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Biomedical subjects

T Shimada

Publications and source records attributed to T Shimada.

At least 325 records · Page 18Linked to original sources
PubMed1998

A simple and efficient purification of transduced cells by using green fluorescent protein gene as a selection marker.

BACKGROUND: Simple and efficient method for the selection of transduced cells would greatly facilitate the clinical utilization of retrovirus vectors. We developed a therapeutic bicistronic retrovirus vector for Gaucher disease, MFG-GC-GFP, which contains the human glucocerebrosidase (GC) gene and the green fluorescent protein (GFP) gene of the jellyfish Aequorea victoria as a vital selection marker, and investigated its applicability as gene therapy for Gaucher disease. METHODS AND RESULTS: A packaging cell line, GP + envAM12, was transfected with MFG-GC-GFP and, thus, produced a high titer recombinant virus (1.0 x 10(6) c.f.u./mL) in the culture supernatant. The expression level of GFP was correlated with the virus production in cells. The recombinant virus infected skin fibroblasts from a Gaucher patient and a sorted fraction of the cells expressing GFP by flow cytometry exhibited almost a six-fold higher activity of GC than normal fibroblasts. CONCLUSIONS: These data indicate that MFG-GC-GFP enables the one-step purification of a transduced fraction of target cells and is, therefore, considered to be a useful therapeutic vector for the experimental gene therapy of Gaucher disease.

Animals↗
PubMed1998

Mesangiolytic glomerulopathy in severe congestive heart failure.

To study the glomerular morphological abnormalities in congestive heart failure (CHF), we analyzed 27 autopsy cases without other causes of renal disease. Their mean age was 59 years, and they showed mild prerenal azotemia. They had generally been treated with digitalis and diuretics, and a few of them with captopril or nifedipine. The abnormal glomerular findings of enlargement, hyperemia, and mesangial thickening were observed at high frequencies (61%, 64%, and 57%, respectively). They characteristically showed mesangiolysis (ML) by the findings of microaneurysms (81%) and mesangial degeneration (70%) such as loose reticular matrix and poor matrix area. In addition, glomerular infiltration of mononuclear leukocytes including macrophages was noted in 70% of the cases. Glomerular enlargement was not correlated with the grade of hyperemia, but it was correlated with the grade of ML index of % glomeruli with microaneurysms (F = 7.22, p < 0.004). There was an inverse relationship between the grades of mesangial thickening and of the ML index (P < 0.005). The number of glomerular leukocytes was positively correlated with mean glomerular size (P < 0.002) and with the ML index (P < 0.03). Notably, the glomerular macrophage-positive cases showed a prominently higher mean ML index than the negative cases (P < 0.005). There was an inverse correlation between the mean glomerular size and the partial oxygen pressure in arterial blood (PaO2; P < 0.01), and a positive correlation between the mean glomerular size and hematocrit (Hct) levels (P < 0.02). The cases positive for mesangiolytic mesangial degeneration showed significantly lower PaO2 values than the cases negative for this lesion (P < 0.04). In the analysis of the various causes of CHF, the patients with congenital cardiac anomalies showed mean levels of the lowest PaO2 (P < 0.02) and the highest Hct (P < 0.03) and histologically the largest mean glomerular size (P < 0.04). There was no difference in the ML index and the glomerular leukocyte number among the subgroups classified by the causes. These results indicate that ML associated with glomerular enlargement is the major glomerular abnormality characteristic in patients with severe CHF and suggest that glomerular infiltration of leukocytes, especially of macrophages, should play an important role in the progression of both ML and glomerulomegaly. The contributions of persistent hypoxia and up-regulated angiotensin II as the causative factors of these glomerular abnormalities in congestive heart failure are discussed.

Adult↗
PubMed1998

Development and evaluation of a multiplex PCR assay for rapid detection of toxigenic Vibrio cholerae O1 and O139.

A multiplex polymerase chain reaction assay was developed for concurrent detection of rfb sequences specific for the O1 and the O139 serogroups of Vibrio cholerae and for ctxA specific sequences. The multiplex PCR assay was found to be highly specific and sensitive and was capable of detecting 65 cfu and 200 cfu per assay of V. cholerae O1 and O139, respectively. Evaluation of the multiplex PCR assay using 121 stool samples from patients admitted to the Infectious Diseases Hospital, Calcutta, showed the assay to be 100% sensitive and 95.2% specific when the culture method was taken as the standard. In addition to the 38 PCR positive stool samples, an additional four samples which were negative by culture method but positive by PCR assay belonged to the O139 serogroup. All the 38 stool samples positive for either O1 or O139 serogroup by PCR assay were also positive for the ctxA amplicon indicating that the O1 and O139 V. cholerae strains have the genetic potential of producing cholera toxin.

