Biomedical subjects

T Yano

Publications and source records attributed to T Yano.

At least 469 records · Page 26Linked to original sources

PubMed1993

Role of an active site residue analyzed by combination of mutagenesis and coenzyme analog.

Asp222 of aspartate aminotransferase is an active-site residue which interacts with the pyridine nitrogen of the coenzyme, pyridoxal 5'-phosphate (PLP). The roles of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase have previously been explored by site-directed mutagenesis. These studies confirmed that a negatively charged residue at position 222 is essential for catalysis, but the reason for this remained speculative. In the present studies, the roles of Asp222 were clarified experimentally by analyzing the mutant D222A enzyme (Asp222 replaced by Ala) reconstituted with the coenzyme analog N(1)-methylated PLP (N-MePLP). Spectroscopic and kinetic analyses showed that Asp222 stabilizes the protonated N(1) of PLP, raising the pKa value of N(1) by more than five units, in the active site of AspAT. The positive charge at N(1) accelerates abstraction of the alpha-proton from the amino acid substrate, stabilizing the transition state by 1.4 to 4.5 kcal.mol-1 in the reaction with aspartate. X-ray crystallographic (2.0 A resolution) and CD spectroscopic studies suggest that the coenzyme analog is not held in a proper orientation within the active site of D222A (N-MePLP). This may account for the finding that the catalytic activity was recovered only partially by the reconstitution of D222A with N-MePLP. These results fully support the following postulated role of Asp222: the negative charge of Asp222 stabilizes the positive charge at N(1) of PLP and thereby enhances the function of PLP as an electron sink.

Aspartate Aminotransferases↗

PubMed1993

Secretory cell immunoscreening assay--a highly sensitive screening method for secretory cells.

We have developed a rapid and sensitive screening method to detect the secretory product from transformed secretory cells. Polyclonal antibodies against the target secretory protein are first adsorbed onto a nitrocellulose membrane and are then called solid-phase antibodies. Then this membrane is placed on top of target protein-secreting adherent animal cell colonies. After trapping the secreted protein on the membrane, the protein is detected using the highly sensitive enhanced chemiluminescence detection system. Thus, this method is named the secretory cell immunoscreening assay (SCISA). We have screened recombinant BHK-21 cells, which secrete human interleukin-6 (hIL-6) for highly productive clones. With the SCISA method, it is possible to detect less than 100 pg of hIL-6 secreted by a single colony. We are also able to select highly productive colonies rapidly and easily from a large sample population of transformed cell colonies.

Animals↗

PubMed1993

Characterization of lung squamous cell carcinoma-derived T-cell suppressive factor.

BACKGROUND: The immunosuppressive state of a tumor-bearing patient is possibly mediated by tumor-derived factor. In this study, the authors characterized lung squamous cell carcinoma-derived immunosuppressive factor (LSCF). METHODS: The immunosuppressive activity of QG56 (a lung squamous carcinoma cell line)-derived LSCF was evaluated by the effect of culture supernatant of QG56 on anti-CD3 monoclonal antibody-induced T-cell, response such as proliferation (3H-thymidine uptake), cytotoxicity (51Cr-releasing assay), and expression of cytokine mRNA (polymerase chain reaction). The LSCF was partially purified with an ion-exchange high-performance liquid chromatography (HPLC) and a gel-filtration HPLC. RESULTS: The LSCF inhibited proliferation, cytotoxicity, and expression of cytokine mRNA of T-cells in a dose-dependent manner. It has a molecular weight of approximately 22 kd, and was sensitive to proteinase K, heating at 60 degrees C, and resistant to treatment with trypsin and pH 3 and 9. These properties appear to be similar to those of transforming growth factor-beta (TGF-beta). However, the activity of the LSCF was not abrogated by anti-TGF-beta sera, and the LSCF did not suppress the proliferation of TGF-beta-sensitive mink lung cells (Mv1Lu). CONCLUSIONS: These data suggest that LSCF may be a novel tumor-derived immunosuppressive protein factor.

