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Biomedical subjects

K Fujisaki

Publications and source records attributed to K Fujisaki.

At least 73 records · Page 4Linked to original sources
PubMed1999

Increased susceptibility to Toxoplasma gondii infection in SAG-1 transgenic mice.

SAG-1, one of the major surface proteins of Toxoplasma gondii, has been reported to play an important role in immune and pathogenic mechanisms of the parasites but its exact function is still unclear. We investigated the time courses of T. gondii infection in B6C3F1 transgenic mice carrying the SAG-1 gene. SAG-1 transgenic mice were infected intraperitoneally with a high virulent RH strain or a low virulent Beverley strain of T. gondii. When infected with RH strain tachyzoites, no significant differences in time courses of survivals between SAG-1 transgenic and wild-type mice were observed. Both groups succumbed to an acute infection within 8 days after infection. However, a lower survival rate (20%) was observed in SAG-1 transgenic mice than in wild-type (80%), when infected with Beverley strain cysts. This result indicates that SAG-1 transgenic mice are more susceptible to T. gondii infection as compared with their wild-type counterpart. ELISA using recombinant SAG-1 protein indicates that SAG-1 transgenic mice do not produce antibodies to the SAG-1 molecule. These findings may provide a critical tool for analysing the molecular mechanisms of pathogenesis and host immune responses during toxoplasmosis.

Animals↗
PubMed1999

Inhibitory effect of monoclonal antibodies on the growth of Babesia caballi.

Monoclonal antibodies (mAbs) were produced against Babesia caballi (USDA strain) to define a species-specific antigen for use in diagnosis and vaccine development. Eight positive clones of B. caballi mAbs determined by indirect immunofluorescent antibody test were selected for purification and further characterisation. Confocal laser microscopy showed that the antigens recognised by the mAbs were located on the surface/cytoplasm, central part, and/or anterior end of B. caballi parasites, with five different reactive patterns. These mAbs seemed to be species-specific, since they did not cross-react with Babesia equi-infected erythrocytes or uninfected erythrocytes. In Western blotting analysis, 18, 20, 34, 36, 48, and 155 kDa proteins of B. caballi merozoites were recognised by six different mAbs. When added to in vitro cultures, four of the mAbs significantly inhibited the in vitro growth of B. caballi parasites. These results provide a rationale for evaluating antigens for the development of diagnostic methods or vaccines.

Animals↗
PubMed1999

Expression of Babesia equi merozoite antigen-2 by recombinant baculovirus and its use in the ELISA.

In this study, the gene encoding Babesia equi merozoite antigen-2 was inserted into a baculovirus transfer vector, and a recombinant virus expressing B. equi merozoite antigen-2 was isolated. Two B. equi merozoite antigen-2-related recombinant baculovirus-expressed peptides of 25 and 30 kDa were detected with a murine anti serum against B. equi merozoite antigen-2; these corresponded to the native B. equi merozoite antigen-2 and were secreted into the supernatants of insect cell cultures. Recombinant B. equi merozoite antigen-2 was not effected by tunicamycin treatment, indicating that B. equi merozoite antigen-2 was not glycosylated. The potential of recombinant B. equi merozoite antigen-2 for use in the ELISA was evaluated by measuring the antibody response to B. equi merozoite antigen-2 in horses experimentally infected with B. equi.

Amino Acid Sequence↗
PubMed1999

Inhibition of GABA system involved in cyclosporine-induced convulsions.

In this study, we attempted to clarify the mechanisms mediating cyclosporine-evoked convulsions. Cyclosporine (50 mg/kg, i.p.) significantly enhanced the intensity of convulsions induced by bicuculline (GABA receptor antagonist), but not those induced by strychnine (glycine receptor antagonist), N-methyl-D-aspartic acid, quisqualic acid or kainic acid (glutamate receptor agonists). Bicuculline plus cyclosporine-induced convulsions were significantly suppressed by an activation of GABAergic transmission with diazepam, phenobarbital and valproate. The GABA turnover estimated by measuring aminooxyacetic acid-induced GABA accumulation in the mouse brain was significantly inhibited by cyclosporine (50 mg/kg, i.p.). When cultured rat cerebellar granule cells were exposed to 1 microM cyclosporine for 24 hr, the specific [3H]muscimol (10 nM) binding to intact granule cells decreased to 53% of vehicle controls. The present study provides the first evidence suggesting that cyclosporine inhibits GABAergic neural activity and binding properties of the GABAA receptor. These events are closely related to the occurrence of adverse central effects including tremors, convulsions, coma and encephalopathy under cyclosporine therapy.

Animals↗
PubMed1999

Cloning and expression of a 48-kilodalton Babesia caballi merozoite rhoptry protein and potential use of the recombinant antigen in an enzyme-linked immunosorbent assay.

