Biomedical subjects

K Mise

Publications and source records attributed to K Mise.

At least 19 recordsLinked to original sources

PubMed2005

Comparison of NSP4 protein between group A and B human rotaviruses: detection of novel diarrhea-causing sequences in group B NSP4.

The human group B rotavirus is a causative agent of severe adult diarrhea. In this study, we analyzed the NSP4 structure of a group B rotavirus strain, CAL-1, and determined whether enterotoxin activity was present in CAL-1 NSP4. CAL-1 NSP4 was comprised of 219 amino acids which was longer than group A and C rotavirus NSP4, and the primary structures of their sequences differed considerably. However, CAL-1 NSP4 had an enterotoxin-like sequence (residues 106-127) that was only 27% identical to the enterotoxin region of NSP4 of KUN (a group A rotavirus strain) at residues 114-135. Interestingly, both of the synthetic peptides, one (residues 99-128) containing the enterotoxin-like sequence and the other (residues 191-219) containing 29 C-terminal amino acids of CAL-1 NSP4, induced diarrhea in 5.5-day-old mice, but not in 17.5-day-old mice, when administered parenterally. Thus, rotavirus "enterotoxin" sequences could be considerably divergent.

Age Factors↗

PubMed2005

Synthesis of infectious in vitro transcripts from Cassia yellow blotch bromovirus cDNA clones and a reassortment analysis with other bromoviruses in protoplasts.

Cassia yellow blotch virus (CYBV), genus Bromovirus, was isolated from the Australian native legume, Cassia pleurocarpa, in western Queensland, and its host range was found to be distinct from other bromoviruses. In this study, CYBV was shown to infect systemically and efficiently a model plant species, Arabidopsis thaliana, as we recently reported for another bromovirus, Spring beauty latent virus (SBLV). We constructed full-length cDNA clones of CYBV genomic RNAs from which infectious in vitro transcripts can be transcribed, and determined their complete nucleotide sequences. CYBV RNA3 contains the box B motif in the intercistronic region, but lacks the subgenomic promoter-like sequence in the 5' noncoding region, as does Brome mosaic virus (BMV). To understand relationships among bromoviruses, we generated reassortants between CYBV and three other bromoviruses, BMV, SBLV and Cowpea chlorotic mottle virus. We found that all reassortants between BMV and CYBV accumulated viral RNAs to detectable levels in protoplasts of Nicotiana benthamiana, even when RNAs 1 and 2, which encode the replication proteins 1a and 2a, respectively, were heterologous. Sequence comparison and reassortment experiments of CYBV and other bromoviruses demonstrated that CYBV is closely related to BMV.

Bromovirus↗

PubMed2005

Coat protein-independent cell-to-cell movement of bromoviruses expressing brome mosaic virus movement protein with an adaptation-related amino acid change in the central region.

The movement protein (MP) of Brome mosaic virus (BMV) depends on the coat protein (CP) to mediate the cell-to-cell movement of BMV and CCMV(B3a), a recombinant Cowpea chlorotic mottle virus (CCMV) expressing BMV MP. Previous studies identified gain-of-function mutations in the central region of BMV MP that enable CCMV(B3a) to adapt to a resistant host. This study demonstrates that all adaptation-related MPs can partially or almost fully mediate the cell-to-cell movement of CCMV(B3a) and BMV without CP. Based on these results, we discuss adaptation mechanisms of CCMV(B3a) and the role of the central region of MP in the determination of virus movement mode.

Adaptation, Physiological↗

PubMed2003

Complete nucleotide sequence of spring beauty latent virus, a bromovirus infectious to Arabidopsis thaliana.

Spring beauty latent virus (SBLV), a bromovirus, systemically and efficiently infected Arabidopsis thaliana, whereas the well-studied bromoviruses brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) did not infect and poorly infected A. thaliana, respectively. We constructed biologically active cDNA clones of SBLV genomic RNAs and determined their complete nucleotide sequences. Interestingly, SBLV RNA3 contains both the box B motif in the intercistronic region, as does BMV, and the subgenomic promoter-like sequence in the 5' noncoding region, as does CCMV. Sequence comparisons of SBLV, BMV, CCMV, and broad bean mottle virus demonstrated that SBLV is closely related to BMV and CCMV.

