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Biomedical subjects

M Oshima

Publications and source records attributed to M Oshima.

At least 73 records · Page 4Linked to original sources

Antibodies and T cells against synthetic peptides of the C-terminal domain (Hc) of botulinum neurotoxin type A and their cross-reaction with Hc.

Seventeen peptides containing T cell and/or antibody (Ab) epitopes previously localized on Hc of botulinum neurotoxin type A were used in SJL and BALB/c mice as immunogens either individually or as an equimolar mixture of groups that contained epitopes of T cells, Abs or both, to determine their abilities to generate T cells and/or Abs that recognize intact Hc. In SJL, peptide 897-915 which included both T cell and Ab epitopes, elicited Abs that cross-reacted very strongly with Hc. In BALB/c, peptides 869-887, 883-901, 981-999 and 1275-1296 which contained Ab epitopes generated Abs that cross-reacted strongly with Hc. A mixture of peptides that contained T cell and Ab epitopes was effective in both strains in eliciting T cells and Abs that cross-reacted with Hc. This mixture form gave a quicker rise (after two injections) in cross-reactive (with Hc) Ab titer as compared to other peptide mixtures or the individual peptides, and sustained in BALB/c a high Ab titer upon further booster injections. Some of the regions that elicited crossreactive immunity to Hc have sequence similarity to other clostridial toxins, suggesting that one or more of these synthetic peptides might provide cross-protection against those toxins.

Amino Acid Sequence↗

Nuclear translocation of beta-catenin in hereditary and carcinogen-induced intestinal adenomas.

The physical interaction between beta-catenin and the adenomatous polyposis coli (APC) gene, and the ability of APC to regulate cytoplasmic levels of beta-catenin suggest a role for beta-catenin in colorectal carcinogenesis. In this study, we found that beta-catenin immunoreactivity was detected exclusively in the cell membrane and cytoplasm of morphologically normal intestinal epithelial cells with predominant distribution in the differentiated nonproliferative cell population. In contrast, beta-catenin was localized predominantly in the nucleus of adenomas from Min/+ mice and transgenic mice expressing a mutant truncated form of the APC gene (Apc(delta716) mice). Beta-catenin was expressed predominantly at the cell membrane and cytoplasm of the nontransformed rat intestinal epithelial (RIE-1) cells in culture, whereas predominantly nuclear localization of beta-catenin was observed in the human colon cancer cell line SW480. In the azoxymethane (AOM) treated rats, overexpression and nuclear localization of beta-catenin was observed in all adenomas. Previous studies have indicated the incidence of APC mutations amongst AOM-induced tumors to be 15% or less. These results demonstrate that nuclear localization of beta-catenin is a common event in colorectal tumorigenesis.

Adenoma↗

Threshold number of provirus copies required per cell for efficient virus production and interference in moloney murine leukemia virus-infected NIH 3T3 cells.

The gag-pol readthrough mutant of Moloney murine leukemia virus, MLV-B(CAG) (T. Odawara, H. Yoshikura, M. Oshima, T. Tanaka, D. S. Jones, F. Nemoto, Y. Kuchino, and A. Iwamoto, J. Virol. 65:6376-6379, 1991), was poorly complemented by a mutant encoding only Gag. This is because with all the genetic elements necessary for env expression present in MLV-B(CAG), insufficient Env protein was produced by the cells expressing MLV-B(CAG) for efficient virus production. Since the env mRNA expression per provirus in the MLV-B(CAG)- and wild-type-MLV-producing cells were the same and since the cells expressing the former contained eightfold fewer proviral copies, the insufficient Env expression by the former was found to be due to insufficient proviral copies in the cells. Examination of the cell clones having various proviral copies of Deltawt MLV (M. Oshima, T. Odawara, T. Matano, H. Sakahira, Y. Kuchino, A. Iwamoto, and H. Yoshikura, J. Virol. 70:2286-2295, 1996) showed that mRNA level was proportional to the number of proviral copies while interference and virus production followed a sigmoid curve with a sharp rise at the threshold number of proviral copies of around four per cell. Multicycle infection probably continues until the threshold level of proviral copies is attained in natural infection too.

