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Biomedical subjects

M Sugitani

Publications and source records attributed to M Sugitani.

At least 37 records · Page 2Linked to original sources

Comparison of amino acid sequences in hypervariable region-1 of hepatitis C virus clones between human inocula and the infected chimpanzee sera.

We analyzed nucleic and amino acid sequences of the hypervariable region (HVR)-1 of the hepatitis C virus (HCV) from inocula and the infected chimpanzee sera. One milliliter each of 10(-5), 10(-3) and 10(-2) dilutions of serum no. 4, and those of 10(-4), 10(-5) and 10(-6) dilutions of serum no. 6 were inoculated to six different chimpanzees. Both inocula nos. 4 and 6 contained 10(7) copies of HCV RNA/ml. The two chimpanzees inoculated with 10(-4) and 10(-5) dilutions of no. 6 were infective, while none of the dilutions from inoculum no. 4 were infective. Our results indicated that HCV RNA titer in sera did not correlate with in vivo infectivity. RNA from both inocula and the infected chimpanzee sera were extracted and transcribed to cDNA. The PCR products amplifying the HCV HVR-1 were incorporated into a vector, followed by transformation. Twenty colonies were picked up randomly from each sample. DNA was extracted and the DNA and amino acid sequences were determined. Genetic variations of 20 clones from inoculum no. 6 were divided into six groups, whereas, 40 clones from infected chimpanzees were completely identical to the major quasispecies of inoculum no. 6 in amino acid sequences. The study suggested that the major quasispecies in the diluted inocula were transmitted and replicated.

Amino Acid Sequence↗

Subdivisions of the guinea-pig accessory olfactory bulb revealed by the combined method with immunohistochemistry, electrophysiological, and optical recordings.

The presence of subgroups in vomeronasal sensory neurons has been known in various animals. To elucidate possible functional subdivisions in the guinea-pig accessory olfactory bulb, the combined studies with GTP-binding protein immunohistochemistry, electrophysiological and optical recordings were carried out. Gi2 alpha and Go alpha proteins were immunohistochemically localized, respectively, in the anterior and posterior regions of the vomeronasal nerve and glomerular layers, indicating that the guinea-pig accessory olfactory bulb receives at least two different inputs. This suggests that an anatomical boundary exists in these two layers. A mapping study of field potentials in sagittal slice preparations demonstrated that stimulation of the anterior vomeronasal nerve layer elicited field potentials with weak oscillatory responses exclusively in the anterior region of the external plexiform layer, whereas shocks to the posterior vomeronasal nerve layer provoked distinct oscillatory responses within the posterior one. The damping factors of oscillations in the anterior and posterior regions were 0.064+/-0.028 and 0.025+/-0.014, respectively. These electrophysiological results suggest that the accessory olfactory bulb consists of two functionally different subdivisions. Real-time optical imaging showed that anterior vomeronasal nerve layer shocks produced neural activity which spread horizontally from anterior to posterior only within the anterior region of the external plexiform and mitral cell layers, whereas shocks to the posterior vomeronasal nerve layer evoked periodic neural activity which spread horizontally from posterior to anterior only within the posterior region. Furthermore, the most posterior extent of the optical response evoked in the anterior region immediately adjoined the most anterior extent of that evoked in the posterior region. The maximal distance of signal propagation in the granule cell layer corresponded to that in the overlying external plexiform and mitral cell layers, indicating that the granule cell layer also has a similar boundary. Thus, these optical imaging studies not only demonstrated a precise boundary in each layer of the accessory olfactory bulb, which was positioned right beneath the boundary defined by GTP-binding protein immunohistochemistry, but also confirmed the observations from electrophysiological mapping that evoked field potentials are independently distributed in each of two subdivisions. The presence of the functional subdivision in each layer leads us to conclude that the accessory olfactory bulb in the guinea-pig is distinctly segregated into the anterior and posterior subdivisions, and to suggest that there are at least two different input output pathways in the vomeronasal system.

Action Potentials↗

[A case of primary fibrous histiocytoma of the lung].

Fibrous histiocytoma is non-epithelial malignant tumor mainly arising from soft tissue in extremities and body. Those derived from lung are rare. A 71-year-old female was admitted to our hospital because of an abnormal shadow on chest X-ray film. Preoperative diagnosis was not obtained by various examination. The tumor was located in right upper lobe (S2) and partial resection of right upper lobe was performed. Pathological diagnosis was fibrous histiocytoma of borderline malignancy. After the operation, adjuvant therapy was not done. The patient is doing well without recurrence and metastasis, during 4 years after the operation. However, careful follow-up should be necessary for long term.