Base Sequence↗

PubMed1993

p53 mutation is associated with progression in follicular lymphomas.

The majority of low-grade follicular lymphomas will eventually transform to an aggressive intermediate, or high-grade lymphoma. The molecular mechanisms responsible for this transformation have not been determined. We studied serial biopsies from 34 patients with follicular lymphomas that underwent histologic transformation, for abnormalities of the p53 tumor suppressor gene by a combination of immunohistochemistry, single strand conformation polymorphism analysis (SSCP), and sequencing. We found overexpression of p53 in 10 of the 34 transformed aggressive lymphomas, 9 of which contained mutations identified by SSCP analysis and subsequent sequencing. Matched pretransformation low-grade follicular lymphoma biopsies were available for 7 of the 10 cases. None of six studied by immunohistochemistry showed overexpression of p53 and only 1 of 4 studied by SSCP/sequencing showed the presence of mutation in the pretransformation biopsy. Interestingly, an eighth p53 positive transformed lymphoma recurred with a clonally related, p53 negative low-grade lymphoma 5 years after the patient had achieved a complete remission. Immunohistochemistry also showed that several pretransformation biopsies from p53 positive transformed cases showed rare p53 positive cells and in one case we could document an increase in their number over time. Twenty-five additional low-grade follicular lymphoma biopsies were also examined. Three patients had lymphomas positive for p53 mutation. One of the three subsequently transformed within a year of the biopsy studied; the second patient had an earlier (unavailable) biopsy at a different site that showed transformed histology. The third patient was treated with ProMACE-MOPP combination chemotherapy and attained a complete remission. We conclude that (1) mutations of p53 are associated with histologic transformation in approximately 25% to 30% of follicular lymphomas and (2) p53 positive cells can be detected before histologic transformation, but do not comprise a significant percentage of the neoplastic cell population (identifiable by SSCP) until late in the disease, just before or after histologic progression. Finally, the data also suggest that p53 positive low-grade lymphomas are at risk for progression and that in this subset, aggressive therapy may be warranted.

Base Sequence↗

PubMed1993

Molecular analysis of the BCL-3 locus at chromosome 17q22 in B-cell neoplasms.

To better understand the role of the BCL-3 locus at chromosome 17q22 in the pathogenesis and progression of leukemias and lymphomas, we examined its genomic configuration in 264 B-cell malignancies and its expression in a smaller subset. Cases studied included 39 chronic lymphocytic leukemias, 58 low-grade follicular lymphomas, 20 mantle cell lymphomas, 30 small noncleaved cell lymphomas, 25 acute lymphoblastic leukemias, 10 acquired immunodeficiency syndrome--related non-Hodgkin's lymphomas, and 44 diffuse mixed- or diffuse large-cell lymphomas. In addition, 38 aggressive lymphomas (transformed follicular lymphomas) derived from previously indolent follicular lymphomas were examined. Southern blot analysis showed BCL-3 locus rearrangement in 4 cases (1.5%), ie, in 3 transformed follicular lymphomas and in 1 indolent follicular lymphoma. All 4 also had BCL-2 rearrangements consistent with their follicular center cell origin. None of the BCL-3 rearranged cases showed MYC gene rearrangement, as reported for the original leukemia that led to the discovery of BCL-3. Pretransformation specimens of all three transformed follicular lymphomas showed the presence of the BCL-3 alteration before histologic progression. In 1 case, serial pretransformation biopsies showed that the BCL-3 rearrangement was acquired during the indolent follicular phase of the patient's disease. Thirty lymphomas, including 2 of the 4 with BCL-3 rearrangement, were also examined for BCL-3 message. All 30, including the 2 with BCL-3 rearrangements, expressed the normal 1.7-kb BCL-3 transcript, at approximately equivalent levels. The data indicate that, although BCL-3 locus alterations are found in only a small fraction of B-cell lymphoid malignancies, they occur primarily in a subset of follicular center cell lymphomas. Interestingly, these alterations appear to be acquired during the indolent (follicular) phase of the disease and they are maintained when histologic transformation takes place. The data also suggest that BCL-3 locus alterations do not result in gross changes of BCL-3 gene expression and do not necessarily involve the MYC gene. Although the preferential involvement of BCL-3 alterations in a small subset of follicular lymphomas that transform suggests a possible link between these abnormalities and progression, further studies are needed to ensure that these alterations are biologically relevant and not simply a manifestation of genomic instability.