Arabidopsis↗

PubMed2003

The movement protein gene is involved in the virus-specific requirement of the coat protein in cell-to-cell movement of bromoviruses.

Brome mosaic virus (BMV) requires the coat protein (CP) for cell-to-cell movement whereas Cowpea chlorotic mottle virus (CCMV), from the same genus, does not. Chimeric viruses created by exchanging the movement protein (MP) gene between the viruses can move from cell to cell. We show that interference in CP expression impaired the movement of the chimeric CCMV with the BMV MP gene but not of the chimeric BMV with the CCMV MP gene. We thus conclude that the MP gene plays a crucial role in determination of the virus-specific CP requirement in bromovirus cell-to-cell movement.

Amino Acid Sequence↗

PubMed2001

Site-specific single amino acid changes to Lys or Arg in the central region of the movement protein of a hybrid bromovirus are required for adaptation to a nonhost.

A hybrid Cowpea chlorotic mottle virus (CCMV) [CCMV(B3a)] in which the CCMV 3a movement protein gene is replaced by the 3a (B3a) gene of Brome mosaic virus cannot infect cowpea systemically. Previously, analysis of RNA3 cDNA clones constructed from cowpea-adapted mutants derived from CCMV(B3a) revealed that a single codon change in the B3a gene allowed CCMV(B3a) to infect cowpea systemically. In this study, to extend the analysis of the CCMV(B3a) adaptation mechanism, we directly sequenced B3a gene RT-PCR products prepared from 28 cowpea plants in which cowpea-adapted mutants appeared, and found seven patterns of a codon change localized at five specific positions in the central region (Ser(118), Glu(132), Glu(138), Gln(178), and Ser(180)). All of the patterns involved an amino acid change to Lys or Arg. Mutational analysis of the B3a gene demonstrated that a single codon change resulting in either Lys or Arg at any of the five positions was sufficient for the adaptation of CCMV(B3a) to cowpea. In contrast, CCMV(B3a) variants with a codon change resulting in Lys or Arg at three other positions (137, 155, and 161) in the B3a gene not only showed lack of systemic infection of cowpea but also showed weakened initial cell-to-cell movement in the inoculated leaves and diminished B3a accumulation in protoplasts. These results suggest that adaptive changes in the B3a gene are site-specifically selected in cowpea plants.

Adaptation, Physiological↗

PubMed2001

Results of breast-conserving therapy for ductal carcinoma in situ: the Kyoto University experiences.

BACKGROUND: The purpose of this study was to evaluate the results of breast-conserving therapy (BCT), defined as the combination of breast-conserving surgery with axillary dissection and definitive radiation therapy for ductal carcinoma in situ (DCIS). METHODS: Between November 1987 and March 1998, 33 patients with DCIS undergoing BCT at our hospital were examined. The mean age was 48. All patients underwent quadrantectomy or wide excision as well as axillary dissection. Radiation therapy consisted of 50 Gy to the ipsilateral whole breast. Boost irradiation of 10 Gy was given to 15 patients with close or positive margins. Nearly all patients received adjuvant chemotherapy with 5-fluorouracil or its derivatives and adjuvant endocrine therapy with tamoxifen for 2 years. RESULTS: The minimum and median follow-up periods were 32 and 80 months, respectively. All patients but one were followed. Only one patient had a non-invasive local recurrence, 23 months after her operation. This patient was salvaged with simple mastectomy. Her prognostic index score was 8. The five-year local control rate was 97%. No serious acute or late complications were noted. CONCLUSION: The results of this retrospective study substantiate favorable data and appear to confirm the efficacy and reasonable local recurrence rate of BCT for the treatment of DCIS.

Adult↗

PubMed2001

Impact of boost irradiation with surgically placed radiopaque clips on local control in breast-conserving therapy.