Aged↗

Clinical analysis of malignant lymphomas of tonsils.

Data of 38 patients with primary tonsil lymphoma, treated during the past 14 years was analysed. All cases were non-Hodgkin lymphomas. There were 11 patients with Stage 1, 14 with Stage II, 8 with Stage III, and 4 with Stage IV tonsillar lymphomas. The applied chemotherapies were CHOP or MACOP-B regimen. The overall 5-year survival rate was 64.4%. Further analysis of the intermediate grade group showed that 5-year survival rates were 72.7%) for patients younger than 60 years old, in contrast to 35.0% for patients aged 60 or older (p 0.0049). Five-year survival rates were 100%) for Stage I, 32.4% for Stage II, 55.6% for Stage III, and 100%) for Stage IV patients (p = 0.0878). In patients with Stage II tonsillar lymphomas, 5-year survival rates were below 100% for CHOP regimen, 100% for MACOP-B regimen, 66.7% for radiation alone, and 0% for radiation followed by chemotherapy (p = 0.1966). In patients with Stage III tonsillar lymphomas, 5-year survival rates were below 100% for MACOP-B regimen, and 0% for initial radiation followed by chemotherapy (p = 0.2568). The factors influencing survival were age, stage, and treatment modality. For Stage I patients without bulky mass, radiation therapy is sufficient. For Stage II patients or Stage I patients with a bulky mass, CHOP regimen (followed by radiation) is the choice of treatment. For Stage III or IV patients,, MACOP-B regimen is promising.

Combined Modality Therapy↗

Discovery of potent cyclic GMP phosphodiesterase inhibitors. 2-Pyridyl- and 2-imidazolylquinazolines possessing cyclic GMP phosphodiesterase and thromboxane synthesis inhibitory activities.

Moderate cyclic GMP phosphodiesterase (cGMP-PDE, PDE V) inhibitor 2-phenyl-4-anilino-quinazoline (1) was identified utilizing MultiCASE assisted drug design (MCADD) technology. Modification of compound 1 was conducted at the 2-, 4-, and 6-positions of the quinazoline ring for enhancement of cGMP-PDE inhibitory activity. The 6-substituted 2-(imidazol-1-yl)-quinazolines are 1000 times more potent in in vitro PDE V enzyme than the well-known inhibitor zaprinast. The 6-substituted derivatives of 2-(3-pyridyl)quinazoline 84 and 2-(imidazol-1-yl)quinazoline 86 exhibited more than 1000-fold selectivity for PDE V over the other four PDE isozymes. In addition, cGMP-PDE inhibitors 64, 65, and 73 were found to have an additional property of thromboxane synthesis inhibitory activity.

3',5'-Cyclic-GMP Phosphodiesterases↗

Damped oscillatory activity in the guinea pig accessory olfactory bulb slice.

Extracellular field potentials were recorded from guinea pig accessory olfactory bulb (AOB) slices following electrical stimulation of the vomeronasal nerve layer (VNL). A single shock of the VNL provoked a characteristic damped oscillatory field potential, which consisted of a compound action potential followed by 6-7 periodic negative peaks (n1, n2, n3, n4, n5, n6 or n7). The average frequency of the oscillation was 32 Hz. The existence of oscillation in the AOB suggests the possibility that the oscillatory activity not only may reflect 'quantal' sampling periods, but also may be dynamically altering the AOB conditions under which incoming pheromone-like information is processed.

Action Potentials↗

[A case of localized fibrous tumor of the pleura with bloody pleural effusion].

A 25-year-old female came to our hospital with the chief complaint of right dorsal pain. On her initial chest X-ray, the tumor (5 x 3 cm) was recognized in the right lower lung field. The tumor grew rapidly during the three month period between the initial X-ray taken on her first visit and her getting admitted in the hospital. A preoperative diagnosis was done by percutaneous aspiration biopsy. Initial diagnosis of spindle cell tumor was made. Partial resection of right lower lobe with chest wall resection was carried out. There were about 500 ml of bloody pleural effusion. Macroscopically, the tumor was encapsulated, measuring 8.0 x 6.0 x 5.0 cm. The cross section was of a yellowish brown solid tumor. The histological diagnosis was of a low grade malignant fibrous mesothelioma. Only 14 cases of localized mesothelioma with pleural effusion including our case have been reported in Japan. She is still alive nine months after surgery. This patient should be carefully followed for the recurrence of the disease.

