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Biomedical subjects

T Mikami

Publications and source records attributed to T Mikami.

At least 19 recordsLinked to original sources
PubMed2001

Serodiagnosis of Toxoplasma gondii infection in cats by enzyme-linked immunosorbent assay using recombinant SAG1.

The gene encoding surface antigen 1 (SAG1, P30) of Toxoplasma gondii (T. gondii) was cloned into the plasmid pGEX-4T-3 and subsequently expressed in Escherichia coli (E. coli) as a glutathione-S-transferase (GST) fusion protein. The recombinant SAG1 (rSAG1) was refolded using 8M urea solution followed by dialysis and thereafter evaluated in an enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of toxoplasmosis. The test sera were adsorbed with GST to block non-specific reactivity to the GST-SAG1 fusion protein. The ELISA with rSAG1 was able to differentiate very clearly between sera from cats or mice experimentally infected with T. gondii and sera from normal cats or mice. The ELISA detected no cross-reactivity with sera from mice experimentally infected with the closely related parasite Neospora caninum (N. caninum). Some 193 cat sera were tested for antibodies to T. gondii, out of which 40 (20.7%) reacted positively by ELISA with the rSAG1 while another 79.3% cats reacted negative to the assay. Both positive and negative sera were confirmed by Western blot analysis. The results of ELISA were in agreement with those of a commercially available latex agglutination test (LAT) kit, although the former had higher titers than the latter.

Animals↗
PubMed2001

Detection of antibodies to Hypoderma lineatum in cattle by Western blotting with recombinant hypodermin C antigen.

The cDNA encoding the entire mature hypodermin C (HC) of Hypoderma lineatum was cloned and expressed in Escherichia coli as a glutathione S-transferase fusion protein using pGEX vector. The recombinant HC protein (rHC) was tested by Western blotting to detect antibodies to H. lineatum in cattle. Western blotting with rHC as antigen clearly differentiated between H. lineatum-infested cattle sera and normal cattle sera. Forty-six out of forty-eight serum samples from cattle in Central Mongolia were positive, whereas all 30 serum samples from cows in Hokkaido, Japan, were negative by Western blotting. The result of Western blotting was identical to that of a previously developed enzyme-linked immunosorbent assay. These data demonstrated that Western blotting, with rHC expressed in E. coli, might be a useful method for the diagnosis of cattle hypodermosis.

Animals↗
PubMed2001

Winter seasonality and rotavirus diarrhoea in adults.

We investigated the aetiological role of group A rotavirus in adults with acute diarrhoea in a 4-year prospective study. Of 683 patients with acute diarrhoea, 97 (14%) shed rotavirus as a sole agent, whereas six (5%) of 115 patients without diarrhoea shed rotavirus. Half of patients with rotavirus diarrhoea required admission to hospital. Unlike rotavirus diarrhoea in children, the occurrence of rotavirus-positive cases did not show a significant winter seasonality. Rotavirus infection should be included in the differential diagnosis of diarrhoeal diseases in adults.

Adult↗
PubMed2001

Mice with only rat mtDNA are required as models of mitochondrial diseases.

We examined the possibility of generation of mice expressing mitochondrial dysfunction by introduction of exogenous mtDNA from different species using mouse mtDNA-less (rho(0)) cells as mtDNA recipients. For determination of how genetically distant species of mtDNA could replicate in cells with only the mouse nuclear genome, we introduced mtDNA of the Syrian hamster (Mesocricetus auratus) into mouse rho(0) cells, and found that its replication was not sufficient to propagate to following generations, probably due to significant incompatibility between mouse-nuclear and Syrian hamster-mitochondrial genomes. On the other hand, rat mtDNA, which propagated stably and expressed mitochondrial dysfunction in mouse cells, also disappeared rapidly by exogenous introduction of mouse mtDNA, suggesting that mouse mtDNA in mouse cells must be excluded completely before introduction of rat mtDNA for generation of mice with rat mtDNA as mitochondrial disease models.

