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Biomedical subjects

M Ueda

Publications and source records attributed to M Ueda.

At least 55 records · Page 3Linked to original sources
PubMed1995

A novel promoter, derived from the isocitrate lyase gene of Candida tropicalis, inducible with acetate in Saccharomyces cerevisiae.

When the isocitrate lyase gene, containing 5'-upstream and 3'-flanking regions, of an n-alkane-assimilating yeast Candida tropicalis was introduced into Saccharomyces cerevisiae, the enzyme was functionally overexpressed in the cells grown on acetate. The amount of the recombinant isocitrate lyase expressed in S. cerevisiae was as much as 30% of the total soluble proteins in the cells, being comparable to that with GAL7 functional under the control of galactose. The expression was also observed when the cells were grown on glycerol, lactate, ethanol or oleate. These facts indicate that the isocitrate lyase gene upstream region (UPR-ICL) contains a strong promoter functional in S. cerevisiae. UPR-ICL is active as a promoter on cheap carbon sources such as acetate and nonconventional carbon sources such as oleate, whereas many conventional strong promoters demand relatively expensive sugars or sugar derivatives. Therefore, it is promising to construct an economical recombinant protein production system by using UPR-ICL.

Acetates↗
PubMed1995

Formation of epithelial sheets by serially cultivated human mucosal cells and their applications as a graft material.

A cultured epithelial sheet can be formed from living mucosal cells in vitro and used as a graft material. In this article, we describe our culturing methods for the preparation of mucosal epithelial sheets as well as the biological characteristics of these sheets compared with those of skin epithelial sheets. A cultured epithelial sheet has 5 to 8 cell layers and sufficient mechanical strength to be used as a graft material. It takes 12 days to form an epithelial sheet from small epithelial segments as compared with 14 days in the case of a skin epithelial sheet. Furthermore, viability of mucosal epithelial sheets was maintained for 30 days in vitro as opposed to 22 days for skin epithelial sheets. Based on the findings from an in vitro study, we applied this cultured mucosal epithelium to humans for reconstruction of skin and mucosal defects and succeeded in repairing the defects. This report also presents an overview of the problems relevant to the use of such methods.

Adult↗
PubMed1995

Early recovery of host-derived hematopoiesis in marrow transplant recipients conditioned with high-dose busulfan and cyclophosphamide.

Three marrow transplant recipients with hematologic malignancies (two AML, one myelodysplastic syndrome) experienced prolonged pancytopenia after allogeneic BMT following conditioning with non-TBI regimens containing high-dose busulfan and cyclophosphamide (Bu/CY), despite the use of G-CSF. Early recovery of host-derived hematopoiesis ensued. Although neutrophil counts in these patients exceeded 500 x 10(6)/l by day 30 after transplant, these cells were of host origin. This early recovery of host-derived hematopoiesis has been observed rarely among patients conditioned with TBI-based regimens. When patients conditioned with Bu/CY show delayed hematologic recovery, mixed chimerism should be considered even in the presence of normal neutrophil recovery.

Adolescent↗
PubMed1995

[Early changes at anastomotic sites of saphenous vein grafts after coronary artery bypass grafting].

Saphenous vein grafts are known to develop intimal fibrocellular proliferation, however, there is little information regarding early changes of intimal fibrocellular proliferation after coronary artery bypass grafting (CABG). This study is based on 6 distal anastomotic sites obtained at autopsy from 3 patients who died within 9 days after CABG. The anastomotic sites were sectioned serially and studied with conventional and immunocytochemical techniques. Endothelial cells at all anastomotic sites showed desquamations. In two cases at 2 and 3 days after CABG, the luminal surfaces of anastomotic sites were covered by fibrin-platelet thrombus with infiltration of T-lymphocytes and macrophages. At these sites, moreover, some spindle-shaped cells were also present. Immunocytochemically, these spindle-shaped cells, which were stained with vimentin but negative with both anti-actin markers HHF35 and CGA7, were considered to be de-differentiated smooth muscle cells. In the case at 9 days after CABG, cellular reactions were mainly composed of macrophages and de-differentiated smooth muscle cells. In conclusion, these observations in human saphenous vein grafts at an early stage suggest that 1) endothelial denudation leads to adhesion of fibrin-platelet thrombus and subsequent cellular response. 2) T-lymphocytes and macrophages may play an important role in an early stage of intimal fibrocellular proliferation.