B-Cell Lymphoma 3 Protein↗

PubMed1993

Ethyl all-cis-5,8,11,14,17-icosapentaenoate modifies the biochemical properties of rat very low-density lipoprotein.

The biochemical properties of serum very low-density lipoprotein (VLDL) were investigated in rats given highly purified all-cis-5,8,11,14,17-icosapentaenoate (EPA), an ethyl-ester derivative (EPA-E). The elution time (gel filtration) of VLDL from EPA-E-treated serum was increased significantly compared with that of the control. EPA-E-treated VLDL isolated by ultracentrifugation exhibited a marked decrease in triglyceride content with a relative increase in cholesterol. In treated VLDL, a significant increase in the ratio of apo E/apo C was observed. There was a remarkable increase in the content of EPA in all the fractions of phospholipids, cholesteryl esters and triglycerides after EPA-E treatment, resulting in n-3 polyunsaturated fatty acid-rich VLDL. EPA-E also reduced the incorporation of [14C]oleate into triglycerides in hepatic microsomes and the rate of hepatic triglyceride secretion. Moreover, lipoprotein lipase activity in heparin-injected plasma was increased in rats given EPA-E without there being an effect on hepatic triglyceride lipase activity. These findings indicate that EPA-E exerts an inhibitory effect on hepatic triglyceride synthesis/secretion and a stimulatory effect on triglyceride degradation, resulting in a reduction in particle size and an increase in the ratio of apo E/apo C. Triglyceride-poor and EPA-rich VLDL may rapidly be converted into the density of intermediate low-density lipoprotein and low-density lipoprotein and/or may be absorbed into the liver rapidly.

Animals↗

PubMed1993

A hydrogen-bonding network modulating enzyme function: asparagine-194 and tyrosine-225 of Escherichia coli aspartate aminotransferase.

The electron distribution within the coenzyme or coenzyme-substrate conjugate needs to be properly regulated during the catalytic process of aspartate aminotransferase (AspAT). Asn194 and Tyr225 may function in regulating the electron distribution through hydrogen-bonding to O(3') of the coenzyme, pyridoxal 5'-phosphate (PLP) or pyridoxamine 5'-phosphate (PMP). The roles of Tyr225 have already been explored by site-directed mutagenesis (Inoue et al., 1991; Goldberg et al., 1991). In the present studies, the mutant enzymes Asn194-->Ala and Asn194-->Ala + Tyr225-->Phe were analyzed kinetically and spectroscopically and were compared with the wild-type and Tyr225-->Phe enzymes. The kinetic studies showed that Asn194 is not essential for AspAT catalysis, although the Kd values for the substrates were increased by 10- to 50-fold upon the replacement of Asn194. The measurements of the absorption and fluorescence excitation spectra revealed that the ratio of an enolimine to a ketoenamine form was considerably increased as a tautomeric form of the protonated PLP in the active site of the double mutant enzyme. The pH-pKd relationship for the binding of maleate to AspAT could be explained by a simple thermodynamic cycle where only one ionizing group (the imine nitrogen of the internal aldimine bond) affects the binding of maleate. The analyses of the pH-pKd curves for the wild-type and mutant enzymes showed that (i) the hydrogen bond between O(3') of PLP and Asn194 is weakened by the binding of maleate to AspAT, while the hydrogen bond between O(3') and Tyr225 is not changed, and that (ii) the replacement of Asn194 causes some effect hampering the binding of maleate.(ABSTRACT TRUNCATED AT 250 WORDS)

Asparagine↗

PubMed1993

Mechanism of the lipid-lowering effect of ethyl all-cis-5,8,11,14,17-icosapentaenoate.