BACKGROUND: The purpose of this study was to determine whether boost irradiation relying on radiopaque clips placed surgically around the resected margin of breast cancer contributes to increasing the local control rate in patients with close or positive margins in breast-conserving therapy (BCT). METHODS: Among 837 patients with breast cancer who underwent BCT between November 1987 and December 1998, 181 patients with close or positive surgical margins received boost irradiation following conventional tangential whole breast irradiation. Since 1994, four radiopaque clips were surgically placed around the resected margin of the breast cancer in 155 patients treated with wide excision. The four clips were clearly and accurately identified with a CT-simulator (CT-S). The boost irradiation field was automatically determined with a safety margin of 3 cm according to one-to-one correspondence of radiopaque clips to pathologically close or positive surgical margins. In the remaining 26 patients treated before 1994, the boost irradiation field was determined according to the skin tattoo of the primary tumor. RESULTS: The median follow-up period of the 155 patients receiving the radiopaque clips was 42 months (range: 19 to 78), and that of the 26 patients without the clips was 87 months. Local recurrence was observed in two of the 155 patients who underwent boost irradiation using the radiopaque clips 39 and 54 months after the surgery, while 4 of the 26 patients developed local recurrence 14, 23, 51, and 76 months after BCT. In three of the four patients without the clips developing local recurrences, local recurrences were observed at the margin of the boost irradiation field. The 5-year local recurrence-free survival rate of patients who received boost irradiation with the radiopaque clips was 97%, and that of patients without the clips was 88%. The difference of local recurrence-free survival rates between the patients with and without the clips was significant (p<0.05). CONCLUSION: Surgically placed radiopaque clips appear to be useful for determining adequate boost field in the BCT using the CT-S and help increase the local control rate.

Adult↗

PubMed2001

Brome mosaic virus replicase proteins localize with the movement protein at infection-specific cytoplasmic inclusions in infected barley leaf cells.

Subcellular localization of the Brome mosaic virus replicase-related 1a and 2a proteins, and the 3a movement protein in infected barley leaves was examined by immunogold electron microscopy. The 1a and 2a proteins colocalized at infection-specific electron-dense cytoplasmic inclusions. The 3a protein was also detected in these inclusions. The inclusions were oval or amorphous, and contained electron-lucent regions. Electron microscopic autoradiography showed that the inclusions were sites of [3H]uridine incorporation, suggesting that they are sites of viral RNA synthesis.

Bromovirus↗

PubMed2001

Inducible high-level mRNA amplification system by viral replicase in transgenic plants.

We have constructed a new system for inducible high-level expression of mRNA for foreign genes in transgenic plants by introducing a glucocorticoid-inducible transcription system into the previously developed "mRNA amplification system" where target mRNA can be amplified as a subgenomic RNA by the replicase of a plant tripartite RNA virus, Brome mosaic virus (BMV). In the new amplification system, the amplification of mRNA is tightly regulated by the expression of a subunit of the BMV replicase. Transgenic Nicotiana benthamiana plants (designated GVG1 x 2FR) were produced that contained cDNA of BMV RNA1 coding a subunit of replicase under the control of a tightly regulated, glucocorticoid-inducible promoter. In addition GVG1 x 2FR plants contain cDNAs of BMV RNA2 coding another subunit of the replicase, and a replicable engineered BMV RNA3 derivative (FCP2IFN) carrying the human gamma interferon (IFN) gene under the control of the Cauliflower mosaic virus 35S promoter. When transgenic plants were treated with dexamethasone (DEX), a strong synthetic glucocorticoid, induction of replication and amplification of the 35S-driven FCP2IFN and synthesis of subgenomic mRNA for IFN were observed. Accumulation levels of amplified FCP2IFN were over 300 times higher than those of the 35S-driven FCP2IFN in the GVG1 x 2FR plant without the treatment and those of the mRNA for IFN were 30-230 times higher than in the previous, non-inducible mRNA amplification system. Without DEX treatment, no subgenomic mRNA for IFN was detected in the GVG1 x 2FR plant. The advantages and potential uses of this system are also discussed.

