Search PubMed⌕ Search

Biomedical subjects

T Toyoda

Publications and source records attributed to T Toyoda.

At least 73 records · Page 4Linked to original sources
PubMed1997

Structure-function analysis of the diphtheria toxin receptor toxin binding site by site-directed mutagenesis.

Diphtheria toxin (DT) binds to the epidermal growth factor (EGF)-like domain of human membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF), the human DT receptor (DTR). DT does not bind to mouse proHB-EGF because of amino acid substitutions within the EGF-like domain. We made 10 independent mutants, replacing a single amino acid within the EGF-like domain of human DTR/proHB-EGF with the corresponding amino acid residue in mouse proHB-EGF. The mutant proteins were transiently expressed in mouse L cells either expressing or not expressing DRAP27/CD9, and DT binding was measured. DT binding activity of GST fusion proteins containing the mutated EGF-like domain was also determined by a cell-free binding assay. The largest effect was seen with E141H, and second largest effects were seen with F115Y and L127F in all of the assay systems. We conclude that Phe115, Leu127, and Glu141 are critical amino acid residues for DT binding. A computer model of the tertiary structure of the EGF-like domain of human DTR/proHB-EGF was made. The model predicts that three amino acid residues critical for DT binding activity, Phe115, Leu127, and Glu141, are all located on the same face of the EGF-like domain, suggesting that this face of DTR/proHB-EGF interacts with the receptor-binding domain of DT.

Amino Acid Sequence↗
PubMed1997

Differential induction of superoxide dismutase in core and penumbra regions after transient focal ischemia in the rat neocortex.

Oxygen free radicals are postulated to participate in the pathogenesis of ischemic brain injury. The present study investigated the response of the endogenous antioxidant enzyme, superoxide dismutase (SOD), in a model of transient focal ischemia in the rat neocortex. SOD activity was increased significantly in the penumbra region at 6-24 h postischemia, while no significant changes in SOD activity were observed in either the core region or striatum. These results indicate that endogenous antioxidant activity is differentially affected by the intensity of ischemic challenge and suggest that the regional effects of oxygen free radicals may vary substantially following ischemia-reperfusion.

Animals↗
PubMed1997

Lead discovery of inhibitors of the dihydrofolate reductase domain of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase.

A three-dimensional structure model of the dihydrofolate reductase (DHFR) domain of the bifunctional DHFR-thymidylate synthase of Plasmodium falciparum was used as a basis for computational screening of commercially available compounds for candidate inhibitors. Compounds which can stably dock to the model with strong ionic hydrogen bonds via protonation by an aspartic acid residue at the bottom of the active site were identified through docking simulation. Among compounds thus identified, 21 were assayed for inhibitory activity towards the recombinant DHFR domain. Two compounds, 2-amino-1,4-dihydro-4,4,7,8-tetramethyl-s-triazino(1,2-a)benzimida zole and Trp-P-2, inhibited the recombinant P. falciparum DHFR domain with Ki values of 0.54 and 8.7 microM, respectively. Kinetic analysis showed that these compounds competitively inhibited the enzyme with respect to the substrate dihydrofolate. These findings support the validity of both the modeled structure and the docking results. Furthermore, these compounds serve as leads for developing new DHFR inhibitors, since their skeletal structures are different from any of known DHFR inhibitors. This paper also reveals a new biological activity of Trp-P-2, a potent mutagen.

Animals↗
PubMed1997

Induction of ischemic tolerance and antioxidant activity by brief focal ischemia.

Three forms of non-injurious ischemic preconditioning were tested for their effects on endogenous antioxidant activity and subsequent ischemic injury. Test ischemia alone, consisting of 1 h occlusion of both carotid arteries (BCAs) and one middle cerebral artery (MCA), elicited an average volume of cerebral infarction of 143.4 +/- 15.0 mm3 (mean +/- s.e.m.). Twenty minutes occlusion of the MCA only or MCA and BCAs (but not BCAs only) 24 h before test ischemia significantly increased the activity of the antioxidant enzyme superoxide dismutase and significantly reduced the cerebral infarction volume. These findings demonstrate that brief focal ischemia can attenuate the injurious impact of subsequent ischemia and that an upregulation of endogenous antioxidant activity may play a key role in this neuroprotective effect.