The effect of highly purified ethyl all-cis-5,8,11,14,17-icosapentaenoate (EPA-E) on cholesterol metabolism in rats was examined to clarify the mechanism of its hypolipidemic action. Pretreatment with EPA-E reduced the increase in plasma radioactivity after oral administration of [14C]cholesterol. The conversion of [14C]3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to [14C]mevalonic acid was significantly inhibited in liver microsomes obtained from rats treated with EPA-E. There was an increase in free cholesterol and a marked rise in the eicosapentaenoic acid (EPA) content of phospholipids in these microsomes. EPA-E restored the suppression of biliary secretion induced by feeding a casein-rich diet to bile duct-cannulated rats. Furthermore, when serum lipoprotein (d < 1.210) from rats given EPA-E was i.v. injected into normal rats, a more rapid elimination of cholesterol was observed as compared to that in rats injected with lipoprotein from EPA-E-untreated rats. This rapid clearance was found in the lipoprotein fractions of d < 1.006 and 1.006 < d < 1.063. These findings suggest that EPA-E has an inhibitory effect on intestinal cholesterol absorption and hepatic cholesterol biosynthesis, and an enhancing effect on hepatic biliary secretion. EPA-E would also seem to cause modification of serum lipoproteins, whereby their clearance from the serum is increased.

Administration, Oral↗

PubMed1993

Treatment and prognosis of primary thymic carcinoma.

From 1972 to 1990, we treated eight cases of thymic carcinoma (6 squamous cell and 2 small cell carcinomas). According to the classification by Masaoka et al., they consisted of one stage I, four stage III, one stage IVa, and two stage IVb. A complete resection of the primary tumour could be done in only three patients; the others had diagnostic biopsy and then radiation treatment. Four of five patients had a prolonged regression of the primary tumors after irradiation at 40-61.2 Gy. Six patients suffered from extrathoracic metastases. All patients received systemic chemotherapy with different regimens to counter either metastatic or locally recurrent lesions. Only two patients (with a regimen including cyclophosphamide, doxorubicin, and vincristine) obtained a partial response. The median survival of the eight patients was 70 months after surgical operation. The identification of an effective drug combination may thus improve the long-term prognosis of thymic carcinoma since radiotherapy is able to control primary lesions, even in the case of unresectable advanced disease.

Adult↗

PubMed1993

Treatment of invasive thymoma with pleural dissemination.

The outcome of eight patients with invasive thymoma accompanying pleural dissemination was investigated. Only two patients had mediastinal tumor resection and pleural disseminated tumor excision. Seven patients underwent radiotherapy to the mediastinum and/or disseminated tumors. A clinical response to radiotherapy was achieved in the six patients with evaluable lesions (complete response in five patients and partial response in one). The estimated 5-year survival rate was 87.5%. Four patients were alive more than 10 years. So far, the mediastinal tumors of seven patients have been controlled for periods ranging from 42 to 154 months. Recurrence in six patients appeared as pleural tumors. Four out of the six patients had five courses of radiotherapy to the recurrent pleural tumors, four of which achieved complete response. No distant metastases were observed at any time. These observations suggest that radiotherapy should be the primary mode of treatment in cases of invasive thymoma with pleural dissemination.

Adult↗

PubMed1993

Inhibition of growth of experimental prostate cancer in rats by LH-RH analogs linked to cytotoxic radicals.