Bacterial Proteins↗
PubMed1998

[Analysis of Vibrio cholerae O1 isolated in Japan by pulsed-field gel electrophoresis].

Vibrio cholerae O1 strains isolated mostly in Japan between 1977 and 1995 were typed according to restriction fragment patterns by cleavage of genomic DNA with Sfi I and Not I and separation by Pulsed-field gel electrophoresis (PFGE). Two hundred sixty five strains from human were divided into 60 PFGE patterns (provisional types). Strains of type 2-3, 3-4, 4-5 and 51-54 were dominant in the Philippines, Thailand, India and Indonesia, respectively. Types 1-1, 2-3, 2-53, and 3-4 were detected over a long period of time in contrast to the other types. Strains of the same type (Types 1-1, 2-3, 2-53 and others) isolated from the Japanese who had never been outside Japan were often found among strains from Southeast Asia. Most strains from humans were cholera-toxin (CT)-positive, while those from the environment and the sea were generally not. 23 strains from the environment and the sea in Japan were divided into 12 types. Strains of the same types as CT-positive strains from humans could not be found in the environment and sea. These results suggest that cholera in Japan is closely related with cholera in Southeast Asia and PFGE is useful for epidemiological analysis of cholera in Japan.

Cholera↗
PubMed1998

Molecular analysis of non-O1, non-O139 Vibrio cholerae associated with an unusual upsurge in the incidence of cholera-like disease in Calcutta, India.

There was an inexplicable upsurge in the incidence of non-O1, non-O139 Vibrio cholerae among hospitalized patients admitted to the Infectious Diseases Hospital, Calcutta, India, between February and March 1996. Of the 18 strains of V. cholerae isolated during this period, 15 belonged to the non-O1, non-O139 serogroups (4 belonged to O144, 3 belonged to O11, 1 each belonged to O6, O8, O12, O19, O39, and O58, and 2 strains could not be typed), 2 belonged to the O139 serogroup, and 1 belonged to the O1 serogroup. Cell-free culture supernatants of 13 representative non-O1, non-O139 V. cholerae strains evoked a distinct cytotoxic effect on CHO and HeLa cells, and the strains examined produced the nonmembrane-damaging cytotoxin. By several PCR assays, it was determined that none of the non-O1, non-O139 strains were positive for the ctxA, zot, ace, and tcpA genes and for the genes representing the heat-labile toxin, heat-stable toxin, and verotoxin of Escherichia coli and the various variants of these genes. Studies on the clonality of non-O1, non-O139 V. cholerae strains by restriction fragment length polymorphism (RFLP) analysis of rRNA genes and of other genes (hlyA, hlyU, hlx, toxR, and attRS1) and by pulsed-field gel electrophoresis (PFGE) collectively indicate that the upsurge which occurred in February and March 1996 was caused by strains belonging to different clones. Overall, there was an excellent correlation between the results of ribotyping, RFLP analysis of various genes, and PFGE, with strains belonging to a particular serogroup showing nearly identical restriction patterns and PFGE profiles. It is clear from this study that some serogroups of V. cholerae can cause diarrhea by a mechanism quite different from that of toxigenic V. cholerae O1 and O139, and we have proposed the nomenclature of enteropathogenic V. cholerae to include these serogroups.

Bacterial Typing Techniques↗
PubMed1998

Combination chemotherapy with G-CSF, M-CSF and EPO: successful treatment for acute myelogenous leukemia without blood transfusion at lower medical costs.

A 55-year-old Jehova's Witness was treated for acute myelogenous leukemia (AML) by intensive chemotherapy with enocitabine, 6-mercaptopurine and daunorubicin. G-CSF, M-CSF and EPO were subsequently administered. Even though no blood transfusion was given for religious reasons, complete remission was achieved without serious infection and hemorrhage. The total cost for induction chemotherapy was less expensive than is the case for elderly AML patients. This case indicates that the administration of cytokines might reduce the incidence of infection and the necessity for blood products, which would result in favorable cost effectiveness for the treatment of elderly patients with AML.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1998

Co-integration, co-expression and co-segregation of an unlinked selectable marker gene and NtFAD3 gene in transgenic rice plants produced by particle bombardment.