3T3 Cells↗

cis Elements required for high-level expression of unspliced Gag-containing message in Moloney murine leukemia virus.

The 441-nucleotide (nt) region (nt 5325 to 5766) around the splice acceptor (SA) site (nt 5491) was found to be necessary for high-level expression of gag-containing unspliced RNA of Moloney murine leukemia virus (M. Oshima, T. Odawara, T. Matano, H. Sakahira, K. Kuchino, A. Iwamoto, and H. Yoshikura, J. Virol. 70:2286-2295, 1996). Detailed genetic dissection of the 441-nt region revealed that the 5'-end 64 nt (nt 5325 to 5389) were necessary for high-level expression of the unspliced RNA when the spliced RNA was not produced, while the 3'-side 301 nt (nt 5466 to 5766) containing the SA site were necessary for producing spliced RNA. When the spliced RNA was produced, the unspliced RNA could be expressed at a high level even when the 5'-end 64 nt were absent. Probably the virus sequence ensuring the splicing could produce an RNA structure able to compensate for the function of the 5'-end 64-nt region responsible for the expression of the unspliced RNA.

3T3 Cells↗

T cell responses in EAMG-susceptible and non-susceptible mouse strains after immunization with overlapping peptides encompassing the extracellular part of Torpedo californica acetylcholine receptor alpha chain. Implication to role in myasthenia gravis of autoimmune T-cell responses against receptor degradation products.

To study the role in myasthenia gravis (MG) of peptides resulting from acetylcholine receptor (AChR) degradation, we examined the ability of AChR peptides to induce T cell responses that are capable of cross-reacting with intact AChR. The studies were carried out in an experimental autoimmune MG (EAMG)-susceptible mouse strain [C57BL/6 (B6)] as well as in two non-susceptible strains [B6.C-H-2bm12 (bm12) and C3H/He]. A set of overlapping peptides encompassing the extracellular part (residues 1-210) of the alpha-chain of Torpedo californica (t) AChR were used, individually or in equimolar mixtures, as immunogens. In B6, immunization with peptides alpha45-60, alpha111-126, alpha146-162 and alpha182-198 gave T cells that responded in vitro to the correlate immunizing peptide. Only the T cells against the latter three peptides cross-reacted with tAChR. Peptide alpha146-162 exhibited the highest in vitro reaction with the immunizing peptide and cross-reaction with tAChR. T cells obtained by immunization of B6 with an equimolar mixture of the peptides responded in vitro to peptides alpha111-126, alpha146-162 and alpha182-198 and cross-reacted very strongly with tAChR. In bm12 and C3H/He, a number of peptides evoked, when used individually as immunogens, strong or moderate T cell responses that recognized in vitro the correlate immunizing peptide but cross-reacted poorly with tAChR. Immunization with the mixture of the peptides gave T cells that recognized several peptides in each strain butdid not cross-react with alpha146-162 or tAChR. The results indicate that the ability to recognize alpha146-162 or AChR by T cells against peptides resulting from receptor degradation can account for the susceptibility to, and aggravation of, MG in B6.

Amino Acid Sequence↗

[Intralobar sequestration associated with pulmonary aspergillosis].

A 49-year-old man with a cough productive of black sputum was admitted to our hospital. A chest CT scan was done. Cystic, estatic changes, and intra-capsula fluid surrounding an enhanced wall in the right S10 were seen. Aspergillus niger was detected by sputum culture. Aortic angiography showed that an aberrant artery originated from the inferior diaphragmatic artery and penetrated the right S10. The preoperative diagnosis was lung sequestration associated with aspergillosis. The operative procedure included right lower lobectomy and ligation of the aberrant artery. Postoperatively, Pryce-type intralobar sequestration infected with aspergillus was diagnosed. Microscopically, the parenchyma was distorted and chronic inflammation was present. Hyphae of aspergillus were found within the lumen of the cyst.

Aspergillosis↗

[Evaluation of clinical utility of 99mTc-MIBI scintigraphy in the localization of hyperfunctioning parathyroid lesions in patients with hyperparathyroidism--a report of multicenter phase III clinical trials].