Aged↗
PubMed1994

Ultrastructural and immunohistochemical characterization of basal cells in three-dimensional culture models of the skin.

Keratinocytes were cultured on fibroblast-free dermal substitutes made of type I collagen film (collagen dermal substitute) and an extracellular matrix gel film (matrix dermal substitute), each of which was laid on a lyophilized type I collagen sponge. The morphology of the basal keratinocytes in these three-dimensional culture models of the skin was studied ultrastructurally and immunohistochemically to assess their differentiation to basal cells. The basal keratinocytes in the artificial epidermis cultured on the collagen dermal substitute showed poorly organized tonofibril networks and desmosomes. Neither the tonofibril-hemidesmosome complex nor the lamina densa were detected along the interface, where many cytoplasmic projections of basal keratinocytes were noted. There were no detectable antigens of type IV or VII collagen, LDA-1, or laminin in the interface. Bullous pemphigoid (BP) and 1-2B7B antigens and integrins were expressed along the cytoplasmic membrane and the projections of the basal keratinocytes. A high molecular weight keratin (keratin 1, 68 kDa, 34 beta B4) was detected only in part of the uppermost layers of this artificial epidermis. In contrast, basal keratinocytes in the artificial epidermis on the matrix dermal substitute developed tonofibril networks radiating to desmosomes and hemidesmosomes, under which a primitive lamina densa was present. Basement membrane zone antigens, such as type IV and VII collagens, LDA-1 and laminin were noted along the interface as were 1-2B7B and BP antigens and integrins. Laminin and type VII collagen were also detected along or in the membrane of the endoplasmic reticulum of basal keratinocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Basement Membrane↗
PubMed1994

M-VEC (methotrexate, vinblastine, epirubicin, and cisplatin) with granulocyte colony-stimulating factor for the treatment of urothelial cancer: an effective and safe chemotherapy regimen.

The toxicity of combination chemotherapy is significant, with the most prominent side effect being myelosuppression. To reduce the toxicity, we used a recombinant human granulocyte colony-stimulating factor (rhG-CSF). A total of 52 patients were enrolled in this study. The sites of tumor involvement included the urinary bladder in 24 patients, the renal pelvis in 5, the ureter in 4, lymph nodes in 11, bone in 4, the lung in 1, and miscellaneous sites in 4 patients. The chemotherapy was given in 21-day cycles as follows: 30 mg/m2 methotrexate was given intravenously on day 1, and approximately 24 h later, 3 mg/m2 vinblastine, 30 mg/m2 epirubicin, and 70 mg/m2 cisplatin were given intravenously. The rhG-CSF (2 micrograms/kg per day) was injected subcutaneously on days 3-16 of each cycle. All patients received full doses of the antineoplastic agents on time according to the protocol design. The response rates were 61% for primary sites, 55% for lymph nodes, 0 for bone, and 67% for miscellaneous sites. Of 42 patients evaluated, 5 (12%) achieved a complete response and 20 (48%) achieved a partial response, for an overall response rate of 60%. Of the 42 patients, 27 (64%) are alive, and the median duration of survival is 14 months. The mean nadir white blood count was more than 5,600 cells/mm3. The incidence of mucositis in the total toxic symptoms was low. There was no cardiac toxicity or drug-related death. These results indicate that the present combination chemotherapy with coadministration of rhG-CSF is an effective and safe regimen for the treatment of urothelial cancer.

Adenocarcinoma↗
PubMed1994

Age and sex-dependent alterations of serum amylase and isoamylase levels in normal human adults.

