatment with GR144053 as indicated above also significantly decreased the SMC proliferating index 1 day after the injury. 5. To examine the potential benefit of the coadministration of the GPIIb/IIIa antagonist with the AT1 receptor antagonist, GR144053 (1.0 mg kg-1 per hour) was combined with post-injury treatment with losartan (10 mg kg-1 per day). This markedly reduced the proliferation of SMCs and significantly decreased the neointimal area (IA/IELA = 31.2 +/- 4.6%) measured 2 weeks following the catheterization. 6. According to the results of a time-dependent study in which GR144053 was given in combination with post injury treatment with losartan for 1, 3, 7 or 14 days, neointima formation could be reduced by treatment with GR144053 for just 7 days. 7. In conclusion, GR144053, a fibrinogen receptor antagonist, enhanced the inhibitory effect of losartan, an AT1 receptor antagonist, on neointima formation in the damaged carotid artery of hamsters.
To determine the effect of phencyclidine (a noncompetitive NMDA receptor antagonist) on expression of Bax and Bcl-2 (apoptosis-regulating proteins) in gerbil hippocampus after transient forebrain ischemia, brain sections were immunohistochemically evaluated 48, 72, 96 h and 7 days following ischemia. In ischemic control animals, the expression of Bax in CA1 neurons was increased with time and peaked at 72 h, then disappeared at 96 h. In the phencyclidine (5 mg kg-1, intraperitoneally)-treated animals, the intensity of Bax expression at 72 h was weaker than that of ischemic control animals. Furthermore, at 96 h, Bax expression was still observed in CA1 neurons. No expression of Bcl-2 in the CA1 neurons was detected in either control or phencyclidine-treated animals. From these results, it is possible that NMDA receptor antagonists exert their preventive effect against delayed neuronal death through inhibition of Bax protein expression, although the precise relationship between the function of Bax protein and delayed neuronal death is still unclear.
Lidocaine and tetracaine suppress superoxide anion (O2-) generation of neutrophils. We examined the effects of eight local anesthetics on O2- generation in human neutrophils and searched for a potential relationship between the biological activities and the physicochemical properties of presently available eight local anesthetics. Human neutrophils incubated with local anesthetic and a Cypridina luciferin analog as a O2(-)-specific chemiluminescence probe were stimulated by phorbol ester. The chemiluminescence development based on O2- generation was monitored by a luminometer. All of the tested local anesthetics suppressed O2- generation in a concentration-dependent manner. The concentration of each of eight local anesthetics that produced 50% inhibition of peak chemiluminescence (IC50) had a rank order of dibucaine < tetracaine < bupivacaine < ropivacaine < procaine < mepivacaine < lidocaine = prilocaine. A linear relationship was obtained between IC50 values and the values of logarithm of partition coefficient (log P) of eight local anesthetics; log (IC50 in molarity) = -1.252 - 0.514 x log P, r2 = 0.891, P < 0.001. Unlike with staurosporine, which inhibits protein kinase C (PKC), no effect was observed on the O2- generation in the presence of tetrodotoxin (TTX), veratridine (VTD), or amiloride. These results suggest that the inhibitory effects of local anesthetics on O2- generation of neutrophils are predicted by physicochemical properties of the drugs, especially partition coefficients.
A non-spore-forming, coryneform bacterium, strain TA-12T, was isolated from spoiled off-flavor orange juice. Growth of this organism occurs at pH 3.2 to 7.5, and optimum growth occurs at pH values between 5.5 and 6.5. This organism produces lactic acid, propionic acid, and acetic acid from glucose. It is catalase negative. The cells are heat resistant and can withstand a temperature of 90 degrees C for 10 min. The DNA G + C content is 66.8 mol%. This strain has as MK-9(H4) respiratory quinone system and contains meso-diaminopimelic acid in its cell wall, and omega-cyclohexyl undecanoic acid is the major cellular fatty acid. The results of a phylogenetic analysis of the 168 rRNA gene of this organism indicated that its highest level of homology is its level of homology with the representative of the classical propionibacteria, Propionibacterium freudenreichii (97.1%). Strain TA-12T is phenotypically similar to P. freudenreichii, but it produces a large amount of lactic acid and has a distinct fatty acid composition, acid tolerance, and heat resistance, which differentiate it from P. freudenreichii and other propionic acid-producing bacteria. On the basis of these findings we propose the name Propionibacterium cyclohexanicum sp. nov. for this organism. The type strain is TA-12 (= IAM 14535 = NRIC 0247).