Phase III clinical study in 78 patients with hyperparathyroidism was performed to determine clinical utility of 99mTc-MIBI in the localization of hyperfunctioning parathyroid lesions. Except slight tremor in one patient, no adverse events were reported. No abnormal changes in clinical laboratories or vital signs were noted. The clinical utility of the agent was evaluated in 70 patients. Out of 108 hyperfunctioning glands, 93 (86%) were detected with 99mTc-MIBI regardless of their histology, numbers, or location. Specifically, single or ectopic lesions were detected with high sensitivity (97% and 100%, respectively). Sensitivity in 53 glands with weight data was 79%, while 94% in 36 glands above 200 mg, which is extremely high compared to the 201T1-99mTc subtraction method. Specificity in a group of PHP patients with single adenoma who underwent surgery was 100% (63/63), though in case of coexistent thyroid disease obviously interfered parathyroid images. Our study indicates that 99mTc-MIBI is a safe and excellent agent for the localization of hyperfunctioning parathyroid tissues. Especially, the fact that 99mTc-MIBI detected ectopic glands with high specificity is a great advantage over the ultrasound or 201Tl-99mTc subtraction method.

Female↗

[Evaluation of clinical utility of 99mTc-MIBI scintigraphy in the localization of thyroid, breast and lung/mediastinum tumors--a report of multicenter phase III clinical trials].

Phase III clinical study in 136 patients diagnosed or strongly suspected of thyroid, lung/mediastinum or breast cancer was performed to determine clinical utility of 99mTc-MIBI in the localization of tumors, and the results in 135 were evaluated. Except taste peculiar in 43 patients and nausea in one, no adverse events were reported. Seven abnormal changes in vital signs and 27 in clinical laboratories were noted, which were all judged as having no or unlikely relationship to the agent. Out of 177 malignant lesions, 156 (88%) were detected with 99mTc-MIBI regardless of their histology or location. Of 6 benign tumors, 3 were detected. Fifteen sites of false positive in 11 malignant tumor patients and 5 sites in 6 patients with non-tumor disease were observed. Our study indicates that 99mTc-MIBI is a safe and excellent agent for the localization of tumors. Especially, the fact that 99mTc-MIBI detected remote metastatic lesions with high sensitivity is a great advantage over the other conventional imaging modalities.

Adult↗

Characterization of room temperature induced apoptosis in HL-60.

We found that exposure to room temperature (RT/21 degrees C) causes apoptosis in HL-60 cells. Here we characterized RT-induced apoptosis in HL-60. After exposure to RT, apoptosis starts within 6 h and more than 80% of the cells underwent apoptosis within 20 h. All cells, however, were committed to apoptosis after 16 h and no viable cells could be recovered. The caspase-1 inhibitor (YVAD-CHO) effectively blocked apoptosis, whereas the caspase-3 inhibitor (DEVD-CHO) did not. About 20% of newly obtained early passage HL-60 cells (passage 10) also underwent apoptosis by RT treatment. These data suggest that some population in HL-60 which responds to RT with apoptosis became dominant during passaging.

Apoptosis↗

CPP32 activation during dolichyl phosphate-induced apoptosis in U937 leukemia cells.

Treatment of U937 cells with dolichyl phosphate led to an increase in the activity of the ICE family protease CPP32, accompanied with cleavage of pre-CPP32 to generate p17. Peptide inhibitors YVAD-cmk and Z-Asp-CH2-DCB (specific to ICE) and DEVD-CHO (specific to CPP32) blocked the dolichyl phosphate-induced apoptosis. The dolichyl phosphate-induced increase of CPP32 activity was inhibited by adenylate cyclase inhibitors, SQ 22536 and 2',5'-dideoxyadenosine. Dolichyl phosphate caused a transient increase of intracellular cAMP concentration. The results suggest that modulation of cAMP synthesis due to the stimulation of adenylate cyclase by dolichyl phosphate plays a critical role in CPP32 activation and apoptosis.

Adenine↗

Early embryonic lethality caused by targeted disruption of the mouse selenocysteine tRNA gene (Trsp).