Analysis of Variance↗
PubMed1997

Proliferative activity of central neurocytoma: measurement of tumor volume doubling time, MIB-1 staining index and bromodeoxyuridine labeling index.

Central neurocytoma is considered to be a benign intracranial neoplasm, but little is known about the biological behavior of this type of tumor. Proliferative activity of central neurocytoma was measured in 10 cases using MIB-1 staining for Ki-67 antigen. The MIB-1 staining value varied from < 0.1% to 5.6%, to indicating that some of these tumors have proliferative potential similar to that of anaplastic astrocytoma or malignant meningioma. The bromodeoxyuridine labeling index (BUdR LI, BrdU LI) was measured in 2 cases and the results correlated well with those of the MIB-1 analysis. Tumor volume doubling time (Td) measured in one case was 358 days which is similar to that of malignant meningioma. In one case, the MIB-1 value taken before and after 58 Gy of radiation treatment decreased markedly from 5.6% to 0.2%. The other 9 cases were also treated by radiation therapy (50-60 Gy) and no tumor recurrence was observed during follow-up periods ranging from 23 to 160 months. Another two patients with partially removed and 3 with subtotally removed tumors showing relatively high MIB-1 values might also have benefited from radiation therapy.

Adult↗
PubMed1997

Crystallization of eukaryotic E3, lipoamide dehydrogenase, from yeast, for exhibiting X-ray diffraction beyond 2.5 A resolution, and preliminary structure analysis.

Lipoamide dehydrogenase, which is a common component of alpha-keto acid dehydrogenase complexes, has been highly purified from yeast (Saccharomyces cerevisiae) to reveal its structure at higher resolution. New crystals obtained by a desalting method exhibited diffraction beyond 2.5 A resolution. The cell dimensions are a = 97.1, b = 158.7, and c = 67.9 A, and the space group is P2(1)2(1)2(1). There is a dimeric enzyme in the asymmetric unit. The crystal structure was solved by means of the molecular-replacement technique and refined in a preliminary manner.

Crystallization↗
PubMed1997

Attenuation of ischemia-reperfusion injury in the rat neocortex by the hydroxyl radical scavenger nicaraven.

OBJECTIVE: Oxygen free radicals are considered important contributors to cerebral ischemia-reperfusion injury. The purpose of this study was to examine the effects of the hydroxyl radical scavenger, (+/-)-N, N'-propylenedinicotinamide (nicaraven), on cerebral injury after focal ischemia-reperfusion. METHODS: A total of 58 male Sprague-Dawley rats was subjected to transient focal ischemia by occluding both carotid arteries and one middle cerebral artery for 3 hours. Animals received continuous infusions of doses of 20 mg/kg per hour or 60 mg/kg per hour of nicaraven beginning either 10 minutes before (pretreatment) or immediately after (posttreatment) ischemia. Infarction volumes were evaluated by staining coronal brain sections with 2% 2,3,5-triphenyltetrazolium chloride (n = 40). In other animals (n = 18), brain edema was evaluated 1 hour after ischemia. RESULTS: A dose of 20 mg/kg per hour of nicaraven elicited small reductions in infarction volume (14.7 and 12.3% for the pre- and posttreatment groups, respectively). Treatment with a dose of 60 mg/kg per hour of nicaraven provided significant reductions in the volume of infarction (18.6% [P < 0.05] and 20.9% [P < 0.01] reductions for the pre- and posttreatment groups, respectively). The reductions in infarction size did not differ significantly between the pre- and posttreatment groups receiving a dose of 60 mg/kg per hour of nicaraven. Posttreatment with either dose of nicaraven significantly reduced brain edema. CONCLUSIONS: This study demonstrates the neuroprotective effects of a hydroxyl radical scavenger when administered systemically during the reperfusion phase after transient focal ischemia. The results provide, to our knowledge, the first evidence that nicaraven is effective against ischemia-reperfusion injury in the brain and demonstrate that oxygen free radicals generated during reperfusion are important triggers of ischemic brain damage. Furthermore, the findings suggest that nicaraven may be a useful agent in limiting brain injury after ischemic stroke.