Recent studies have suggested that apoptosis is a key phenomenon in the chemopreventive action of nonsteroidal antiinflammatory drugs (NSAIDs), which exhibit cancer-preventive and tumor-regressive effects in the human colon. The effect of NS-398, N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide, which is a selective inhibitor of cyclooxygenase-2 (COX-2), on the induction of apoptosis in two human colorectal cancer cell lines (Colo320 and THRC) was determined. The apoptotic ratios (-fold vs. control value) of Colo320 in the presence of 100 microM indomethacin and NS-398 were 3.3 +/- 1.5 and 9.0 +/- 0.94, and those of THRC were 2.3 +/- 0.46 and 7.4 +/- 0.87, respectively. The ability of NS-398 to induce apoptosis is greater than that of indomethacin. Both indomethacin and NS-398 reduced the cell proliferation in a concentration-dependent manner. The IC50 values of NS-398 (54.8 +/- 3.6 and 77.2 +/- 4.9 microM) were significantly lower than those of indomethacin (206.3 +/- 43.0 and 180.3 +/- 22.6 microM) at P < 0.01 in Colo320 and THRC cell lines, respectively. These findings suggest that NS-398, a selective inhibitor of COX-2, is a possible candidate for a chemopreventive agent with a potent apoptosis-inducing effect and low ulcerogenic activity.
We have purified L-sorbose dehydrogenase (SDH) and L-sorbosone dehydrogenase (SNDH) from Gluconobacter oxydans T-100 that showed an ability to convert D-sorbitol to 2-keto-L-gulonate (2-KLGA). A genomic library of Gluconobacter oxydans T-100 was screened with a probe, a 180-bp PCR product which was obtained from degenerate oligodeoxyribonucleotides based on the elucidated sequence of the purified SDH (used as primers) and the genomic DNA of G. oxydans T-100 (used as a template). From sequencing of the DNA from a clone positive to the probe, the SNDH and the SDH were estimated to be coded in sequential open reading frames with 1,497 and 1,599 nucleotides, respectively, which was confirmed by expression of the DNA in Escherichia coli that showed both enzymatic activities. The DNA was introduced to a shuttle vector which was prepared from a plasmid of G. oxydans T-100 and pHSG298 to obtain an expression vector designated pSDH155. The production of 2-KLGA by pSDH155 in G. oxydans G624, an L-sorbose-accumulating strain, was improved to 230% compared to that of G. oxydans T-100. Chemical mutation of the host strain to suppress the L-idonate pathway and replacement of the original promoter with that of E. coli tufB resulted in improving the production of 2-KLGA. Consequently, high-level production from D-sorbitol to 2-KLGA (130 mg/ml) was achieved by simple fermentation of the recombinant Gluconobacter.
Silkworm larvae plasma (SLP) reagent is activated by peptidoglycan (PG), a fragment of both the gram-positive and gram-negative bacterial cell wall, as well as beta-glucan (BG), a component of fungi. It is possible to measure contamination of gram-positive bacteria quantitatively by combining the conventional limulus amebocyte lysate (LAL) and PG measurement methods. Therefore, a more highly accurate analysis of dialysate can be made using both SLP and LAL methods to detect endotoxin (ET) and/or PG contamination. We studied the effects of contaminated dialysate on human peripheral blood mononuclear cells (PBMC) by producing various cytokines in vitro. Muramyl dipeptide (MDP) was used as the biologically active minimum constituent of PG. A total of 54 dialysate samples were obtained under sterile conditions from 4 sites: (1) reverse osmosis water unit; (2) proportioning unit; (3) multiple dialysate preparation console, and (4) personal dialysate preparation console, at 9 dialysis facilities. To detect bacterial contamination, the samples were measured with LAL(C), LAL(G) and SLP methods. PBMC were collected from 10 healthy controls and from 10 hemodialysis patients and cultured for 24 h with ET, MDP, ET + MDP and contaminated dialysate. IL-1 receptor antagonist (IL-1Ra), IL-1 beta and TNF-alpha in the culture medium supernatants were measured using the ELISA method. PG was not detected in dialysate from sites 1 or 2. However, dialysate from the inlet of the dialyzer at the bedside monitor of the central supply and personal console showed 4.1 +/- 6.1 ng/ml for site 3 (in 7 of 18 samples) and 3.3 +/- 4.6 ng/ml for site 4 (in 3 of 18 samples). Contamination by PG alone and complex contamination by PG and ET were also detected. Furthermore, IL-1Ra, IL-1 beta and TNF-alpha production by PBMC increased in accordance with the concentrations of MDP. Cytokine production was enhanced 5-10 times more where MDP and ET coexisted than where either MDP or ET existed alone, showing the synergic effects of MDP and ET. Based on these results, there is a high possibility that PG may also be a pyrogen in the dialysate prior to this study. ET had been considered the only pyrogen in dialysate. Therefore, it is essential to recognize the existence of both ET and PG in investigating dialysate contamination.
