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T Matsuba

Publications and source records attributed to T Matsuba.

At least 37 records · Page 2Linked to original sources
PubMed1996

Yeast expression of the cytokine receptor domain of the soluble interleukin-6 receptor.

The complex of the soluble interleukin-6 receptor (sIL-6R) and IL-6 (IL-6) is a potent agonist on cells expressing the signal transducing protein gp 130. In contrast, IL-6 alone only stimulates cells which express a membrane bound form of the IL-6R and gp 130. The natural occurring sIL-6R is generated by shedding of the membrane receptor and to a lesser extend by alternative splicing. We have inserted the coding sequence of the 323 amino acid residues of the human sIL-6R into an expression/secretion vector suitable for the methylotrophic yeast Pichia pastoris. We obtained, however, no detectable expression and secretion of the recombinant protein. When we used only the coding sequence of the cytokine receptor domain of the sIL-6R for the construction of an expression plasmid, this truncated version of the sIL-6R accumulated in the supernatant to 1-5 mg/l. The protein was purified by a single affinity chromatography step using a monoclonal antibody directed against the human IL-6R. Following the same approach, we expressed a truncated splice variant of the sIL-6R. Both, the secreted truncated sIL-6R and the splice variant showed full agonistic biological activity on human hepatoma cells. The described expression strategy will be useful for large scale production of biologically active sIL-6R and might be adapted for the expression of other members of the hematopoietic cytokine receptor family.

Antigens, CD↗
PubMed1996

Expression of cathepsin E in pancreas: a possible tumor marker for pancreas, a preliminary report.

Ductal cancers of the pancreas frequently express markers of gastrointestinal epithelial cells. Cathepsin E (CTSE) is a non-secretory, intracellular, but non-lysosomal proteinase found in the highest concentration in the superficial epithelial cells of the stomach. The aims of our study were to examine the expression of CTSE in the pancreas, to establish an assay system of CTSE and to evaluate the diagnostic usefulness of CTSE in the pancreatic juice. Eleven patients with pancreatic ductal adenocarcinoma, 10 with mucin-producing adenoma, 3 with intraductal papillary hyperplasia and 43 with chronic pancreatitis were examined. Surgically resected pancreatic tissues were subjected to immunohistochemistry for CTSE. Pancreatic juice was collected from the patients and subjected to sandwich ELISA and Western analysis for detecting CTSE. Positive staining for CTSE was observed in pancreatic ductal adenocarcinoma by immunohistochemistry. CTSE was also expressed in mucin-producing adenoma, intraductal papillary hyperplasia and mucinous hyperplasia. CTSE in the pancreatic juice was present in 8 of 11 patients with pancreatic ductal adenocarcinoma, 5 of 10 patients with mucin-producing tumor, 1 of 3 patients with intraductal papillary hyperplasia and 4 of 43 patients with chronic pancreatitis. The detection frequency of CTSE in the pancreatic juice was significantly higher in the patients with pancreatic ductal adenocarcinoma than in the patients with chronic pancreatitis. Our findings suggest that the expression of CTSE is associated with the pathogenesis of pancreatic ductal adenocarcinoma, that CTSE in the pancreatic juice seems to be a useful marker for a definitive diagnosis and that CTSE may be expressed at a relatively early stage of multistep carcinogenesis in pancreatic lesions.

Adult↗
PubMed1995

Expression of human cathepsin E in methylotrophic yeast, Pichia pastoris.

The human gastric cathepsin E (CTSE) was expressed in the methylotrophic yeast Pichia pastoris by placing the CTSE cDNA under the control of the methanol-inducible alcohol oxidase promoter. The human CTSE expressed in P. pastoris was efficiently secreted into the culture medium as an active enzyme directed by its native signal sequence, whereas CTSE has been shown to be retained in mammalian tissue cells. The recombinant human CTSE was secreted as a 90-kDa molecule and then converted via an 84-kDa intermediate to an 82-kDa molecule. The 90-kDa molecule and the 82-kDa molecule were considered to be the proenzyme and the mature enzyme as dimeric forms, respectively.

Amino Acid Sequence↗
PubMed1995

Purification and characterization of recombinant human cathepsin E.