Animals↗
PubMed1997

Role of gamma delta TCR+ lymphocytes in the augmented resistance of trehalose 6,6'-dimycolate-treated mice to influenza virus infection.

Trehalose 6,6'-dimycolate (TDM), an immunomodulator, potentiates non-specific resistance in mice to influenza virus infection. When mice were injected intravenously with TDM, the striking proliferation of a minority of T-lymphocytes bearing gamma/delta T-cell receptors (gamma delta T-cells) that accumulated in granulomatous lungs was thought to be associated with the maintenance of acquired resistance to lethal influenza virus infection. To clarify the cellular basis of the defence against influenza virus, mice were depleted of gamma delta T-cells, alpha/beta (alpha beta) T-cells, or natural killer (NK) cells by in vivo administration of corresponding antibodies prior to influenza virus infection. The depletion of gamma delta T-cells significantly abrogated the augmented resistance of TDM-treated mice to infection, as did depletion of either alpha beta T-cells or NK cells. To gain insight into the functional ability of gamma delta T-cells, we evaluated the cytotoxic activity of this T-cell subset against a panel of target cell lines that were stably transfected with the influenza virus haemagglutinin (HA) gene from A/PR/8/34(H1N1) and A/Aichi/2/68(H3N2) strains. The gamma delta T-cells from TDM-treated mice showed profound cytotoxicity against the target cells expressing HA of either the H1 or H3 subtype, in a non-major histocompatibility complex-restricted manner. Taken together, these results indicate that gamma delta T-cells play a non-specific role, in conjunction with alpha beta T-cells and NK cells, in protecting mice against influenza virus infection, and that the recognition and destruction of HA-expressing target cells by the activated gamma delta T-cells is one of the steps involved in this anti-influenza virus immunosurveillance.

Adjuvants, Immunologic↗
PubMed1997

[Clinical evaluation of a reagent for detection of DNA of Mycobacterium tuberculosis complex using the ligase chain reaction (LCR) method].

We evaluated the clinical efficacy of LCR MTB, a reagent developed by Abbott in the USA, in the full automatic ligase chain reaction (LCR) for detection of DNA of M. tuberculosis complex using a thermostable ligase. Using 458 samples isolated from patients with tuberculosis, LCR was compared with a smear method and with a culture method, and was also compared with two other methods of gene amplification, MTD and Amplicor, using 340 and 200 of the 458 samples, respectively. The LCR method detected M. tuberculosis in 49.8% (228/458) of the samples, and was superior to the smear method (31.9%, 146/458) and the culture method (39.1%, 179/458) in sensitivity. The LCR method was also superior to the MTD and Amplicor methods; sensitivity were 37.9% (129/340) for MTD vs. 47.6% (162/340) for LCR, and 56.5% (113/200) for Amplicor vs. 59.5% (119/200) for LCR. These favorable results and the convenience of the LCR method, which enables rapid detection of target genes with a high degree of sensitivity, strongly suggest that LCR MTB is useful as a reagent for detection of M. tuberculosis using nucleic acid amplification.

Adolescent↗
PubMed1997

Prevention and reversal of cerebral vasospasm by an endothelin-converting enzyme inhibitor, CGS 26303, in an experimental model of subarachnoid hemorrhage.