Adult↗

Immunocytochemical detection of lung cancer cells with monoclonal antibodies to 14-3-3 proteins.

Murine monoclonal antibodies were raised against 14-3-3 proteins, the antigen of human monoclonal antibody AE6F4 which had been shown potentially useful for the immunochemical diagnosis of lung cancer via sputum cytology. Enzyme-linked immunosorbent assays of the murine anti-14-3-3 monoclonal antibodies with isolated bovine brain 14-3-3 isoforms showed that the antibodies were classified into four different profiles of isoform reactivity. The comparison of 14-3-3 isoform and lung cancer tissue on the reactivity with murine monoclonal antibodies indicated that beta isoform can be responsible for cancer recognition, whereas human monoclonal antibody AE6F4 showed preferential binding to zeta isoform. No murine monoclonal antibody of the same isoform specificity as human monoclonal antibody AE6F4 was obtained. Since murine monoclonal antibodies with different isoform specificities could immunostain lung cancer cells in sputum successfully, the combination use of murine monoclonal anti-14-3-3 antibodies with human monoclonal antibody AE6F4 is potentially useful for facilitating the sputum cytodiagnosis of lung cancer.

14-3-3 Proteins↗

Signal propagation from piriform cortex to the endopiriform nucleus in vitro revealed by optical imaging.

Optical signals were recorded from the posterior piriform cortex slices of guinea pigs stained with a voltage-sensitive dye to analyse spatio-temporal spread of neural activity evoked by electrical stimulation of afferent fibers. After propagation of activity along layers II and III, an isolated island of activity appeared deep to the layer III and moved caudally. Histological inspection revealed that the area where the island appeared corresponded well to the endopiriform nucleus. The present results provided an evidence for one of the main outflows of olfactory information from the posterior piriform cortex.

Animals↗

Optical imaging of the in vitro guinea pig piriform cortex activity using a voltage-sensitive dye.

The spatio-temporal patterns of signal processing in guinea pig piriform cortex (PC) slices were analyzed by optical imaging using a voltage-sensitive dye. Slices (400 microns thick) were cut in a plane parallel to the lateral olfactory tract and perpendicular to the cortical surface. In all the anterior PC and the majority of the posterior PC preparations, neural activity elicited by electrical stimulation of layer Ia propagated along the same layer, then it invaded into layers II and III and propagated along them. In addition to the above pattern, invasion of activity into the deeper area than layer III was observed in some posterior PC preparations. Real-time imaging of an active zone evoked by Ia shocks and its spatio-temporal behavior will contribute to resolving olfactory information processing.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

Molecular cloning and heterogeneity of the human hepatitis C virus (HCV) genome.

The Japanese variant of the hepatitis C virus (HCV-N) genome, consisting of 9440 nucleotides in length, was cloned from a small amount (2 ml) of plasma from a single Japanese carrier by using RT-PCR and modified RT-PCR. The HCV-N genome has a long open reading frame that encodes a 3014 amino acid polyprotein with 340 and 57 bases of 5' and 3' non-coding sequences, respectively. HCV-N has a 4-amino-acid insertion in the NS5 region as compared to other HCV isolates, but this insertion is found to be very rare upon direct sequencing of that region. Comparative sequence analysis of all the complete and partial HCV sequences that were reported indicates that HCV can be subdivided into at least 4 groups. The HCV-N isolate has a high homology with HCV-J and HCV-BK (> 90%) and so belongs to group II, but shows less similarity to HCV-1 (> 78%, group I) and least to HC-J6 (> 67%, group III). Among these HCV isolates, the 5' non-coding region was the most conserved (> 93%) since it plays an important role in replication. The RT-PCR assay to detect HCV-RNA, using the primers deduced from this region, was very sensitive and specific. The putative core protein could become an important target for immunoassay because of a high degree of amino acid sequence similarity in that region. A high degree of diversity and a low similarity between each HCV isolate in the putative envelope protein play an important role in the chronicity of HCV infection and development of immunopreventive agents, such as immunoglobulin and vaccine for that infection.