A cDNA expression library prepared from Babesia caballi merozoite mRNA was screened with a monoclonal antibody BC11D against the rhoptry protein of B. caballi merozoite. A cDNA encoding a 48-kDa protein of B. caballi was cloned and designated BC48. The complete nucleotide sequence of the BC48 gene had 1,828 bp and was shown to contain no intron. Southern blotting analysis indicated that the BC48 gene contained more than two copies in the B. caballi genome. Computer analysis suggested that this sequence contained an open reading frame of 1,374 bp with a coding capacity of approximately 52 kDa. The recombinant protein expressed by the vaccinia virus vector in horse cells had an apparent molecular mass of 48 kDa, which was the same as that of the native B. caballi 48-kDa protein. Moreover, recombinant proteins expressed by the pGEX4T expression vector in Escherichia coli as glutathione S-transferase fusion proteins were used for antigen in an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate very clearly between B. caballi-infected horse sera and B. equi-infected horse sera or noninfected normal horse sera. These results suggest that this simple and highly sensitive test might be applicable to the detection of B. caballi-infected horses in the field.

Amino Acid Sequence↗
PubMed1999

Expression of recombinant Toxoplasma gondii P24.

The gene encoding Toxoplasma gondii P24 has been reported previously. To determine the function of P24 against immune systems in the near future, we prepared recombinant P24 antigens using Escherichia coli, insect cells infected with recombinant baculovirus and mammalian cells infected with recombinant vaccinia virus. The P24 antigens derived from E. coli, insect cells and mammalian cells were detected with mouse immune sera against P24 or T. gondii homogenates by Western blot analysis; these corresponded to the authentic P24 and secreted into the supernatants of the insect and mammalian cell cultures. These proteins were not effected by tunicamycin treatment in cultured cells, indicating that recombinant P24 did not contain N-linked sugars. Recombinant P24 was separated by two-dimensional electrophoresis and analyzed by Western blotting. From these results, P24 was acidic protein and had identical isoelectric point with the authentic P24.

Animals↗
PubMed1999

DNA polymorphism of srRNA gene among Eimeria tenella strains isolated in Japan.

DNA polymorphism in twelve starains of Eimeria tenella isolated from various places in Japan was examined using 1.l kb small subunits ribosomal RNA amplified by PCR. Genetic variation was evaluated by random amplification of polymorphic DNA (RAPD) analysis. DNA fingerprint patterns were grouped into two, indicating that at least two DNA polymorphisms exist in Japanese E. tenella strains.

Animals↗
PubMed1998

Inter-species differentiation of benign Theilerias by genomic fingerprinting with arbitrary primers.

A method was developed to obtain reproducible DNA fingerprints from five distinct purified benign Theileria genomic DNAs by PCR-based amplification. Randomly amplified polymorphic DNA (RAPD) profiles were obtained from 10 randomly designed 12-mers. However, nine of the 10 primers could generate the difference in RAPD-PCR profiles which allowed discrimination of Theileria species. The method has advantage of being simple, fast and sensitive for diagnosis and characterization of the parasites since it does not require prior DNA sequence information to construct species-specific probes or primers. The results are also beneficial for a proper understanding of the epidemiology and designing rational control programmes for Theileriosis in Asian and South-East Asian countries.

Animals↗
PubMed1998

Circulating vascular endothelial growth factor in patients with colorectal cancer.

OBJECTIVE: The expression of vascular endothelial growth factor (VEGF), a glycoprotein that selectively promotes proliferation of endothelial cells, has been associated with cancer development. The aim of the present study was to determine whether serum levels of VEGF correlate with disease progression in patients with colorectal cancer. METHODS: VEGF levels were measured by a highly sensitive enzyme-linked immunosorbent assay in sera from 67 patients with colorectal cancer, 14 patients with colorectal adenomas, and 72 healthy volunteers, and in tissue homogenates from 10 patients with colorectal cancer. RESULTS: Serum VEGF levels were significantly higher in patients with colorectal cancer than in patients with colorectal adenomas or in normal controls (p < 0.01). In patients with colorectal cancer, serum VEGF levels were significantly associated with Dukes stage (p < 0.01) and with carcinoembryonic antigen levels (r = 0.725, p < 0.001). Patients with hepatic and/or lymph node metastasis had higher serum VEGF levels than those without. Surgical resection of the colorectal tumor led to a decrease in serum VEGF levels whether or not metastasis was present (p < 0.05). The tumor-bearing tissue contained significantly more VEGF than normal-appearing mucosa (p < 0.05). CONCLUSIONS: VEGF is involved in the development of colorectal cancer. Measurement of VEGF in the serum may be a useful noninvasive clinical marker for evaluating the disease status.

Adenoma↗
PubMed1998

Usefulness of the high-frequency ultrasound probe in pretherapeutic staging of superficial-type colorectal tumors.

We evaluated the usefulness of the high-frequency ultrasound probe (HFUP, 20 MHz) to determine the depth of tumor invasion in 45 patients with superficial colorectal tumors. The correct diagnostic rate was 66% (30/45) when the depth of tumor invasion was classified into the following 6 layers: mucosa (m), upper 1/3 (sm1), middle 1/3 (sm2), and lower 1/3 (sm3) areas of the submucosa, muscularis propria (mp), and the subserosa or deeper areas (s). However, when the depth of tumor invasion was evaluated in 3 layers (m-sm1, sm2-sm3, and mp-deeper layer), which is the classification used to select cases for endoscopic mucosal resection, the correct diagnostic rate was 88.9% (40/45). These results suggest that the HFUP is useful to determine the depth of invasion to select treatment for superficial colorectal tumors.