Delayed cerebral ischemia due to cerebral vasospasm is a major cause of morbidity and mortality in patients with aneurysmal subarachnoid hemorrhage (SAH). Increasing evidence implicates the potent vasoconstrictor peptide endothelin (ET) in the pathophysiology of cerebral vasospasm. In the present study the authors examined the therapeutic value of blocking the production of ET-1 by inhibiting the conversion of its relatively inactive precursor, Big ET-1, to a physiologically active form. An inhibitor of ET-converting enzyme (ECE), CGS 26303, was injected intravenously after inducing SAH in New Zealand white rabbits. Injections of CGS 26303 were initiated either 1 hour after SAH (prevention protocol) or 24 hours after SAH (reversal protocol). One of three concentrations (3, 10, or 30 mg/kg) of CGS 26303 was injected twice daily, and all animals were killed by perfusion fixation 48 hours after SAH occurred. Basilar arteries were removed and sectioned, and their cross-sectional areas were measured in a blind manner by using computer-assisted videomicroscopy. Treatment with CGS 26303 attenuated arterial narrowing after SAH in both the prevention and reversal protocols. The protective effect of CGS 26303 achieved statistical significance at all dosages in the prevention protocol and at 30 mg/kg in the reversal protocol. These findings demonstrate that inhibiting the conversion of Big ET-1 to ET-1 via intravenous administration of an ECE inhibitor can be an effective strategy for limiting angiographic vasospasm after SAH. Moreover, the results demonstrate that treatment with the ECE inhibitor is capable of reducing vasospasm even when initiated after the process of arterial narrowing has begun. Finally, the results provide further support for the role of ET in the establishment of cerebral vasospasm. The ECE inhibitor CGS 26303 thus represents a promising therapeutic agent for the treatment of cerebral vasospasm following aneurysmal SAH.

Animals↗
PubMed1997

[Combination use of second generation cephem and isepamicin for the treatment of post-surgical infection of the lower digestive tract].

Effects of combination use of a second generation cephem and an aminoglycoside antibiotic isepamicin (ISP), for the treatment of post-surgical infections of the lower digestive tract were examined from a laboratory and clinical perspective. Thirty-three out of total 35 cases were included in the analysis of the test, while the other 2 cases did not meet the criteria for analysis. ISP was administered in combination with one of the second generation cephems among cefotiam (CTM), cefmetazole (CMZ), or cefuroxime (CXM) for 26 cases of wound infection and 7 cases of peritonitis. The overall efficacy rate was 88%; 92% in wound infection and 71% in peritonitis individually. There was no significant difference in efficacy among different groups. Bacteriological study showed the combination use of any one of the second generation cephems and ISP was very effective in all 14 cases of infections with a single species of bacterium, the efficacy rate was 100%. It was also effective in 9 out of 10 cases with mixed infections of 2 species of bacteria (90%), but effectiveness was diminished in only 6 out of 9 cases with mixed infections of 3 species (67%). Sixty-nine strains were isolated from the 35 cases, more than half of which (44 strains) were Gram-negative; 14 strains of E. coli (20%), 13 strains of E. faecalis (13%) and 6 strains of P. aeruginosa (9%). The MIC50 and MIC90 were, respectively, 1.56 micrograms/ml and 100 micrograms/ml for ISP, 6.25 micrograms/ml and 100 micrograms/ml for CTM, 12.5 micrograms/ml and 100 micrograms/ml for CMZ, and 25 micrograms/ml and 100 micrograms/ml for CXM. The MICs in any combination uses decreased synergistically according to ISP concentration. Adverse events were observed in 3 out of 35 cases, but they were not severe. The results indicated that the combination use of a second generation cephem and ISP was useful and should be one of the choices for the treatment of post-operative infections of the lower digestive tract.

Adult↗
PubMed1997

[Structure-function relationship of the influenza virus RNA polymerase subunits].