The human cathepsin E was purified from the culture supernatant of Pichia pastoris strain transformed with a human cathepsin E expression plasmid. Purification was performed by a three-step procedure, TSKgel Phenyl-5PW, Toyopearl HW55S and TSKgel DEAE-5PW column chromatographies. The purified recombinant cathepsin E had the molecular mass of around 82-kDa with the amino-terminal sequence started with Ile37 of the predicted amino acid sequence, suggesting the human cathepsin E was accumulated in the culture suparnatant as the mature dimer enzyme. The result of endoglycosidase-H digestion followed by Western blot analysis of the purified recombinant cathepsin E suggested that the human cathepsin E expressed in Pichia pastoris received N-linked high-mannose type glycosylation.

Cathepsin E↗
PubMed1995

Expression of a 32 kilodalton Theileria sergenti piroplasm surface protein by recombinant baculoviruses.

Previous studies detected a single amino acid substitution (Ala196 to Gly196) between cDNA clones encoding a 32 kDa antigen (p32) of Theileria sergenti (Chitose stock) obtained from a persistently infected calf. In this study, 2 different recombinant baculoviruses (pAc/p32-Ala196 and pAc/p32-Gly196) were constructed for the expression of p32. Molecular masses of the polypeptides produced in Spodoptera frugiperda cells infected with the recombinant baculoviruses were the same as that of authentic p32. pAc/p32-Ala196 produced additional polypeptides, with molecular masses higher than 32 kDa, which resulted from differential N-glycosylation as revealed by endo N-glycosidase treatment. The results indicate that a single amino acid substitution may lead to a conformational change in p32 which affected post-translational modification of recombinant products.

Alanine↗
PubMed1995

Expression of recombinant human thyrotropin receptor in myeloma cells.

Starting with a previously isolated cDNA for human thyrotropin receptor (TSHR), we established a transformed myeloma cell line, SP56, which expresses human TSHR on its cell surface. Binding analysis showed that SP56 bears 1.1 x 10(5) TSHR per cell with a Kd of 2.2 x 10(-10) M. Using the purified cellular membrane, we established a TSH binding inhibition immunoglobulin (TBII) assay for autoantibodies against TSHR. We compared it with the TBII assay utilizing porcine thyroid membranes expressing porcine TSHR, which has been widely used for TBII assay, by using 96 serum samples from patients with autoimmune thyroid disease and normal individuals. Our TBII assay was more sensitive than the one using porcine TSHR: of 38 sera of patients which were judged negative for autoantibodies to TSHR (TBII value below 10%) by the latter assay, 28 were positive (above 20%) in our assay. By using a perfusion culture system, we obtained as many as 3 x 10(10) SP56 cells, from which 3,450 mg protein of the membrane could be purified; this is sufficient for 15,000 assays. The results indicate that the membrane of the myeloma cell line SP56 is more suitable for use in the TBII assay than the porcine thyroid membrane, in terms of sensitivity to autoantibodies against TSHR in human sera.

Animals↗
PubMed1994

Secretion of human intracellular aspartic proteinase cathepsin E expressed in the methylotrophic yeast, Pichia pastoris and characterization of produced recombinant cathepsin E.

The human gastric cathepsin E (CTSE), a dimeric aspartic proteinase, was expressed in the methylotrophic yeast Pichia pastoris by placing the CTSE cDNA under the control of the methanol inducible alcohol oxidase promoter. The human CTSE expressed in P. pastoris was secreted into the culture medium as an active enzyme directed by its native signal sequence despite its intracellular localization in mammalian cells. The time course analysis of the culture supernatant of the P. pastoris transformant expressing human CTSE revealed that the recombinant human CTSE was secreted as a 90 kDa molecule and then converted via an 84 kDa intermediate to an 82 kDa mature molecule. A large-scale culture of the transformant was performed in a high cell density fermentor and the recombinant human CTSE was highly purified from the culture supernatant. The purified recombinant cathepsin E had the molecular mass of 82 kDa with the amino-terminal sequence starting with Ile37 of the sequence deduced from its cDNA sequence, suggesting that the human cathepsin E was accumulated in the culture supernatant as mature dimeric enzyme. The result of endoglycosidase-H digestion followed by Western blot analysis of the purified recombinant cathepsin E suggested that the human cathepsin E expressed in P. pastoris received N-linked high-mannose type glycosylation. The enzymatic properties of the recombinant enzyme were comparable to those of natural human CTSE.