Total amylase and salivary- and pancreatic-type isoamylase levels were assayed in sera from 606 apparently healthy adults of different sex and age groups. There were significant differences in both total amylase and isoamylase levels, depending on age and sex, one of the characteristics being that levels of these three enzymes were significantly higher in the elderly group in both men and women than in other age groups. Another feature was that all of these enzyme levels were significantly greater in women in the third and fourth decade than in men. Age and sex differences should be taken into consideration in the evaluation of mild hyperamylasemia.

Adolescent↗
PubMed1994

Evidence that glutamate is released from capsaicin-sensitive primary afferent fibers in rats: study with on-line continuous monitoring of glutamate.

The aim of this study is to elucidate whether the excitatory amino acid glutamate is released from capsaicin-sensitive primary afferent fibers, and to compare the releasing effect of capsaicin on glutamate with that on substance P. The release of glutamate was measured using a fluorometric on-line continuous monitoring system, in which the immobilized glutamate dehydrogenase column was connected to an in vitro superfusion system. In the presence of 0.3 microM tetrodotoxin, 2-min application of capsaicin produced an increased outflow of glutamate, as well as an increase in the release of immunoreactive substance P from dorsal horn slices of the rat. The release of glutamate was concentration-dependently increased by capsaicin at concentrations in the range of 0.1-3 microM, and the release evoked by 10 microM capsaicin was not higher than that evoked by 3 microM. On the other hand, capsaicin at concentrations of 1-10 microM produced a concentration-dependent increase in the release of immunoreactive substance P, without effect at 0.1 microM. The amount of glutamate release evoked by 3 microM capsaicin was about 42.8 pmol.mg-1 protein, and 290 times that of immunoreactive substance P. The release of glutamate by 3 microM capsaicin was suppressed by the depletion of calcium from the superfusate. Capsaicin at 3 microM failed to increase the release of glutamate from the dorsal horn slices of the rats made an L4-L6 dorsal rhizotomy. These results suggest that capsaicin evoked the release of glutamate from primary afferent fibers in the dorsal horn and that glutamate may play an important role in pain transmission between primary afferent fibers and dorsal horn neurons.

Animals↗
PubMed1994

SENCAR mouse skin tumors produced by promotion alone have A to G mutations in codon 61 of the c-rasHa gene.

SENCAR mice, developed by selective breeding for high susceptibility to skin carcinogenesis by initiation with 7,12-dimethylbenz[a]anthracene and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA), form squamous papillomas in approximately 20% of animals treated repeatedly with TPA, without chemical initiation. DNA from eight skin tumors produced by a TPA-only protocol and four cell lines derived from these tumors was amplified by polymerase chain reaction and analyzed by discriminative oligonucleotide hybridization using oligomers specific for various c-rasHa gene codon 61 sequences. Five tumors and three cell lines had CAA (wild-type) to CGA mutations. In addition, one tumor had a CAA to CTA mutation, for a total of six of eight tumors having an activating mutation at this codon. Two tumors and one cell line had no codon 61 mutations detectable by this method. Since tumors derived from promotion-only protocols presumably originated from constitutively initiated cells, we examined tumor-free skins of untreated newborn and eight-month-old retired breeders and of 78-88-week-old SENCAR mice of both sexes, which were treated with TPA for 10 weeks starting at age 16-28 weeks and were untreated thereafter. Only the wild-type c-rasHa gene codon 61 sequence was seen, suggesting that the constitutively initiated cell population, if present, is below the limit of detection by this method.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1994

Lack of low Km diazepam N-demethylase in livers of poor metabolizers for S-mephenytoin 4'-hydroxylation.