Base Sequence↗

PubMed2001

The C terminus of brome mosaic virus coat protein controls viral cell-to-cell and long-distance movement.

To investigate the functional domains of the coat protein (CP; 189 amino acids) of Brome mosaic virus, a plant RNA virus, 19 alanine-scanning mutants were constructed and tested for their infectivity in barley and Nicotiana benthamiana. Despite its apparent normal replicative competence and CP production, the C-terminal mutant F184A produced no virions. Furthermore, virion-forming C-terminal mutants P178A and D182A failed to move from cell to cell in both plant species, and mutants D181A and V187A showed host-specific movement. These results indicate that the C-terminal region of CP plays some important roles in virus movement and encapsidation. The specificity of certain mutations for viral movement in two different plant species is evidence for the involvement of host-specific factors.

Alanine↗

PubMed2001

Conversion in the requirement of coat protein in cell-to-cell movement mediated by the cucumber mosaic virus movement protein.

Plant viruses have movement protein (MP) gene(s) essential for cell-to-cell movement in hosts. Cucumber mosaic virus (CMV) requires its own coat protein (CP) in addition to the MP for intercellular movement. Our present results using variants of both CMV and a chimeric Brome mosaic virus with the CMV MP gene revealed that CMV MP truncated in its C-terminal 33 amino acids has the ability to mediate viral movement independently of CP. Coexpression of the intact and truncated CMV MPs extremely reduced movement of the chimeric viruses, suggesting that these heterogeneous CMV MPs function antagonistically. Sequential deletion analyses of the CMV MP revealed that the dispensability of CP occurred when the C-terminal deletion ranged between 31 and 36 amino acids and that shorter deletion impaired the ability of the MP to promote viral movement. This is the first report that a region of MP determines the requirement of CP in cell-to-cell movement of a plant virus.

Bromovirus↗

PubMed2001

Staging in untreated patients with small cell lung cancer.

In order to describe the real biological behavior of the small-cell lung cancer we have analyzed survival rates of 66 patients with small-cell lung cancer who did not receive any specific anti cancer therapy. Also, objective of this study was to evaluate the staging system of the small-cell lung cancer. Untreated small-cell lung cancer patients with limited stage disease had statistically significant (p < 0.05) better survival rates in comparison to patients with extensive stage disease. T and N factor of the TNM classification did not influence the survival in untreated small-cell lung cancer patients. It appears that the TNM staging system is not predicting survival probabilities of untreated patients with small-cell lung cancer, while the two-stage system appeared very well based on survival probabilities of these patients.

Adult↗

PubMed2000

Double-labeled donor probe can enhance the signal of fluorescence resonance energy transfer (FRET) in detection of nucleic acid hybridization.

A set of fluorescently-labeled DNA probes that hybridize with the target RNA and produce fluorescence resonance energy transfer (FRET) signals can be utilized for the detection of specific RNA. We have developed probe sets to detect and discriminate single-strand RNA molecules of plant viral genome, and sought a method to improve the FRET signals to handle in vivo applications. Consequently, we found that a double-labeled donor probe labeled with Bodipy dye yielded a remarkable increase in fluorescence intensity compared to a single-labeled donor probe used in an ordinary FRET. This double-labeled donor system can be easily applied to improve various FRET probes since the dependence upon sequence and label position in enhancement is not as strict. Furthermore this method could be applied to other nucleic acid substances, such as oligo RNA and phosphorothioate oligonucleotides (S-oligos) to enhance FRET signal. Although the double-labeled donor probes labeled with a variety of fluorophores had unexpected properties (strange UV-visible absorption spectra, decrease of intensity and decay of donor fluorescence) compared with single-labeled ones, they had no relation to FRET enhancement. This signal amplification mechanism cannot be explained simply based on our current results and knowledge of FRET. Yet it is possible to utilize this double-labeled donor system in various applications of FRET as a simple signal-enhancement method.