Aged↗
PubMed1997

Expression of a 70-kDa heat-shock-related protein during transformation from free-living infective larvae to the parasitic stage in Strongyloides venezuelensis.

The in vitro transformation system of Strongyloides venezuelensis has been established which induces free-living infective larvae to transform into the parasitic stage by a temperature shift from 25 to 37 degrees C. Comparison of the profiles of proteins labeled with [35S]-methionine in infective larvae at 25 and 37 degrees C with marked morphological transformation revealed an increase in the 70-kDa protein, thus showing these proteins to be related to the family of heat-shock proteins. In addition, the new appearance of two complexes between 16-kDa and 22-kDa proteins was observed. The 70-kDa protein cross-reacted with monoclonal antibody against human heat-shock protein 70, which was constitutively expressed. These proteins, synthesized during the transformation of S. venezuelensis from the infective larval stage to the parasitic stage, might be crucial for biochemical events that regulate the infectivity of the parasite for the host.

Animals↗
PubMed1997

A polymorphism observed in the experimentally successful peptide vaccine sequence derived from Theileria sergenti piroplasm major surface antigen (p33).

A polymorphism in the experimentally successful peptide vaccine sequence (EVVWKEKKEVKDLDA, amino acids 134-148) derived from the 33 kDa piroplasm major surface antigen (p33) of Theileria sergenti was examined. The vaccine sequences obtained by PCR amplification and sequencing of the p33 gene from a total of 15 parasite-infected cattle blood samples collected from 4 prefectures through Hokkaido to Kumamoto revealed the two major sequences (Ikeda and Chitose stock types) either of which was identified in all samples. Since the peptide vaccine develops the parasite species- or stock-specific immunity in the animals, an application of the two major peptide sequences as cocktailed vaccine should be evaluated for a practical use of this strategy to controlling T. sergenti infection in Japan.

Amino Acid Sequence↗
PubMed1997

Discrimination of eight chicken Eimeria species using the two-step polymerase chain reaction.

A method was developed for the discrimination of 8 Eimeria species of chickens, i.e., E. acervulina, E. brunetti, E. mitis, E. maxima, E. necatrix, E. praecox, E. tenella, and E. hagani using the 2-step polymerase chain reaction (PCR). In the first PCR, the small subunit ribosomal RNA (srRNA) gene was amplified from the parasite genome using conserved sequences for the Apicomplexa srRNA gene as the primers. The srRNA gene amplified from the parasite genome was discriminated in the second step by random-amplified polymorphic DNA (RAPD) PCR using 10 arbitrary primers. Each arbitrary primer produced species-specific RAPD patterns that provided a simple method for species identification from the srRNA genes of the 8 Eimeria species. This method should be useful for discrimination of the parasite species for diagnosis or epidemiological surveys of chicken coccidiosis.

Animals↗
PubMed1996

Specialty choice and understanding of primary care among Japanese medical students.

To assess specialty choice and understanding of primary care among Japanese medical students, all students from seven Japanese medical schools (three public and four private) were surveyed, using a written questionnaire. A total of 3377 students provided data for the study. Of the students surveyed, 89.8% wanted to become clinicians, and 79.3% wanted to have general clinical ability. About half of the respondents, 54.9%, replied that they had some, or great, interest in primary care, but it was found that their understanding of primary care was inadequate. Almost half (56.3%) of the students answered that they had some idea of what a general practitioner did. This proportion was nearly the same through all years of medical school. While 1245 (36.9%) students (most of them in the fifth or sixth year) replied that they had received some clinical training while working in hospitals, only 203 (6.0%) students had worked in private clinics (the sites where most primary care is still provided), and 129 (3.8%) students had experience in providing home visits and home care. An even greater number, 64.3%, replied that they had inadequate information about the career options available to them. The study found that although many Japanese medical students want to obtain broad clinical competence, their understanding of primary care is insufficient. In order to increase the number of primary care providers the system of medical education in Japan must provide primary care doctors to act as role models, and must make available information about postgraduate primary care programmes. These programmes need to be increased, as do rewarding positions for programme graduates.

Career Choice↗
PubMed1996

Experimental transmission of Babesia ovata oshimensis n. var. of cattle in Japan by Haemaphysalis longicornis.

Transovarial transmission of a newly isolated large intraerythrocytic parasite, Babesia sp. 1 by Haemaphysalis longicornis was experimentally demonstrated. Larvae of H. longicornis were transovarially infected with the parasite by feeding as adults on the calf which had been experimentally infected with B. sp. 1. Piroplasms of B. sp. 1 were observed in peripheral blood of the calf which was infested with the parasite-infected larvae. Based on the transmissibility of the parasite with vector ticks, this parasite was suggested to be a variety of B. ovata. Thus, we propose a new variety name for B. sp. 1 as B. ovata oshimensis n. var.

Animals↗