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Biomedical subjects

W Watanabe

Publications and source records attributed to W Watanabe.

At least 19 recordsLinked to original sources
PubMed2001

Establishment of CHO cell lines expressing four N-methyl-D-aspartate receptor subtypes and characterization of a novel antagonist PPDC.

To develop an assay system that allows the N-methyl-D-aspartate (NMDA) receptor subtype-selective antagonistic potency of drugs, we have established Chinese hamster ovary cell lines expressing the four NMDA receptor subtypes (GluRepsilon1/zeta1-GluRepsilon4/zeta1) heat-indelibly. Using these clonal cells, we found that a novel antagonist, (1S,2R)-1-phenyl-2[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide, was less selective for the GluRepsilon1/zeta1: the IC(50) values for the GluRepsilon1/zeta1-GluRepsilon4/zeta1 were 41.7, 13.3, 12.6 and 11.5 microM, respectively, while two well-known antagonists, DL-2-amino-5-phosphonovaleric acid and ifenprodil, showed the known potency and selectivity for each subtype. Thus, the established clonal cells are of use in characterizing the pharmacological properties of drugs that act on NMDA receptors.

Animals↗
PubMed2001

Autologous bone grafting without screw fixation for tibial defects in total knee arthroplasty.

We evaluated 30 knees with autologous bone grafts, performed without screw fixation, for tibial defects in total knee arthroplasty (TKA). The tibial defects were classified into three types: contained, flat peripheral, and slant peripheral. The resected femoral condyle was fixed with a combination of bone cement and the tibial component, without using screws. The patients were followed for an average of 6 years and 10 months. In all knees except 1, the grafted bone united and formed good continuity with the tibial floor. Autologous bone grafting without screw fixation is a simple and effective method to deal with the tibial defects in primary TKA, especially for contained and flat peripheral defects.

Aged↗
PubMed2001

A rare anatomic variant of the superior glenohumeral ligament.

The attachment of the superior glenohumeral ligament (SGHL) to the upper pole of the glenoid is variable and 3 types have been described. We report an anatomic variant of SGHL attachment to the upper pole of the glenoid that has not heretofore been reported in the literature. In this case, the SGHL overrode the biceps origin, continued to the superior labrum posteriorly, and had no attachment to the middle glenohumeral ligament or the anterior labrum. This variant was detected during routine arthroscopic examination undertaken before surgery on a rotator cuff tear.

Adult↗
PubMed2001

Mechanism of selective inhibition of respiratory syncytial virus by a benzodithiin compound (RD3-0028).

RD3-0028, a compound with a benzodithiin structure, was found to be a potent inhibitor of respiratory syncytial virus (RSV) replication. Its action is specific; no activity is seen against influenza A virus, measles virus, herpes simplex virus type 1 or 2, or human cytomegalovirus. A time-dependent drug addition experiment indicated that the antiviral activity occurs in the late stage of the RSV replication cycle, since this compound completely inhibited syncytium formation even when added up to 16 hr after the infection of cell monolayers at an MOI of 3. RD3-0028 had no direct virucidal effect on RSV. Western blotting analysis showed that RD3-0028 significantly decreased the amount of RSV proteins released into the cell culture medium. Moreover, five independent isolates of the RSV long strain were selected for growth in RD3-0028 (5-20 microg/ml). These resistant viruses were more than 80-fold less sensitive to RD3-0028 than the long strain. The F gene segment of each of these viruses was sequenced and in each case the mutant RNA segment contained at least one sequence alteration, converting asparagine 276 to tyrosine (F1 protein). These results suggest that RD3-0028 inhibits RSV replication by interfering with intracellular processing of the RSV fusion protein, or a step immediately thereafter, leading to loss of infectivity.

Amino Acid Sequence↗
PubMed2001

Inhibitory effect of low density lipoprotein on the inflammation-inducing activity of calcium pyrophosphate dihydrate crystals.

OBJECTIVE: It has been proposed that low density lipoprotein (LDL) plays a role in the self-limiting nature of pseudogout inflammation. We investigated changes of LDL concentration in rat air pouch fluid during periods of acute and subsiding inflammation to evaluate whether LDL contributes to inhibiting inflammation of pseudogout. We examined whether LDL binds to calcium pyrophosphate dihydrate (CPPD) crystals as a possible mechanism for reduction of inflammation. METHODS: In this in vivo study, 5 mg suspensions of CPPD crystals and saline were injected into the rat air pouch. Fluid samples were taken from rat air pouch at 0, 3, 6, 12, 24, and 48 h after injection. White blood cells in the samples were counted; the remaining fluid was centrifuged and concentrations of beta-glucuronidase and PGE2 in the supernatant were measured as inflammatory markers. LDL in the supernatant was immunochemically identified by Western blotting, then pellets containing crystals were examined by the same technique. RESULTS: LDL was identified in the air pouch 3 h after CPPD crystal injection, and its concentration increased and reached a peak level after 24 h. Inflammatory markers reached maximal level from 6 to 12 h, then decreased after 24 h. In the pellets containing crystals, LDL could not be identified in every specimen. CONCLUSION: LDL in the rat air pouch increased during the inflammatory course induced by CPPD crystal and the inflammation subsided as the LDL increased. Since some reports indicate LDL was related to reduction of crystal induced inflammation such as gout or pseudogout, we concluded that LDL could contribute to the resolution of acute pseudogout arthritis in vivo with or without binding to CPPD crystals.