Animals↗
PubMed2001

Specific IgA antibody response to coproantigens of Cryptosporidium parvum in serum and saliva of calves after experimental infection.

The antibody response to coproantigens of Cryptosporidium parvum was examined in saliva and sera of calves experimentally infected with C. parvum. Coproantigens of C. parvum with approximate molecular masses of 17, 15 and less than 14kDa were found in the feces of infected calves on day 3 or later, and 60 and 23kDa coproantigens observed between days 4 and 9 post-infection, respectively. The antibody reactivity to the coproantigens was mainly attributable to IgA class antibodies in saliva and was detectable during the convalescent phase of infection. A 15kDa protein isolated from the feces of infected calves by immunoaffinity adsorption using a monoclonal anti C. parvum antibody was recognized by IgA antibodies present in the saliva during the convalescent phase of infection. These results suggest that this coproantigen may be released from C. parvum sporozoites and may induce IgA antibody production in the mucosal immune system of infected calves.

Animals↗
PubMed2001

Platinum(IV) complexes with dipeptide. X-ray crystal structure, 195Pt NMR spectra, and their inhibitory glucose metabolism activity in Candida albicans.

Three dipeptide complexes of the form K[Pt(IV)(dipep)Cl3] and two complexes of the form K[Pt(IV)(Hdipep)Cl4] were newly prepared and isolated. The platinum(IV) complexes containing the dipeptide were obtained directly by adding KI to H2[PtCl6] solution. The reaction using KI was rapidly completed and provided analytically pure yellow products in the form of K[Pt(dipeptide)Cl3] for H2digly, H2gly(alpha)-ala, H2alpha-alagly and H2di(alpha)-ala. The K[Pt(IV)(digly)Cl3] complex crystallizes in the monoclinic space group P2(1)/c with unit cell dimensions a = 10.540(3) A, b = 13.835(3) A, c = 8.123(3) A, beta = 97.01(2) degrees, Z = 4. The crystal data represented the first report of a Pt(IV) complex with a deprotonated peptide, and this complex has the rare iminol type diglycine(2-) coordinating to Pt(IV) with the bond lengths of the C2-N1 (amide) bond (1.285(13) A). The 195Pt NMR peaks of the K[Pt(IV)(dipep)Cl3] and the K[Pt(IV)(Hdipep)Cl4] complexes appeared at about 270 ppm and at about -130 ppm, respectively, and were predicted for a given set of ligand atoms. While the K[Pt(IV)(x-gly)Cl3] complexes, where x denotes the glycine or alpha-alanine moieties, were easily reduced to the corresponding platinum(II) complexes, the K[Pt(IV)(x-alpha-ala)Cl3] complexes were not reduced, but the Cl- ion was substituted for OH- ion in the reaction solution. The K[Pt(digly)Cl3] and K[Pt(gly-L-alpha-ala)Cl3] complexes inhibited the growth of Candida albicans, and the antifungal activities were 3- to 4-fold higher than those of cisplatin. The metabolism of glucose in C. albicans was strongly inhibited by K[Pt(digly)Cl3] and K[Pt(gly-L-alpha-ala)Cl3] but not by the antifungal agent fluconazole.

Antifungal Agents↗
PubMed2001

A 14-3-3 protein homologue is expressed in feline enteroepithelial-stages of Toxoplasma gondii.

Fourteen cDNA clones encoding epitopes of proteins of Toxoplasma gondii feline enteroepithelial-stages parasites were isolated and expressed in Escherichia coli in an effort to determine the antigenecity of the parasites. Sequence analysis showed that four of the cDNA clones had a 930-bp open-reading frame encoding a product showing similarity to the 14-3-3 protein mRNA sequence.(1) Southern hybridization of DIG-labeled positive clone with T. gondii genomic DNA cleaved with EcoRI, BamHI and HindIII resulted in one or two bands in each case. In an immunofluorescence assay, polyclonal and monoclonal antibodies raised against the expressed protein showed strong reactivity with feline enteroepithelial-stages parasites and sporozoites. In a complementation assay in which a plasmid carrying the protein-coding region of the isolated cDNA was introduced into a Saccharomyces cerevisiae mutant, strain DS9-22, the expressed protein showed complementation of the function of the 14-3-3 protein in yeast transformants. These findings suggest that T. gondii parasites produce a protein showing partial homology with members of the 14-3-3 protein family and this protein is expressed in feline enteroepithelial-stages parasites.