Base Sequence↗
PubMed1994

Analyses of antigenic and genetic diversities of Theileria sergenti piroplasm surface proteins.

Monoclonal antibodies (MoAbs) against Theileria sergenti Fukushima stock, an intraerythrocytic protozoan parasite of cattle, were produced. The MoAbs reacted with 32 kDa or 23 kDa piroplasm surface protein (p32 or p23) in Western blot analysis. Using a panel of the MoAbs, antigenic analysis of the stocks and field isolates distributed in Japan was carried out. The results revealed antigenic diversity of T. sergenti isolates, but diversity did not appear to be correlated with the geographical region of the isolates. N-terminal amino acids sequences analysis revealed substitutions of a few amino acids between the p23 of two T. sergenti stocks. The sequences couldn't be found in amino acid sequence of the p32 deduced from cDNA, which indicated that the p23 is not a proteolytic product of the p32. Genetic diversity of T. sergenti isolates was also observed by Southern blot analysis using a cDNA of the p32 as a probe.

Amino Acid Sequence↗
PubMed1994

Acute pneumonitis presumed to be silicone embolism.

A 39-year-old housewife who underwent intramammary injections of a proprietary silicone fluid mixture showed clinical and novel transbronchial lung biopsy (TBLB) findings. She presented with complaints of progressive dyspnea, dry cough, and pleuritic chest pain 2 days after the last silicone injections. The chest X-ray and CT scan showed diffuse interstitial infiltrates. TBLB demonstrated translucent, presumably silicone globules embolized within the pulmonary capillaries. The documentation of intramammary injections, the clinical and radiographic features of acute pneumonitis, and the histopathologic evidence by TBLB, may support the causal relationship between illicit injections and the silicone embolism. We discuss the pathogenesis and urge that this potentially toxic source of pulmonary embolism be removed.

Acute Disease↗
PubMed1993

Changes in the hybridization patterns of populations of Theileria sergenti during infection.

Restriction fragment length polymorphisms (RFLPs) of Theileria sergenti DNA were analysed using probes of a genomic DNA fragment (pTs 2) and a cDNA corresponding to this genomic probe (C-Ts 2). Each of the probes detected RFLPs in DNA from different stocks of Theileria sergenti. Additionally, using these probes, alterations in hybridization patterns were observed in samples of the parasites harvested at different times after individual calves had been infected with Theileria sergenti. This result suggests that the Theileria sergenti stocks used were mixed parasite populations.

Animals↗
PubMed1993

Analysis of mixed parasite populations of Theileria sergenti using cDNA probes encoding a major piroplasm surface protein.

The gene for the 32 kDa surface protein (p32) of Theileria sergenti was cloned into lambda gt11 and its nucleotide sequence was determined. The gene encodes a protein of 283 amino acids as deduced from its nucleotide sequence with a 22 residue N-terminal signal peptide. Using this cDNA as a probe we have isolated another two clones from a cDNA library with a CDM8 vector system derived from the same parasite stock. Comparison with three cDNA clones revealed differential polyadenylation and differences in sequences of non-coding regions. Within the coding regions, there were nucleotide transitions which affected the Pst I-restriction site, and one of the transitions was also accompanied by an amino acid substitution (Ala to Gly). Southern blot analysis showed hybridization pattern changes among the parasites isolated from individual calves at different times after infection. From these results, we conclude that at least 3 genetically different parasite populations may coexist, and the transition to predominant parasite populations might occur during persistent infections in a host, possibly to evade the host immune responses.

Amino Acid Sequence↗
PubMed1993

Detection of Theileria sergenti infection in cattle by polymerase chain reaction amplification of parasite-specific DNA.