Animals↗
PubMed2000

Intra-articular membranous interposition detected by MRI in developmental dysplasia of the hip.

Intra-articular membranous interposition was detected by MRI in the hip joint with residual subluxation of a girl aged 5 years 10 months. This structure, which had low signal intensity on both T1- and T2-weighted images, separated the femoral head from the acetabulum. Histological examination revealed chondrometaplasia, which suggested that this interposition might be transformed to a surface cartilaginous tissue of the secondary acetabulum often observed in residual subluxation of the hip.

Acetabulum↗
PubMed2000

Distribution of acid sphingomyelinase in human various body fluids.

Enzyme activities of acid sphingomyelinase (ASM) were determined in various human cell-free body fluids, serum, cerebrospinal fluid, urine, salivary fluid, tear fluid, and synovial fluid, using assay buffers with or without Zn2+ -cation. Although ASM activity was not detected in the cerebrospinal fluid, the other fluids demonstrated significant enzyme activities of ASM. All ASMs detected in the fluids were stimulated by the addition of Zn2+ -cation, suggesting that those enzymes are secretory ASM derived from ASM gene. We suggest a possible enzymatic diagnosis of Niemann-Pick disease types A and B using those body fluids. Interestingly, salivary and tear fluids showed much higher activities of ASM than those of the other fluids. Because sphingolipids, especially sphingomyelin, are major constituents of a normal diet, especially, milk, eggs, and meat products, we suggest that ASM in the salivary gland may play an important role in the digestion of sphingomyelin in a normal diet.

Adult↗
PubMed1999

Efficacy of RD3-0028 aerosol treatment against respiratory syncytial virus infection in immunosuppressed mice.

RD3-0028, a benzodithiin compound, has antiviral activity against respiratory syncytial virus (RSV) in cell culture. We used a mouse model of RSV infection to determine the in vivo effect of RD3-0028. Cyclophosphamide (CYP)-treated, immunosuppressed mice were inoculated intranasally. The lungs of the mice were removed on day 4. The virus titers of the lungs of RD3-0028-treated mice were compared to the virus titers of the lungs of virus-inoculated, untreated control mice. In an effort to increase the therapeutic effectiveness of this compound, RD3-0028 was administered by aerosol to RSV-infected mice by using a head-exposure system. Aerosols generated from reservoirs containing RD3-0028 (7 mg/ml) administered for 2 h twice daily for 3 days significantly reduced the pulmonary titer of RSV-infected mice. It is clear that the minimal effective dose of RD3-0028 for RSV-infected mice is significantly less than that of ribavirin, the only compound currently available for use against RSV disease. Furthermore, the RD3-0028 aerosol administration appeared to protect the lungs of infected, CYP-treated mice against tissue damage, as evidenced by the preservation of the lung architecture and a reduction in pulmonary inflammatory infiltrates. RD3-0028 aerosol was not toxic for mice at the therapeutic dose. The present study demonstrates the effectiveness of aerosol administration of RD3-0028 for RSV-infected mice.

Aerosols↗
PubMed1999

Mouse model of respiratory syncytial virus infection to evaluate antiviral activity in vivo.

In this study, we have developed a practical mouse model for evaluating in vivo the antiviral activity of compounds against respiratory syncytial virus (RSV) infection. BALB/c mice are not particularly susceptible to RSV infection; however, infection rates were improved by pretreatment with the immunosuppressive agent cyclophosphamide (CYP). When mice were inoculated intranasally with RSV A2 strain, the pulmonary RSV titres of CYP-pretreated 10-week-old mice were higher than those of untreated 10-week-old and 28-week-old mice, peaking on days 4 and 5 post-infection. Sections of lung from RSV-infected mice pretreated with CYP, taken on day 4 post-inoculation, showed widespread evidence of interstitial pneumonia and other significant pathological changes. We also confirmed that ribavirin, a representative antiviral agent, significantly reduced the pulmonary RSV titres of mice pretreated with CYP when administered intraperitoneally.

Animals↗
PubMed1998

Novel anti-respiratory syncytial(RS) viral compounds: benzodithiin derivatives.