Using the scutellar tissue of rice mature embryos as a target tissue, a selectable marker gene, bar, and an unselectable gene, fatty acid desaturase gene from tobacco (NtFAD3), on separate plasmids, were introduced by particle bombardment. Co-integration, co-expression and inheritance of these genes were analyzed as well as seed fertility of the transgenic plants. Twenty-three out of 32 bialaphos-resistant plants integrated the NtFAD3 gene, which was confirmed by Southern-blot analysis of R0 plants, and showed one to more than 20 hybridizing bands of exogenous DNA, indicating a 72% (23/32) co-integration frequency. However, the frequency of the transgenic plants containing the 1.4-kb fragment of NtFAD3 gene was 34% (11/32). Northern-blot analysis revealed that seven out of ten fertile transgenic rice plants which had a 1.4-kb fragment of NtFAD3 cDNA expressed NtFAD3 mRNA. The NtFAD3 gene under the control of CaMV35S promoter stably expressed in the transgenic rice plants and modified the proportions of linoleic acid (18:2) and linolenic acid (18:3) in fatty acids; the content of 18:2 decreased and that of 18:3 increased. Fourteen out of 32 (44%) transgenic plants set seeds and 18 (56%) showed low fertility or sterility. Molecular analysis of the selfed progeny indicated that all copies in almost all R0 plants were inherited as a single dominant hemizygous locus.

Fatty Acid Desaturases↗
PubMed1998

Genetic mapping of RAPD markers linked to the densonucleosis refractoriness gene, nsd-1, in the silkworm, Bombyx mori.

In the silkworm, Bombyx mori, nonsusceptibility to B. mori densonucleosis virus type-1 (BmDNV-1) is controlled by a recessive gene, nsd-1 (nonsusceptibility to DNV-1), located on the twenty-first chromosome. We investigated genetic linkage between five random amplified polymorphic DNA (RAPD) markers and the +nsd-1 gene. Initially, we constructed the CSD-1 strain (nsd-1/+) which is congenic to strain C137 (nsd-1/nsd-1) for the twenty-first chromosome, starting with a female of C137 and a male of strain J137 (+nsd-1/+nsd-1). For the crossing over experiment, a female of C137 was crossed with a male (nsd-1/+) of CSD-1. Segregation analysis showed that the most closely linked RAPD marker mapped 3.0 cM distant from +nsd-1. A more specific marker for +nsd-1 was made by converting this RAPD band into a sequence characterized amplified region (SCAR) using a series of newly designed primer pairs based on its DNA sequence.

Animals↗
PubMed1998

Identification of novel random amplified polymorphic DNAs (RAPDs) on the W chromosome of the domesticated silkworm, Bombyx mori, and the wild silkworm, B. mandarina, and their retrotransposable element-related nucleotide sequences.

Genomic DNAs were compared between males and females of the domesticated silkworm, Bombyx mori, strains C108, C137, J137, p50, and WILD-W (constructed by crossing a wild silkworm, B. mandarina, female with a male of strain C108) by polymerase chain reaction (PCR) with 700 arbitrary 10-mer primers. Four female-specific RAPDs (W-Kabuki, W-Samurai, W-Kamikaze, and W-Yamato) were found. The sex chromosome formulas of B. mori and B. mandarina are ZW (XY) for the female and ZZ (XX) for the male. The four female-specific RAPDs are assumed to be derived from the W chromosome because the other chromosomes are shared by both sexes. A computer search for deduced amino acid sequences of these four RAPDs revealed that all of them showed homology to previously reported amino acid sequences encoded in known retrotransposable elements from various organisms.

Amino Acid Sequence↗
PubMed1998

Molecular structure of the copia-like retrotransposable element Yokozuna on the W chromosome of the silkworm, Bombyx mori.

We discovered a novel retrotransposable element, designated Yokozuna, on the W chromosome of Bombyx mori. The size of this element is 4738 bp, including a 208-bp long terminal repeat (LTR) on one side and a 183-bp LTR on the other. This retrotransposable element is flanked by a 5-bp target site duplication, TAATT. Yokozuna contains a single long open reading frame (ORF) and the whole deduced amino acid sequence of ORF reveals strong homology with copia of Drosophila. Moreover, an alignment analysis of the reverse transcriptase (RT) sequences suggested that the Yokozuna element is the first Bombyx retrotransposable element belonging to the Ty1-copia group. The number of Yokozuna per haploid genome is approximately four copies. Although Yokozuna was discovered on the W chromosome, it is not specific for the W chromosome.

Amino Acid Sequence↗
PubMed1998

A complete full-length non-LTR retrotransposon, BMC1, on the W chromosome of the silkworm, Bombyx mori.

In the silkworm, Bombyx mori, a non-long terminal repeat (non-LTR) retrotransposon, BMC1, is considered to be a LINE (long interspersed nuclear element)-like element. So far, a BMC1 containing two intact open reading frames (ORFs) has not been found. However, we discovered a complete full-length BMC1 on the W chromosome. This BMC1 is 5091 bp and contains a 5' untranslated region (5'-UTR), two intact ORFs, and 3'-UTR which terminates in a poly(A) tail. ORF1 encodes a putative nucleic acid-binding protein, while ORF2 encodes a protein containing an endonuclease domain and a reverse transcriptase domain.

Amino Acid Sequence↗
PubMed1998

Frequent expression of the c-kit proto-oncogene in canine malignant mammary tumor.