Selenoprotein biosynthesis is mediated by tRNASec, which inserts selenocysteine at UGA codons in a complex, context-specific manner. This opal suppressor serves in the conversion of serine to selenocysteine as well. The mouse tRNASec gene (Trsp) maps to a proximal segment of chromosome 7. We constructed mice carrying a targeted deletion of the Trsp gene. The heterozygous mutants were viable, fertile, and appeared normal. Although the level of tRNASec was reduced to about 50%-80% of the wild type in most organs, one of the selenoproteins, glutathione peroxidase, remained unaffected in the levels of its mRNA, protein, and enzyme activity, indicating that the haploid amount of tRNASec is not limiting in its biosynthesis. In contrast, the homozygous mutants died shortly after implantation, and the embryos were resorbed before 6.5 days post coitum. When the preimplantation embryos were placed in culture, however, the trophoectoderm cells showed outgrowths and the inner cell mass cells of the homozygous embryos were able to proliferate. These results indicate that Trsp expression is essential for early development of the embryo, and its lack causes peri-implantation lethality. However, the lethality does not appear to be due to a cell-autonomous function of tRNASec.

Animals↗

Morphological and molecular processes of polyp formation in Apc(delta716) knockout mice.

Mutations in the human adenomatous polyposis coli (APC) gene are responsible for not only familial adenomatous polyposis but also many sporadic cancers of the digestive tract. Using homologous recombination in embryonic stem cells, we recently constructed Apc gene knockout mice that contained a truncation mutation at codon 716 (Apc(delta716)). The heterozygous mice developed numerous intestinal polyps. All microadenomas dissected from nascent polyps had already lost the wild-type allele, indicating the loss of heterozygosity (M. Oshima et al., Proc. Natl. Acad. Sci. USA, 92: 4482-4486, 1995). We also demonstrated that cyclooxygenase 2 is induced in the polyps at an early stage and plays a key role in polyp development (M. Oshima et al., Cell 87: 803-809, 1996). We have analyzed the process of polyp development in these mice both at morphological and molecular levels. A small intestinal microadenoma is initiated as an outpocketing pouch in a single crypt and develops into the inner (lacteal) side of a neighboring villus forming a double-layer nascent polyp. The microadenoma then enlarges and gets folded inside the villus. When it fills the intravillous space, it expands downward and extends into adjoining villi, rather than rupturing into the intestinal lumen. During this course of development, the basement membrane remains intact, and the labeling index of the microadenoma cells is similar to that of the normal crypt epithelium. As in the crypt cells, neither transforming growth factor beta1 nor its receptor type II is expressed in the microadenoma cells. No hot spot mutations in the K-ras gene are found in the microadenoma tissue during these early stages of polyp development. Essentially, the same results have been obtained for the colonic polyps as well. These results suggest that early adenomas in the Apc(delta716) polyps are very similar to the normal proliferating cells of the crypt except for the lack of directed migration along the crypt-villus axis.

Activin Receptors, Type I↗

Down-regulation of epidermal growth factor receptors by nerve growth factor in PC12 cells is p140(trk)-, Ras-, and Src-dependent.

Nerve growth factor (NGF) treatment causes a profound down-regulation of epidermal growth factor receptors during the differentiation of PC12 cells. This process is characterized by a progressive decrease in epidermal growth factor (EGF) receptor level measured by 125I-EGF binding, tyrosine phosphorylation, and Western blotting. Treatment of the cells with NGF for 5 days produces a 95% reduction in the amount of [35S]methionine-labeled EGF receptors. This down-regulation does not occur in PC12nnr5 cells, which lack the p140(trk) NGF receptor. However, in PC12nnr5 cells stably transfected with p140(trk), the NGF-induced heterologous down-regulation of EGF receptors is reconstituted in part. NGF-induced heterologous down-regulation, but not EGF-induced homologous down-regulation of EGF receptors, is blocked in Ras- and Src-dominant-negative PC12 cells. Treatment with either pituitary adenylate cyclase-activating peptide (PACAP) or staurosporine stimulates neurite outgrowth in PC12 cell variants, but neither induces down-regulation of EGF receptors. NGF treatment of PC12 cells in suspension induces down-regulation of EGF receptors in the absence of neurite outgrowth. These results strongly suggest a p140(trk)-, Ras- and Src-dependent mechanism of NGF-induced down-regulation of EGF receptors and separate this process from NGF-induced neurite outgrowth in PC12 cells.