14-3-3 Proteins↗
PubMed2001

Prevention of vertical transmission of Neospora caninum in BALB/c mice by recombinant vaccinia virus carrying NcSRS2 gene.

Neospora caninum infection is the major cause of bovine abortion. To develop a vaccine against N. caninum infection, recombinant vaccinia viruses carrying NcSRS2 and NcSAG1 genes (vv/Nc-p43 and vv/Nc-p36, respectively) were constructed and were tested in a mouse model. Vaccination of dams with vv/Nc-p43 appeared to confer effective protection against vertical transmission to offspring, though that with vv/Nc-p36 only provided partial protection. Moreover, the vv/Nc-p43 vaccination provoked cellular immune responses and antibody production against N. caninum. In conclusion, it is expected that vv/Nc-p43 can be used as an effective live vaccine to prevent vertical transmission of N. caninum in natural hosts.

Animals↗
PubMed2001

Radical fringe negatively modulates Notch signaling in postmitotic neurons of the rat brain.

Fringe was originally identified as a novel secreted signaling protein with a key role in wing formation of Drosophila. Three vertebrate fringe homologues, Radical, Lunatic and Manic fringe, were also identified, and have been shown to play major roles in neurogenesis during development. However, the expression and roles of vertebrate fringe homologues in the adult brain remain to be elucidated. We isolated the cDNA encoding rat Radical fringe (334 amino acids) from rat embryos, and found its mRNA to be most abundantly expressed in the adult rat brain by Northern blotting analysis. The localization of Radical fringe mRNA in the adult rat brain was also examined by in situ hybridization. The mRNA was abundantly expressed in most neurons, but not glial cells, throughout the brain. Notch signaling was shown to negatively modulate the stability of neurites and connections in postmitotic primary neurons. Furthermore, genetic evidence indicated that fringe modulated the Notch signaling pathway. Therefore, we examined the effects of Radical fringe on the Notch signaling pathway in primary rat neurons of the cerebral cortex using recombinant rat Radical fringe protein. Radical fringe protein significantly inhibited expression of the Notch effector Hes1 mRNA in primary neurons. These results indicated that Radical fringe functions by inhibiting Notch signaling in postmitotic neurons of the brain.

Amino Acid Sequence↗
PubMed2001

CD44 variant overexpression in gallbladder carcinoma associated with tumor dedifferentiation.

BACKGROUND: Recent studies have suggested a correlation between increased or decreased expression of CD44 variant molecules and tumor metastasis. CD44 expression in gallbladder carcinoma was examined and compared with tumor differentiation. METHODS: Eighty-three samples of gallbladder carcinoma, 17 gallbladder adenoma samples, and 66 normal control mucosa samples were stained immunohistochemically for CD44 standard form (CD44s), variant 3 (CD44v3), and variant 6 (CD44v6). RNA extracted from nine patients with carcinoma also was investigated with reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and Southern blot hybridization (SBH) for the CD44 gene. RESULTS: Normal gallbladder mucosa showed strong, membranous staining for CD44s but not for CD44v3 or CD44v6. In gallbladder tumors, CD44s was stained as strongly as it was in normal mucosa, but immunoreactivity for CD44v3 and CD44v6 also was significant. In well differentiated, advanced adenocarcinomas (n = 38), CD44s immunoreactivity was significantly lower in the invasive component than in the intramucosal component of the tumors (P = 0.0048). Immunoreactivity for CD44v3 and CD44v6 in moderately and poorly differentiated areas was significantly higher than in well differentiated areas (P < 0.0001 and P = 0.0378, respectively). RT-PCR and SBH signals for CD44v3 and CD44v6, including exons 7 and 10, were strong in carcinoma samples but weak in normal samples, in line with the results of immunohistochemistry. The prognosis of patients with gallbladder carcinoma was not associated significantly with altered expression of CD44s, CD44v3, or CD44v6. CONCLUSIONS: The current study demonstrated that CD44 variant overexpression in patients with gallbladder carcinoma was linked closely with histologic dedifferentiation rather than clinicopathologic factors, including prognosis.