The mammary tumor is one of the popular neoplastic diseases in female dogs. In the present study, the expression of canine c-kit proto-oncogene in mammary tumor specimens was investigated to evaluate its potential usefulness as a tumor marker. By comparing the homology among the nucleotide sequences reported for human mouse, rat and feline c-kit c-DNA, a pair of primers was synthesized for the reverse transcriptase-polymerase chain reaction (RT-PCR) method. The RT-PCR product of canine spleen total RNA was shown to have 756 bp in size and to be highly homologous to the corresponding sequences reported for the mammalian species. The expression of c-kit transcript was detected in 11 mammary tumors of different histopathology including adenocarcinomas, benign and malignant mixed tumors. The level of the transcription in adenocarcinomas was significantly higher than those in malignant mixed tumors. Fifteen canine tumor specimens originated from various tissues were also tested for their c-kit transcript. In all of the mastocytoma samples examined, high expression of the mRNA was detected. Of other 12 tumors, only low level of RT-PCR products were detected in 5 samples, whereas no apparent amplification was observed in 7 tumors. These results indicate that the high expression of c-kit transcript is helpful for the diagnosis of canine mammary tumors.

Animals↗
PubMed1998

Rapid and progressive CD4+ decline in a monkey infected with an SIV+HIV-1 chimeric virus.

We previously constructed a simian immunodeficiency virus+human immunodeficiency virus type 1 (HIV-1) chimeric virus, NM-3rN to generate a pathogenic HIV-1 in macaque monkeys. During the in vivo passage of this virus in several monkeys, a viral strain, R43-56 was obtained which acquired a better replication ability in vivo. MM121, one of the three monkeys inoculated with the R43-56, showed weight loss, diarrhea and a rapid and continuous decrease in CD4+ lymphocytes at the moribund stage. An autopsy revealed generalized lymphadenopathy, dehydration, and ileocecal intussusception. In situ hybridization showed that the virus infection was in systemic lymphoid organs. We are presently monitoring the survivors to obtain candidates for a more virulent virus. R43-56 may be a better challenge virus and useful tool for human acquired immunodeficiency syndrome research.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Cytotoxicity induced by recombinant human tumor necrosis factor-alpha dependent on the types of its receptors on canine cells.

Based on the recent findings that show how recombinant human tumor necrosis factor (rh-TNF)-alpha has potent antitumor activity on human cancer patients when it locally administrated, we have tested the cytotoxicity of rh-TNF-alpha on 3 canine cultured cells: (1) canine kidney carcinoma (CKCa-1), (2) mastocytoma and (3) Mardin Darby canine kidney cells (MDCK). The cell surface expression of TNF-alpha receptors on these canine cells was also determined with anti-human TNF RI and RII polyclonal antibodies. Our data shows that on CKCa-1 which has TNF RI receptors rh-TNF-alpha induced cytotoxicity. By contrast, it exhibited no toxicity on canine mastocytoma which has mainly RII receptors. The data also suggest actinomycin D (ACT-D), an anticancer antibiotic, enhanced the cytotoxicity of rh-TNF-alpha. Combined with ACT-D, rh-TNF-alpha showed the cytotoxicity on MDCK which possessed both TNF RI and RII receptors. The results indicate that the cytotoxicity of rh-TNF-alpha depends on the presence of TNF RI receptors on canine tumor cells.

Animals↗
PubMed1998

Expression of the mutant (1735T-DEL) tissue-nonspecific alkaline phosphatase gene from hypophosphatasia patients.

Hypophosphatasia (HOPS) is an inherited disorder characterized by defects in skeletal mineralization due to the deficiency of tissue-nonspecific alkaline phosphatase (TNSALP). To date, various mutations in the TNSALP gene have been identified. Especially, a deletion of T at position 1735 (1735T-del) located in exon 12 has been detected in three genetically unrelated Japanese patients, which seems to be one of the hot spots among the causative mutations in Japanese HOPS patients. 1735T-del causes a frame shift downstream from codon 503 (Leu), and consequently the normal termination codon at 508 is eliminated. Since a new inframe termination codon appears at codon 588 in the mutant DNA, the resultant protein is expected to have 80 additional amino acids. Expression of the mutant TNSALP gene using COS-1 cells demonstrated that the protein translated from the mutant 1735T-del had undetectable ALP activity, and its molecule size was larger than normal, as expected. Interestingly, an immunoprecipitation study of patients' sera using antibody against TNSALP revealed an abnormal protein which corresponded in size to the mutated TNSALP expressed by COS-1 cells, suggesting that the abnormal TNSALP is made by HOPS patients. The detection of TNSALP in cells transfected with 1735T-del using an immunofluorescent method exhibited only a faint signal on the cell surface, but an intense intracellular fluorescence after permeabilization.

Alkaline Phosphatase↗