Metabolism of diazepam was studied in vitro to identify the forms of cytochrome P450 (CYP) responsible for N-demethylation (nordazepam formation) and 3-hydroxylation (temazepam formation), using liver microsomes obtained from extensive (EM) and poor metabolizers (PM) for S-mephenytoin 4'-hydroxylation. Involvement of at least two P450 forms in diazepam N-demethylation was suggested by a biphasic pattern in Lineweaver-Burk and Eadie-Hofstee plots from the EM, whereas a monophasic pattern was observed from the PM liver microsomes. The kinetic parameters for the N-demethylation in the EM group were: Km 1, 19.4 +/- 0.4 microM; Vmax 1, 0.27 +/- 0.04 nmol min-1 per mg protein; Km 2, 346 +/- 34 microM; Vmax2, 1.82 +/- 0.63 nmol min-1 per mg protein (n = 3, mean +/- SD). The PM group showed the mean values of Km and Vmax (Km, 319 +/- 30 microM; Vmax, 1.49 +/- 0.62 nmol min-1 per mg protein) (n = 3) similar to those of Km2 and Vmax2 in the EM group. An antibody raised against CYP2C9 (anti-human CYP2C) strongly inhibited diazepam N-demethylation in EM liver microsomes at a low substrate concentration (20 microM). However, the anti-human CYP2C showed no clear inhibition of N-demethylation in EM liver microsomes at a high substrate concentration (200 microM). Diazepam N-demethylation in PM liver microsomes was not clearly inhibited by the anti-human CYP2C at either the low or high substrate concentrations. These data suggest that different P450 forms mediated diazepam N-demethylation in EM and PM liver microsomes, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies↗
PubMed1994

A case of testicular teratoma located in the opposite side of the upper abdominal wall.

We report a case of a testicular teratoma located in the opposite side of the upper abdominal wall. A 2-year-old boy presented with a non-palpable right testis and underwent right inguinal exploration for right cryptorchidism. During surgery, the tumor was found in the opposite side of the upper abdominal wall across the midline. Histological investigation of the tumor revealed a mature teratoma occurring in the intra-abdominal testis. No such case has been previously reported.

Child, Preschool↗
PubMed1994

M-VEC (methotrexate, vinblastine, 4'-epirubicin and cisplatin) combined with glycosylated recombinant human granulocyte colony-stimulating factor (rhG-CSF) for the treatment of transitional cell carcinoma of urothelium: reduction in toxicity produced by rhG-CSF.

Forty-six patients with urothelial cancer were treated with a systemic chemotherapeutic regimen consisting of methotrexate, vinblastine, 4'-epirubicin and cisplatin (M-VEC) in conjunction with glycosylated recombinant human granulocyte colony stimulating factor (rhG-CSF); then 33 were evaluated for response. Complete response was observed in 7 patients (21%) and partial response in 13 (39%). As far as the toxic effects of this treatment are concerned, mucositis of a minimum grade and leukopenia greater than grade 3 occurred in 5% and 10% of the patients, respectively; there were no cases of nadir sepsis and drug-related death. Minor toxicity such as nausea vomiting occurred in 81% of patients, and no patient required either dose-reduction or a delay of more than 5 d before starting of the second cycle. Thus, it may be concluded that M-VEC chemotherapy combined with rhG-CSF is useful in the treatment of urothelial cancer, especially when used as a neoadjuvant.

Adult↗
PubMed1994

Isolation of naturally processed peptides from a Toxoplasma gondii-infected human B lymphoma cell line that are recognized by cytotoxic T lymphocytes.

Naturally processed peptides derived from Toxoplasma gondii (T. gondii) were acid extracted from T. gondii-infected cells and detected by cytotoxic T lymphocytes (CTL) derived from peripheral blood lymphocytes of a patient with chronic toxoplasmosis. The CTL lines were obtained by weekly in vitro stimulation with a T. gondii-infected human B cell lymphoma line, ARH, which shares HLA-A2 and -Cw4 determinants with the patient. The lytic activity of these CTL lines against T. gondii-infected ARH and ARH pulsed with fraction 29 of a reversed-phase high-performance liquid chromatography (HPLC) extract from T. gondii-infected ARH was inhibited by an anti-HLA-A, B, C monoclonal antibody (mAb) and an anti-HLA-A2 mAb. Anti-HLA-DR mAb failed to block the lytic activity. Thus, the presentation of peptides by T. gondii-infected cells for CTL is mediated by HLA-A2 molecules. Interestingly, antigen presentation of ARH pulsed with naturally processed HPLC fraction 29 peptides was not inhibited by treatment with brefeldin A. The amino acid sequence of the HLA-A2-bound peptide in fraction 29 was in part consistent with the predictive algorithm of HLA-A2-binding peptide motifs.