Adenocarcinoma↗
PubMed2001

Protective efficacy of vaccination by recombinant vaccinia virus against Neospora caninum infection.

The recombinant vaccinia viruses expressing the surface protein of Neospora caninum tachyzoite, NcSAG1 or NcSRS2, were constructed. The vaccination with these recombinant viruses could protect effectively the parasite invasion in a mouse model system. The vaccine efficacy of NcSRS2 was higher than that of NcSAG1. The present study indicated that a high level of IgG1 Ab production to parasite is important for clearance of parasite at the early stage of infection and that T cell response has a crucial role for protection against the intracellular infection at the late stage of infection. The recombinant vaccinia viruses might be applicable as vaccine against N. caninum infection in a natural host.

Animals↗
PubMed2001

More frequent beta-catenin exon 3 mutations in gallbladder adenomas than in carcinomas indicate different lineages.

To clarify the contribution of beta-catenin, which is related to cell adhesion and intranuclear transcription, to gallbladder carcinogenesis, we investigated its expression using immunohistochemistry, and beta-catenin exon 3 mutations by DNA direct sequencing, in 18 gallbladder adenomas and 82 adenocarcinomas. Membranous expression was significantly lower in moderately and poorly differentiated than in well-differentiated adenocarcinoma cases (P < 0.001). The gallbladder adenomas showed significantly stronger expression in the cytoplasm and the nucleus than carcinomas (P < 0.05 and P < 0.001, respectively), and exon 3 mutations were observed in 62.5% (10 of 16) of adenomas, but only 4.8% (1 of 21) of carcinomas. With beta-catenin as a molecular marker, the adenoma-carcinoma sequence can be considered to be a minor pathway in gallbladder carcinogenesis.

Adenoma↗
PubMed2001

Treatment modalities for oral verrucous carcinomas and their outcomes: contribution of radiotherapy and chemotherapy.

BACKGROUND: This clinical study focused, firstly, on the results of treatment and, secondly, on the anaplastic transformation, of oral verrucous carcinomas (OVCs) diagnosed and treated from 1981 to 1997 at the Department of Oral and Maxillofacial Surgery at Shimane Medical University Hospital. METHODS: We analyzed the treatment modalities and outcomes for 15 patients with OVC. RESULTS: Excluding the results for 4 palliatively treated patients, the disease-free survival rates of the patients after the initial treatments, were 82% at 5 years and 66% at 10 years; for all 15 patients, these rates were 57% and 46%, respectively. Surgery alone and surgery combined with other treatments (such as radiotherapy and chemotherapy) appeared to yield disease-free survival rates to those achieved superior with other treatments whether single or combined; (78% vs 33% for 5-year disease-free survival; 52% vs 33% for 10-year disease-free survival); however, the difference was not significant (P = 0.47). Well differentiated squamous cell carcinomas (W-SCCs) (n = 5) as well as spindle cell carcinoma (n = 1) were found in subsequent operative or biopsy specimens. CONCLUSION: Surgery was the most reliable treatment method for OVC; however, radiotherapy combined with chemotherapy was the next most preferable treatment when surgery was not undertaken. We also found that highly malignant transformation (anaplastic transformation) occasionally occurred during treatments for OVC.

Aged↗