Benzodithiin derivatives are highly potent and specific inhibitors of respiratory syncytial virus (RSV) replication in vitro. The most potent and selective congener of a benzodithiin derivative is 1,4-dihydro-2,3-benzodithiin(RD3-0028). According to the modified 3-(4,5-dimethylthiazole-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) assay developed in our laboratories, this compound has a 50% effective concentration of 4.5 microM and a 50% cytotoxic concentration of 271.0 microM, which is superior to that of ribavirin. This compound also inhibits RSV strain subgroups A and B and clinical isolates. RD3-0028, however, does not inhibit the replication of influenza A virus, measles virus, herpes simplex virus types 1 and 2, or human cytomegalovirus. Two other benzodithiin derivatives [1,4-dihydro-6-methyl-2,3-benzodithiin (RD3-0270) and 1,4-dihydro-5-methyl-1-2,3-benzodithiin (RD3-0284)] also inhibit RSV replication at a selectivity index greater a factor of 20. These results suggest that the benzodithiin skeleton is an important structure for inhibitory activity against RSV replication.

Antiviral Agents↗
PubMed1997

Inducible expression of N-methyl-D-aspartate (NMDA) receptor channels from cloned cDNAs in CHO cells.

To develop a drug screening system, we introduced expression vectors carrying the mouse N-methyl-D-aspartate (NMDA) receptor channel epsilon1 and zeta1 subunit cDNAs under the promoter of the Drosophila heat shock protein hsp70 into Chinese hamster ovary (CHO) cells. We selected clonal cell lines by means of RNA blot hybridization and fura-2 fluorometry. One of these cell lines, ZE1-1, optimally expressed the epsilon1 and zeta1 subunit mRNAs when induced by an incubation at 43 degrees C for 2 h. Heated ZE1-1 cells exhibited the NMDA-induced intracellular Ca2+ elevation, whereas unheated they showed no such response. NMDA and L-glutamate, but not alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate, induced an increase in the intracellular Ca2+ concentration. The response to the agonists was marginal in the absence of glycine, and diminished by Mg2+ and NMDA receptor antagonists. Furthermore, exposure to agonists of ZE1-1 cells expressing the epsilon1/zeta1 NMDA receptor channel resulted in the release of lactate dehydrogenase (LDH) activity in the culture medium indicating agonist-induced cell death. NMDA receptor antagonists inhibited the LDH activity release. These results suggest that ZE1-1 cells will provide a useful screening system for novel drugs acting on the epsilon1/zeta1 NMDA receptor channel.

Animals↗
PubMed1995

Use of lactate dehydrogenase to evaluate the anti-viral activity against influenza A virus.

The detection of lactate dehydrogenase (LDH) can be used to evaluate efficiently anti-influenza A virus agents. LDH levels in the virus-infected Madin-Darby canine kidney cell cultures were significantly higher than in controls, were in proportion to the degree of virus infection, and corresponded to a decrease in mitochondrial dehydrogenase activity as assayed using a tetrazolium colorimetric assay (MTT method). The EC50 value and cytotoxicity of ribavirin, 3-deazaguanine, pyrazofurin, and carbodine against influenza A virus as measured by the LDH detection method was equivalent to that derived by the MTT method.

Amides↗
PubMed1995

Aberrant insulinoma of the duodenal bulb.

A rare insulin-immunoreactive neuroendocrine tumor of the duodenum in a 54 year old male is reported. The incidentally identified tumor was located on the anterior free wall of the duodenal bulb and measured approximately 6 mm in diameter. Uncomplicated endoscopic resection of the tumor was carried out. The lesion exhibited classic histologic features of insulinoma of the beta-islet cell type with stromal amyloid deposition. In addition to positive reactivities of chromogranin A, neuron-specific enolase, synaptophysin, Leu 7 (CD57), cystatin C, CA15-3 and cytokeratin, the non-argyrophilic tumor cells were strongly immunoreactive for insulin and C-peptide. The stromal amyloid was clearly labeled for amylin. A few cells were stained for somatostatin, whereas other hormones were negative. Interestingly, a few isolated insulin-positive cells were identified in the non-neoplastic duodenal mucosa in the proximity of the tumor. Immunoelectron microscopy using paraffin sections disclosed insulin-immunoreactive secretory granules in the cytoplasm. The patient exhibited no signs or symptoms of hypoglycemia. Serum insulin levels were not measured prior to resection. No tumors were demonstrated in the pancreas. Magnetic resonance imaging revealed a 1 cm asymptomatic pituitary mass, in association with moderately elevated serum prolactin levels. The patient is currently being followed up in the outpatient clinic.

Duodenal Neoplasms↗
PubMed1995

A colorimetric LDH assay for the titration of infectivity and the evaluation of anti-viral activity against ortho- and paramyxoviruses.