Amino Acid Sequence↗
PubMed1993

Inhibitory effect of staphylokinase on platelet aggregation.

We investigated the effect of staphylokinase (SAK), which has specific thrombolytic properties, on human platelet aggregation. Platelet aggregation induced with collagen was observed following preincubation of platelets in platelet-rich plasma (PRP) or washed platelet suspension (WP) with SAK at 37 degrees C for 30 min. SAK inhibited platelet aggregation in PRP only at the highest examined concentration (1 x 10(-4) g/ml). Although SAK did not inhibit platelet aggregation in WP which contained fibrinogen, it did when the platelets had been preincubated with SAK and plasminogen. The most effective concentration in WP was 1 x 10(-6) g/ml. The effect could be inhibited by adding aprotinin or alpha 2-antiplasmin. The highest generation of plasmin in the same preincubation fluid was detected at 1 x 10(-6) g/ml SAK. We concluded that SAK can inhibit platelet aggregation in WP by generating plasmin and/or fibrinogen degradation products, but is only partially effective in PRP because of the existence of alpha 2-antiplasmin.

Amino Acid Sequence↗
PubMed1993

Parotid swelling. Does it affect serum isoamylase level?

Serum levels of total amylase and P-type, and S-type isoamylase were measured in 57 patients with chronic parotid swelling (41 men and 16 women; mean age 60.4) and the same number of age- and sex-matched control individuals without parotid swelling (41 men and 16 women; mean age 60.3). None of the three enzyme measurements differed significantly between patients with parotid swelling and control subjects. It is concluded that chronic enlargement of parotid glands does not affect serum isoamylase level.

Adult↗
PubMed1993

Interspecies-specific ovarian autoantigens involved in neonatal thymectomy-induced murine autoimmune oophoritis.

PROBLEM: Thymectomy of mice on day 3 after birth (3d-Tx) gives rise to the appearance of a particular type of ovarian lesion known as experimental autoimmune oophoritis (AIO). METHOD: In the present study, the spleen cells from BALB/c mice having undergone 3 day-Tx AIO were fused with X63-Ag8-6.5.3 myeloma cells, and a number of hybridoma clones producing autoantibodies against ovarian tissue were established. RESULTS: On the basis of immunohistochemical and protein-chemical analysis, two independent clones, designated at T2.2 and S1.6, were found to react with interstitial tissues surrounding the follicles. These monoclonal antibodies showed broad cross-species reactivity, in that they recognized similar antigenic macromolecules in the rat, pig, human, and mouse. The antigenic determinants were strongly resistant to heat and acid, especially to the treatment of periodic acid, indicating that the antigenic determinants had no relation with carbohydrate components. CONCLUSIONS: By antibody affinity chromatography, two kinds of autoantigens were identified. SDS-PAGE, under reduced or nonreduced conditions, revealed an 80/85 kDa protein for T2.2, and an 82 kDa for S1.6, respectively.

Animals↗
PubMed1993

Expression of human erythropoietin in cultured tobacco cells.

Human erythropoietin (Epo) cDNA was engineered for expression in cultured tobacco cells (Nicotiana tabacum L. cv. BY2). Two plasmid DNAs were constructed: pCEP, which contained Epo cDNA under control of the cauliflower mosaic virus-derived 35S RNA promoter and terminator, and pNSEP, which contained signal sequence-deleted Epo cDNA under control of the 35S RNA promoter and terminator. By using the electroporation method, each of these plasmid DNAs was transferred into the protoplasts of BY2 cells together with a plasmid, pNR35, which conferred G418-resistance on the cells. Four G418-resistant clones were obtained from protoplasts transfected with pNSEP and pNR35, and only one of them, named 11N, survived in suspension culture. Integration of pNSEP DNA into the genome of 11N cells was confirmed by Southern blot and PCR analyses. Production of Epo mRNA was shown by Northern blot analysis. Epo protein was shown to be expressed in 11N cells by colorimetric enzyme immunoassay. The productivity of Epo in the 11N cells (1 pg/g of wet cells) was very low.

Amino Acid Sequence↗