The effects of hybrid cytotoxic LH-RH analogs, produced by linking anthraquinone or methotrexate to carrier LH-RH agonist [D-Lys6]LH-RH, were evaluated in Copenhagen-Fisher F1 rats bearing Dunning R-3327H prostate adenocarcinoma. The two cytotoxic LH-RH analogs T-98 [(D-Lys6)LH-RH coupled to glutaryl-2-(hydroxymethyl)anthraquinone (G-HMAQ)], and AJ-04 [(D-Lys6)LH-RH linked to methotrexate (MTX)], carrier [D-Lys6]LH-RH, or the free cytotoxic compounds MTX and G-HMAQ were administered from Alzet Osmotic minipumps for 7-8 weeks. The cytotoxic LH-RH analogs caused somewhat greater tumor growth inhibition than the carrier peptide, while anthraquinone or methotrexate alone, administered in equimolar doses, were ineffective. The inhibition of androgen sensitive organs (testes, ventral prostates, and seminal vesicles) was pronounced with both carrier and cytotoxic analogs, showing the latter to be fully hormonally active in suppressing the pituitary-gonadal axis. Histological changes were also evaluated. The inhibition of mitosis and the frequency of apoptosis were higher in tumors treated with AJ-04, T-98, [D-Lys6]LH-RH, or by castration than in those of controls. Serum hormone levels were lowered by both carrier peptide and cytotoxic analogs, LH being substantially depressed, and testosterone not detectable. These results and other findings indicate that LH-RH analogs containing cytotoxic radicals anthraquinone or methotrexate retain their hormonal activity after administration in vivo, and can effectively inhibit tumor growth. Extensive further studies are required on this new class of compounds, but apparent binding of cytotoxic LH-RH analogs to tumors such as prostate cancer, which have receptors for LH-RH, could greatly reduce peripheral toxicity of chemotherapeutic agents. This approach, based on targeted chemotherapy, might be of practical therapeutic importance for the management of advanced prostate cancers, which eventually relapse after palliative hormonal therapy.

Adenocarcinoma↗

PubMed1993

Alpha-fetoprotein-producing esophageal adenocarcinoma: report of a case.

This paper describes a rare case of adenocarcinoma located in the middle portion of the esophagus with liver metastasis. An 80-year-old man was admitted to our hospital with dysphagia and vomiting, following which an upper gastrointestinal series and esophagoscopy located an elevated-type carcinoma in the middle thoracic esophagus. Computed tomography revealed an esophageal tumor invading the left atrium and aorta, and multiple intrathoracic lymph node swellings, and an ultrasonograph of the liver showed multiple liver metastases. The serum carcinoembryonic antigen, carbohydrate antigen 19-9, and squamous cell carcinoma-related antigen levels were normal, but the serum alpha-fetoprotein (AFP) level was 351.5 ng/ml. The patient died 124 days after undergoing an esophageal bypass operation. On post-mortem histological examination, the original esophageal tumor was diagnosed as a poorly differentiated adenocarcinoma without a squamous component and immunohistochemical staining for AFP showed positive granules in the cytoplasm. All the metastatic nodules, including the lymph nodes, liver, spleen, and lungs, showed the same histological type and AFP-staining pattern as the original esophageal tumor. To our knowledge, this is the first case of AFP-producing esophageal carcinoma to be reported in Japan.

Adenocarcinoma↗

PubMed1993

An intrathoracic vagus nerve schwannoma invading the trachea.

A patient with a schwannoma of the intrathoracic vagus nerve is presented. The tumor invaded the trachea and caused dyspnea. It was successfully excised by a segmental resection of the trachea with end-to-end anastomosis. This instance of an intrathoracic vagus tumor invading the trachea is, as far as we know, the first reported in the world.

Cranial Nerve Neoplasms↗

PubMed1993

MRI visualization of complete bilateral optic nerve involvement by pituitary adenoma: a case report.

With large pituitary adenomas, the optic nerves and chiasm usually lie on the tumour capsule and are displaced superiorly. We report a large invasive pituitary adenoma, with complete involvement of both optic nerves. Review of the preoperative MR images demonstrated the optic nerves, with signal intensity close to that of cerebral white matter, and different from the flow void of the basal cerebral arteries. Correlation of this observation with intraoperative findings is discussed.

Adenoma↗

PubMed1993

Characteristics of the energy-transducing NADH-quinone oxidoreductase of Paracoccus denitrificans as revealed by biochemical, biophysical, and molecular biological approaches.