A rapid and precise screening assay was developed for in vitro evaluation of anti-orthomyxo- and anti-paramyxovirus agents. The procedure is spectrophotometrical assessment for viability of cells via extracellular leakage of lactic dehydrogenase (LDH). HMV-II cells, a human melanoma cell line was found to be suitable for the titration of virus infectivity and screening of anti-viral agents for orthomyxo- and paramyxoviruses. Comparative titration of infectivity of stock viruses by the LDH and the MTT in site reduction of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) methods with HMV-II cells as well as plaque titration with MDCK, Vero and HeLa cells was carried out. The LDH method was comparable or more sensitive for influenza viruses (FLUV)-A, B, C, parainfluenza viruses (PFLUV)-1, 2 and less sensitive for PFLUV-3, mumps virus (MPSV), measles viruses (MLSV) and respiratory syncytial virus (RSV) than the plaque titration. The 50% effective concentration (EC50) of 1-beta-D-ribofuranosyl-1, 2, 4-triazol-3-carboxamide (ribavirin) and 5-ethynyl-1-beta-D-ribofuranosyl-imidazole-4-carboxamide (EICAR) against orthomyxo- and paramyxoviruses were examined comparatively by the LDH, MTT and plaque reduction (PR) methods. The EC50 values of FLUV-C and PFLUV-1 were able to be evaluated only by the LDH but not by the MTT and PR methods. The LDH method with HMV-II cells simplifies the assay procedure and permits the evaluation of a large number of compounds for anti-orthomyxo- and anti-paramyxoviruses activity in vitro.

Antiviral Agents↗
PubMed1994

MTT colorimetric assay system for the screening of anti-orthomyxo- and anti-paramyxoviral agents.

A rapid and sensitive method was developed for screening potential antiviral agents against orthomyxo- and paramyxoviruses, using the MTT method with cell culture suspensions. The cell lines used for the assay were as follows: MDCK cells for the influenza A virus (Fluv. A), HeLa cells for the respiratory syncytial virus (RSV), and Vero cells for the measles virus (MSV). Test compounds were diluted and plated in 96-well round-bottomed microtiter plates. Trypsinized cell suspensions and viruses were added to each well, the plates were then centrifuged (700 x g, 5 min, room temperature), and incubated for several days. The MTT assay was carried out after the degeneration of virus-infected cells became evident. The optical density (OD) of formazan was determined using a computer-controlled microplate reader. With this assay system, the EC50 values of Ribavirin (used as the reference compound) were 3.7 micrograms/ml for Fluv. A, 4.5 micrograms/ml for RSV, and 12.3 micrograms/ml for MSV, respectively. These EC50 values were equivalent to those obtained using the plaque reduction assay. The confluent cell culture system was inadequate for antiviral assays against RSV and MSV when the MTT method was used, because the inhibition of formazan formation was not observed in viral-infected cells. Moreover, the suspension method is more sensitive to the cytotoxicity of antiviral agents than the confluent cell culture system.

Amides↗
PubMed1993

[A case with severe neurological involvement due to vitamin B1 deficiency associated with megaduodenum].

We reported a case of 4-year-old boy with multiple vitamin deficiencies, especially vitamin B1 deficiency. He had megaduodenum associated with membranous stenosis on upper jejunum. He showed recurrent vomiting at his infantile period, and recently intermittent neurological symptoms. When he was admitted to our hospital, he could not walk and showed masked face, absent deep tendon reflexes, horizontal and vertical nystagmus, proximally dominant muscle weakness and multiple vitamin deficiencies. Oral administration of small doses of vitamin B1 (20 mg/day) could make remarkable clinical improvements. At three weeks after the treatment he could walk and run. Before the admission he had febrile convulsions and showed transiently striatal low density on CT image. We concluded that his neurological symptoms were due to vitamin B1 deficiency associated with megaduodenum. When a patient with intestinal anomaly shows neurological symptoms, we should think of vitamin deficiency.

Child, Preschool↗
PubMed1992

Comparison of the acute inflammation induced by calcium pyrophosphate dihydrate, apatite and mixed crystals in the rat air pouch model of a synovial space.

Pure monoclinic or triclinic calcium pyrophosphate dihydrate (CPPD) crystals, apatite crystals or mixtures of these crystals were injected into the synovial-like space created by the rat air pouch to compare the acute inflammation induced by these crystals. Fluids were withdrawn 6 h after injection and examined for leukocyte counts, protease, prostaglandin E2 (PGE2) and tumor necrosis factor (TNF) levels. CPPD crystals (especially monoclinic CPPD) induced higher numbers of leukocytes, and more protease, PGE2 and TNF than apatite. CPPD seemed to play a predominant role in the acute inflammation induced by mixed crystals.

Animals↗