A pair of synthetic oligonucleotide primers, designed from the gene encoding a 32-kDa intraerythrocytic piroplasm surface protein of Theileria sergenti, were used to amplify parasite DNA from the blood of T. sergenti-infected cattle by means of the polymerase chain reaction (PCR). PCR-amplified DNA was examined by electrophoresis and by dot blot or microplate hybridization using a parasite-specific cDNA probe. PCR was specific for T. sergenti, since no amplification was detected with DNA from Anaplasma centrale, Babesia ovata, uninfected erythrocytes, and leukocytes. This method was sensitive enough to detect about 4.5 parasites per microliters of blood with a 10-microliters sample volume. Moreover, of 66 specimens from grazing cattle, 40 were microscopically positive, whereas PCR revealed that 54 samples were positive. Therefore, PCR provides a useful diagnostic tool for detecting T. sergenti-infected cattle, and it is significantly more sensitive than the current methods.

Animals↗
PubMed1993

[A study of knowledge, attitude and health behavior toward tuberculosis among non-immigrant Korean people in Japan].

Knowledge, attitude and health behavior toward tuberculosis among Non-immigrant Korean people in Japan was researched by using questionnaire because of increasing the number of the tuberculosis patients among those group. The Korean member of protestant churches in Tokyo were subjects for the survey. Immigrant Korean people and their descendants were excluded. The questionnaire form was written in Korean language under the guidance of native Korean tuberculosis specialists. Proportion of Response was 53.1%, or 251 among 473 from 10th January to 30th June in 1992. The knowledge of tuberculosis among them was revealed to be higher than among ordinary native Korean people. It was different statistically by generation, namely, younger subjects aged less than 40 years old tended to answer that tuberculosis was a minor illness. The mass screening system in Japan was well known by the subjects, as shown by the fact that 72.4% of them answered that they knew about it. But only 56.6% of them replied that they actually took the mass screening. The source of its information was different statistically by sex, occupation, and generation. As for their health behavior, nearly two third (63.7%) of them visited the hospital or dispensary quickly when they fell sick. A small number of them answered that they could not visit a doctor because of their problems with the Japanese language. More than 80% of them possessed a National Health Insurance certificate. This proportion varied according to the period of stay in Japan. That is to say, The group which stayed in Japan less than one year was significantly the lowest because they were limited in their ability to enter National Health Insurance.

Adult↗
PubMed1993

[A case of adenocarcinoma presenting as a cavitary lesion with niveau formation].

Primary pulmonary adenocarcinoma rarely shows cavitation with a fluid level on chest roentgenograms. Herein we describe such a case misdiagnosed as pulmonary tuberculosis. The patient was a 63-year-old, female who had never smoked. Chest roentgenograms revealed a cavitary lesion in the left lower lobe, possessing a prominent fluid level. Fiberoptic bronchoscopic aspirate was positive for acid-fast bacilli on stains. Since there was no improvement with antituberculous chemotherapy, a left lower lobectomy was performed. The present case is of interest in the light of cavity formation in pulmonary carcinoma. The diagnosis and roentgenographic features are discussed.

Adenocarcinoma↗
PubMed1992

Genomic analysis of Theileria sergenti stocks in Japan with DNA probes.

Restriction fragment length polymorphisms of Theileria sergenti DNA from 18 different infections of cattle in 14 locations in Japan were analyzed by Southern blotting using T. sergenti genomic DNA fragments as probes. Probe pTs 2 hybridized with four fragments in BamHI digested piroplasm DNA, at 8.0, 7.3, 6.0 and 3.4 kb. Probe pTs 11-D1 hybridized with multiple fragments. With each probe, polymorphisms were observed among stocks from different locations. However, there was no correlation between the patterns of hybridization bands and the locations where parasites were collected. Analysis of the hybridization patterns of stocks obtained from individual cattle in the same grazing areas showed an almost identical pattern.

Animals↗
PubMed1992

Activation of bovine peripheral blood monocyte and its suppressive effect on parasitemia in Theileria sergenti infected calves.

Activation of bovine peripheral blood monocytes and its suppressive effect on parasite growth was examined in Theileria sergenti-infected calves by using a rosette assay that detects changes in Fc receptor expression and by luminol-dependent chemiluminescence response. Monocyte activation preceded the peak of parasitemia but was depressed parallelly with the growth of the parasites. When four calves were treated with prednisolone, three showed a good correlation between the suppression of monocyte activity and an increase of parasitemia.

Animals↗