A comparison of the mitochondrial NADH-ubiquinone oxidoreductase and the energy-transducing NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans revealed that both systems have similar electron-transfer and energy-transduction pathways. In addition, both complexes are sensitive to the same inhibitors and contain similar electron carriers, suggesting that the Paracoccus NDH-1 may serve as a useful model system for the study of the human enzyme complex. The gene cluster encoding the Paracoccus NDH-1 has been cloned and sequenced. It is composed of 18,106 base pairs and contains 14 structural genes and six unidentified reading frames (URFs). The structural genes, URFs, and their polypeptides have been characterized. We also discuss nucleotide sequences which are believed to play a role in the regulation of the NDH-1 gene cluster and Paracoccus NDH-1 subunits which may contain the binding sites of substrates and/or electron carriers.

Bacterial Proteins↗

PubMed1993

Sexing of in vitro-fertilized preimplantation mouse embryos by the PCR method.

The applicability of the dual PCR method to embryo sexing was examined with the aim of establishing a noninvasive method of preimplantation diagnosis for human genetic disorders. Mouse pre-embryos obtained by in vitro fertilization were studied. The Sry gene sequence and the myogenin sequence were amplified as the Y-specific and internal control sequences, respectively. Amplification of as little as 10 pg of mouse genomic DNA was possible with the dual PCR method, the sensitivity being 10-fold greater than that of the single PCR method. The sex was identified in 100% (24/24) and 96% (23/24) of the pre-embryos tested at the 16- and 4-cell stages, respectively. In addition, the sex of all four single blastomeres dissociated from 4-cell pre-embryos agreed in 76% (16/21) of the specimens tested and 94% (79/84) of dissociated blastomeres could be sexed. The sex of single blastomeres biopsied from pre-embryos at the 8-cell stage could be identified. After transfer of 13 male and 25 female sexed pre-embryos, six viable fetuses were obtained. Histological examination showed that all these fetuses were of the predicted sex. Sexing of biopsied single blastomeres by the dual PCR method was rapid and reliable, suggesting its feasibility for preimplantation diagnosis of in vitro fertilized human pre-embryos.

Animals↗

PubMed1993

Deletion detection for diagnosis of Duchenne muscular dystrophy in the Japanese population--comparison between the polymerase chain reaction and the Southern blot analysis.

We compared the efficacy of the multiplex PCR with that of the cDNA analysis for detection of deletions of the DMD gene in the Japanese patients. Thirty males with DMD from 27 Japanese families were studied by the multiplex PCR, and 24 of them were also investigated by Southern blot analysis. We used five dystrophin cDNA probes for deletion analysis. A total of 19 regions were amplified by the PCR to detect deletions, 9 regions by the method of Chamberlain et al. and another 10 regions by the method of Beggs et al. Deletions were detected in 14 (52%) out of 27 DMD families by the PCR. Southern blot analysis detected deletions in 14 (64%) out of 22 families. Thirteen (93%) of the 14 DMD families with deletions detected by Southern blotting were also confirmed by the multiplex PCR. Provided care is taken in cases where the deletion is limited to a single exon, the multiplex PCR appears to be an efficient and useful alternative to conventional Southern blot analysis for detecting deletions during the prenatal and postnatal diagnosis of DMD.

Asian People↗

PubMed1993

Cytomegalovirus mononucleosis--associated gastric ulcers in normal host.

A case of cytomegalovirus (CMV) mononucleosis associated with gastric ulcers is reported in a normal non-immunocompromised host. The demonstration of intranuclear inclusion bodies in a few gastric glandular epithelial cells contributed to the diagnosis of CMV gastritis. Extraction of DNA from the gastric biopsy specimens and the amplification of CMV-DNA by the polymerase chain reaction (PCR) showed the presence of CMV-DNA in the gastric mucosa. In situ hybridization with a probe derived from CMV genomic regions demonstrated the numerous glandular cells with CMV-DNA at the nuclear region. Treatment with a proton pump inhibitor to suppress gastric acid secretion was useful to mitigate the epigastralgia and to hasten the ulcer healing. The value of highly sensitive PCR and the in situ hybridization method to detect the CMV-DNA were emphasized for rapid and sensitive diagnosis of CMV gastritis.

Adult↗