Influenza virus RNA polymerase with the subunit structure PB1-PB2-PA is involved in both transcription and replication of the RNA genome. PB1 is the catalytic subunit and binds both to PB2 in its N-terminus and to PA in its C-terminus. PB2 is required for priming with cap and binds to PB1 in its N-terminus. PA is required for cRNA-->vRNA synthesis of genome replication and binds to PB1 in its C-terminus. Therefore, PB1 is the core subunit not only for RNA polymerase activity but also complex formation of the enzyme. Protease activity is identified in the N-terminal region of PA, but its role on viral proliferation remains unknown.

DNA-Directed RNA Polymerases↗
PubMed1997

[An observation on tuberculosis associated with HIV infection in Japan].

OBJECTIVE: To observe the reported cases of tuberculosis (TB) with HIV infection in Japan, in terms of their main clinical features and related factors. METHODS: A voluntary reporting network has been organized by the authors who are specialists of TB or respiratory medicine in tuberculosis institutions located roughly all over the country. The members have been encouraged to report not only their own cases but cases seen by their friends or in other institutions. RESULTS: By the end of 1996, a total of 71 cases have been reported of which 59 were TB and 12 NTM cases. Nationality of the cases were; Japan 48, Other Asian countries 16, Others 7. All of the NTM cases were Japanese. 30% of the cases were aged less than 30 years, 24% were thirties, 24% forties, 17% fifties and 6% were those aged 60 years or older. The cases were clearly younger than the TB cases in the national TB registry, and older than HIV-infected persons as known from the HIV surveillance system. 97% of the TB cases were bacteriologically confirmed cases. Eight of NTM cases were positive for MAC, others for M.kansasii. 42% of the cases had extra-pulmonary disease, including disseminated infections seen among 19%. Of TB cases 25% were excreting bacilli resistant to any of the anti-TB drugs which was higher than in the case of general TB population (10-15%). 11% of TB cases had past history of TB treatment. The cases had severe immunological impairment, 79% of the cases having CD4+ cell count less than 100. The route of HIV infection were; 51% heterosexual, 13% homosexual, 13% through blood preparations, etc. DISCUSSION: Although there may be many cases not included in this observation, it is considered to well reflect the real situation of the problem of Japan. More attention should be paid to HIV infection of the patients in the clinical practice of TB in Japan.

AIDS-Related Opportunistic Infections↗
PubMed1997

X-ray analysis of DNA dodecamer containing 2'-deoxy-N6-methoxyadenosine.

Oxyamines have been known as a mutagen which attacks amino groups of DNA bases. It is expected that the modified adenine derivative has a tautomer which can form a stable base pair not only with thymine but also with cytosine. For establishing such tautomerization mutagenesis, we have solved a crystal structure of DNA dodecamer containing 2'-deoxy-N6-methoxyadenosine. It is shown that the N6-methoxyadenine takes a imino form to form a Watson-Crick type pairing with cytosine in the DNA duplex.

Base Composition↗
PubMed1996

Molecular dissection of influenza virus RNA polymerase: PB1 subunit alone is able to catalyze RNA synthesis.

Influenza virus RNA polymerase with the subunit structure PB1-PB2-PA is involved in both transcription and replication of the RNA genome. Enzyme reconstitution experiments indicated that all three P proteins are required for RNA synthesis in vitro (Kobayashi, et al. Virus Res 22, 235-245, 1992). Nuclear extracts of HeLa cells infected with three kinds of the recombinant vaccinia virus, each carrying one of the three P protein cDNAs, exhibited the activity of complete replication, that is, vRNA-sense RNA-directed and cRNA-sense RNA-directed RNA synthesis in the absence of primers. The nuclear extract from cells singly infected with the virus carrying PB1 cDNA exhibited a significant level of model v-sense RNA-directed RNA synthesis activity. Thus we conclude that PB1 is the catalytic subunit of influenza virus RNA polymerase and that under certain conditions, PB1 alone is able to catalyze RNA synthesis in vitro.

Base Sequence↗