In an attempt to develop gene therapy for thyroid carcinomas, the present studies were undertaken to evaluate in vitro and in vivo therapeutic efficacy and toxicity of herpes simplex virus thymidine kinase (HSV-tk) gene and ganciclovir (GCV) treatment, a widely used prodrug/suicide gene therapy, in human thyroid carcinoma cell lines, FRO and WRO cells, using a means of retrovirus-mediated gene transduction. In vitro experiments demonstrated dose- and time-dependent cell killing by transduction of the HSV-tk gene followed by GCV treatment. The IC50 (the concentration required to elicit 50% growth inhibition) shifted from 250 to 0.5 mg/liter in FRO cells, and from 3,000 to 0.09 mg/liter in WRO cells with therapeutic indexes of 500 and 33,000, respectively. Treatment with 30 mg/liter GCV for 4 days led to complete cell death in HSV-tk tumor cells. Nontransduced cells mixed with transduced cells were also effectively killed by GCV (bystander effect). Low concentrations of GCV, which alone showed little cytotoxicity, enhanced radiation-induced cytotoxicity (radiosensitization). In vivo sc FRO-tk tumor models in nude mice also showed dose- and time-dependent tumor regression. The IC50 was less than 2 mg/kg, and treatment with 100 mg/kg GCV for 2 weeks completely eradicated all tumors. The bystander effect and radiosensitization were also obtained in vivo. These results suggest that the HSV-tk/GCV approach to human thyroid carcinoma cells appears to be very efficacious, with a wide therapeutic range, and exerts a bystander effect and radiosensitization both in vitro and in vivo. Thus, HSV-tk/GCV system, alone or in combination with radiotherapy, may be a promising suicide gene therapy for thyroid carcinomas.
Patients with antiphospholipid antibody syndrome manifesting as neurological symptoms (one patient with twice repeated cerebral ischemic symptoms in the left parietal and frontal lobes, and the other patient with cerebral infarction of the right temporal lobe) and non-neurological symptoms (one patient with occlusion of the superior mesenteric artery and the other patient with thrombophlebitis) underwent cerebral blood flow (CBF) studies using xenon-enhanced computed tomography. The patients with neurological symptoms had decreased CBF at the affected sites. The patients with non-neurological symptoms had decreased CBF in the left parietal and temporal lobes and/or corona radiata. Subtle preclinical thrombosis may cause chronic cerebral hypoperfusion in patients with antiphospholipid antibody syndrome.
Three patients presenting with subarachnoid hemorrhage due to aneurysmal rupture underwent cerebral blood flow (CBF) measurements before and after cisternal injection of papaverine hydrochloride. One patient showed prominent increases in CBF in the frontal lobe and basal ganglia after injection of papaverine, but paradoxical decreases in the parietal lobe and corona radiata. The other two patients had poor CBF response. Dense clotting in the basal cisterns may have prevented diffusion of the agent so that only the proximal trunks of the internal carotid, anterior cerebral, and middle cerebral arteries were dilated in the former case. The dilation of proximal trunks of cerebral arteries might steal blood flow from the parietal lobe and corona radiata, where the intraparenchymal arteries were maximally dilated and cerebrovascular reserve capacity was poor.
The effect of glycerol on blood flow in tumoral and peritumoral tissue was measured in 32 patients with brain tumor, 17 gliomas and 15 meningiomas. Blood flow before and after the administration of glycerol was measured by stable xenon-enhanced computed tomography. The tumor part of glioma was significantly hypoperfused. In contrast, the tumor part of meningioma was significantly hyperperfused. Peritumoral edema of both glioma and meningioma was hypoperfused. After the administration of glycerol, blood flow increased in all regions except for the tumor part of glioma. Vascular responses to glycerol may be different in these two tumor types. The steal phenomena of blood flow might occur in cases of glioma.
The antithrombotic and restenosis-preventing effects of FK633, an inhibitor of platelet aggregation via binding to the glycoprotein (GP) IIb/IIIa receptor, were studied, IC50 value of FK633 against platelet aggregation ex vivo induced by 2.5 microM adenosine diphosphate (ADP) was 5.4 x 10(-7) M as determined using hamster platelet rich plasma. The inhibitory effect was also investigated in vivo on thrombus formation at the carotid arterial wall injured by a modified catheter. As a control, the left carotid artery was injured and the time required to develop a thrombotic occlusion (3.9 +/- 1.1 min, mean +/- S.E.M., n = 18) was determined. Then, the right carotid artery of the same animal was injured while a continuous intravenous (i.v.) infusion of FK633 was administered at doses of 0 (saline), 0.1, 0.3 or 1.0 mg/kg/h. The time to occlusion was dose-dependently prolonged. In a separate experiment, 10% of the total tPA dose (0.52 mg/kg) was injected into the injured artery as a bolus and the remaining was infused i.v. at a constant rate for 30 min. When FK633 (0.3 or 1.0 mg/kg/h) was infused together with tPA, late patency of the reperfused artery was much improved as compared with that of treatment with tPA alone. Bleeding time, measured at the end of the tPA infusion, was markedly prolonged when the higher dose of FK633 (1.0 mg/kg/h) was coadministered, however coadministration of the lower dose of FK633 (0.3 mg/kg/h) was almost without prolongation on the bleeding time, despite a significant effect on the vascular patency after thrombolysis. Next, neointima formation was evaluated 2 weeks after the vascular injury. When FK633 (0.3 mg/kg/h) was continuously infused i.v. by an implanted osmotic pump for 3, 7 or 14 days after the vascular injury, the neointimal area formation was significantly suppressed in the treatment groups for 7 or 14 days. These findings suggest that FK633 inhibits platelet activation in the injured artery and improves vascular patency after thrombolysis with tPA with a concomitant suppression of neointima formation.