Base Sequence↗

Sensitivity of serological assays to identify blood donors with hepatitis C viraemia.

Blood donors at high risk of hepatitis C virus (HCV) infection were tested for viraemia by the polymerase chain reaction (PCR). PCR results were accepted as positive only if reactive in 3 of 4 tests and if confirmed in an independent laboratory. The sera were also tested by 6 different assays to determine the ability of current serological assays to detect viraemic blood donors. Of 19 PCR-positive sera, only 13 (68%) were detected by the most sensitive of the serological assays. If these results are confirmed, automated PCR assays may be required for blood-donor screening to prevent transmission of HCV.

Blood Donors↗

Actions of cholinergic agonists and antagonists on the efferent synapse in the frog sacculus.

Intracellular recordings were made from hair cells in the frog saccular epithelium isolated with its innervating nerves. Inhibitory post-synaptic potentials (IPSPs) were recorded from hair cells when the efferent fibers were activated by electrical stimulation. The effects of acetylcholine (ACh), cholinomimetics, and cholinergic antagonists on the efferent synapse were studied in a preparation where the IPSPs can be observed directly. ACh or carbachol (CCh) produced a transient membrane hyperpolarization with a decrease in input resistance followed by an abolition or reduction of the IPSP. In a low Ca2+ medium where efferent synaptic activity was abolished, ACh or CCh still induced hyperpolarization, though the response appeared to be smaller than that in normal medium. Neither nicotinic (dimethyl-4-phenyl-piperazinium (DMPP), phenyltrimethylammonium (PTMA) and nicotine) nor muscarinic (muscarine, methacholine, bethanechol and oxotremorine) agonists induced the membrane hyperpolarization, but the former drugs inhibited the IPSPs while the latter drugs did not. Both d-tubocurarine and atropine inhibited the IPSP, but the d-tubocurarine was more potent, causing inhibition even at a dose of 0.5 microM while 2 microM or more atropine was needed. The ACh- or CCh-induced hyperpolarization was inhibited completely by d-tubocurarine (5 microM), but only slightly by atropine (5 microM). These results may indicate that the IPSP and the effects of ACh or CCh are based on a direct interaction between ACh or CCh and ACh receptors on the hair cells.

Acetylcholine↗

Genomic structure of the human prototype strain H of hepatitis C virus: comparison with American and Japanese isolates.

Genomic RNA from the human prototype strain H of the hepatitis C virus (HCV-H) has been molecularly cloned and sequenced. The HCV-H sequence reported consists of 9416 nucleotides including the 5' and 3' untranslated regions. HCV-H shows 96% amino acid identity with the American isolate HCV-1 but only 84.9% with the Japanese isolates HCV-J and HCV-BK. In addition to the hypervariable region (region V) previously identified in the putative E2 domain, three other variable domains were identified: region V1 (putative E1), region V2 (putative E2), and region V3 (putative NS5). These regions appear rather conserved (86-100%) among the American isolates (HCV-1 and HC-J1) or among various Japanese isolates (HCV-J, HCV-BK, HCV-JH, and HC-J4) but show striking heterogeneity when the two subgroups are compared (42-87.5% amino acid difference). A structural similarity between the 5'-terminal hairpin structure of HCV and of poliovirus was observed. This study further suggests the existence of at least two genomic subtypes of HCV and confirms a distant relationship between HCV and pestiviruses.

Amino Acid Sequence↗

ONO-5046, a novel inhibitor of human neutrophil elastase.

ONO-5046, N-[2-[4-(2,2-Dimethylpropionyloxy)phenylsulfonylamino] aminoacetic acid, competitively inhibited human neutrophil elastase (IC50 = 0.044 microM, Ki = 0.2 microM). It also inhibited leukocyte elastase obtained from rabbit, rat, hamster and mouse. However, ONO-5046 did not inhibit trypsin, thrombin, plasmin, plasma kallikrein, pancreas kallikrein, chymotrypsin and cathepsin G even at 100 microM. In in vivo studies, ONO-5046 suppressed lung hemorrhage in hamster (ID50 = 82 micrograms/kg) by intratracheal administration and increase of skin capillary permeability in guinea pig (ID50 = 9.6 mg/kg) by intravenous administration, both of which were induced by human neutrophil elastase.

Animals↗