Animals↗

A human UDP-GalNAc: polypeptide, N-acetylgalactosaminyltransferase type 1 gene is located at the chromosomal region 18q12.1.

The cDNA clone encoding human UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-1 (GalNAc-T1) was isolated from colon tissue by a reverse transcriptase-polymerase chain reaction (RT-PCR). Using fluorescence in situ hybridization, the position of the GalNAc-T1 gene was shown to be localized at the human chromosomal region, 18q12.1.

Chromosome Banding↗

Immune recognition of botulinum neurotoxin type A: regions recognized by T cells and antibodies against the protective H(C) fragment (residues 855-1296) of the toxin.

Botulism toxicity is caused by botulinum neurotoxins (BoNTs), a group of protein neurotoxins produced by Clostridium botulinum. Recent studies have shown that immunization with a C-terminal fragment [H(C), residues 855-1296] of BoNT type A (BoNT/A) affords excellent protection against BoNT/A toxicity. The present work was carried out in order to map the molecular and cellular immunological recognition of H(C). We have previously described the synthesis of 31 overlapping peptides encompassing the entire H(C)-fragment of BoNT/A. These peptides were employed in this study to localize the continuous regions recognized by T cells and by antibodies (Abs) generated in two mouse strains against H(C). T cells from SJL that had been primed with H(C) gave a strong proliferative response to challenge in vitro with each of the six peptides spanning residues 897-985 and a lower response to peptide 1051- 1069. While H(C)-primed T cells of BALB/c recognized three regions residing within residues 939-957, 1009-1027 and 1135-1153 (strong). Recognition regions by Abs in SJL or BALB/c anti-H(C) antisera essentially overlapped. However, the level of Abs bound to each region differed between the two strains. These common or similar recognition regions by the two strains were: 855-915 (SJL) or 855-901 (BALB/c); 939-957; 967-1013 (BALB/c) or 981-1013 (SJL); 1051-1069; 1079-1111 (BALB/c) or 1093-1125 (SJL); 1177-1195; and 1275-1296. In addition, BALB/c recognized region 1135-1153. Some of these regions show considerable sequence similarity in BoNT types B and E and, therefore, H(C) of these two BoNTs might offer protection against the correlate clostridial toxins.

Amino Acid Sequence↗

Suppression of intestinal polyp development by low-fat and high-fiber diet in Apc(delta716) knockout mice.

Most epidemiological and animal studies show a positive correlation of the dietary intake of fat with the incidence of colon cancer, whereas an inverse correlation of the dietary intake of fiber. In rats fed a diet low in fat and high in wheat bran fiber and calcium, a significant decrease was reported in the number of azoxymethane-induced aberrant crypt foci compared with those fed a high-fat, low-fiber and low-calcium diet. Mutations in the human APC gene play a key role, not only in familial adenomatous polyposis, but also in many sporadic cancers of the entire digestive tract. We previously constructed a mouse strain Apc(delta716), carrying a truncation mutation at codon 716 of the Apc gene, the homolog of human APC (10). The heterozygous mice developed numerous intestinal polyps, and all microadenomas dissected from the earliest polyps had already lost the wild-type allele, indicating the loss of heterozygosity. Using these Apc(delta716) knockout mice, we have investigated the effect of a low-fat and high-fiber diet (LRD for 'low-risk' diet) on intestinal polyposis, and compared it with that of a high-fat and low-fiber diet (HRD for 'high-risk' diet). The mice were fed either diet for 7 weeks, and the number and size of intestinal polyps were scored. The LRD-fed mice had fewer polyps than the HRD-fed mice, by 36% in the small intestine and by 64% in the colon. As for the polyp size distribution, there was no significant difference between the HRD- and LRD-fed mice. These results indicate that LRD can suppress intestinal polyposis compared with HRD which does not, and suggest that its suppression is at the initiation of polyp formation. This is likely to be due to a decreased frequency of loss of heterozygosity, rather than a retarded growth of the polyp adenomas.

Animals↗