DNA Probes↗

PubMed2000

Cosmetic results and complications after breast conserving therapy for early breast cancer.

BACKGROUND: The cosmetic and functional results of breast conserving therapy for early breast cancer were evaluated. These are important endpoints in the assessment of breast conserving therapy in addition to tumor control and survival. The factors suspected to influence cosmesis were also analyzed. METHODS: In 206 patients with stage I and II breast cancer treated by wide excision and axillary dissection followed by radiation therapy, the cosmetic results and complications were analyzed. The cosmetic outcome was assessed by a scoring method and breast retraction assessment (BRA). As complications, arm edema and restriction of shoulder movement and late skin reactions were analyzed. RESULTS: Of the 206 patients 92% showed an excellent to good cosmetic score before radiation therapy. The score deteriorated, but gradually improved and stabilized after 1 year. Eighty-one percent of the patients had an excellent to good cosmetic score at 3 years. The BRA of the 206 patients was 1.8 cm on average before radiation therapy. It increased to 2.3 cm after termination of radiation therapy, and did not change thereafter. Tumor size over 2 cm (p = 0.005) and tumors in the inner quadrant (p = 0.003) were factors which negatively affected the cosmetic score at 3 years. Tumor size over 2 cm (p = 0.003), tumors in the upper quadrant (p = 0.005), or a nipple-tumor distance of more than 2 cm (p = 0.01) were also negative factors for the BRA at 3 years. Arm edema, restriction of shoulder movement, and late skin reaction were generally mild, and were observed in 12%, 0% and 34% of patients at 3 years, respectively. CONCLUSIONS: The overall cosmetic results of breast conserving therapy are acceptable and the complication rate is low. Tumor characteristics, tumor size, location and nipple-tumor distance are factors that affect cosmesis.

Adult↗

PubMed2000

Bromovirus movement protein conditions for the host specificity of virus movement through the vascular system and affects pathogenicity in cowpea.

Previously, we reported that CCMV(B3a), a hybrid of bromovirus Cowpea chlorotic mottle virus (CCMV) with the 3a cell-to-cell movement protein (MP) gene replaced by that of cowpea-nonadapted bromovirus Brome mosaic virus (BMV), can form small infection foci in inoculated cowpea leaves, but that expansion of the foci stops between 1 and 2 days postinoculation. To determine whether the lack of systemic movement of CCMV(B3a) is due to restriction of local spread at specific leaf tissue interfaces, we conducted more detailed analyses of infection in inoculated leaves. Tissue-printing and leaf press-blotting analyses revealed that CCMV(B3a) was confined to the inoculated cowpea leaves and exhibited constrained movement into leaf veins. Immunocytochemical analyses to examine the infected cell types in inoculated leaves indicated that CCMV(B3a) was able to reach the bundle sheath cells through the mesophyll cells and successfully infected the phloem cells of 50% of the examined veins. Thus, these data demonstrate that the lack of long-distance movement of CCMV(B3a) is not due to an inability to reach the vasculature, but results from failure of the virus to move through the vascular system of cowpea plants. Further, a previously identified 3a coding change (A776C), which is required for CCMV(B3a) systemic infection of cowpea plants, suppressed formation of reddish spots, mediated faster spread of infection, and enabled the virus to move into the veins of inoculated cowpea leaves. From these data, and the fact that CCMV(B3a) directs systemic infection in Nicotiana benthamiana, a permissive systemic host for both BMV and CCMV, we conclude that the bromovirus 3a MP engages in multiple activities that contribute substantially to host-specific long-distance movement through the phloem.

Biological Transport↗

PubMed2000

The Colletotrichum lagenarium MAP kinase gene CMK1 regulates diverse aspects of fungal pathogenesis.

The infection process of Colletotrichum lagenarium, the causal agent of cucumber anthracnose disease, involves several key steps: germination; formation of melanized appressoria; appressorial penetration; and subsequent invasive growth in host plants. Here we report that the C. lagenarium CMK1 gene encoding a mitogen-activated protein (MAP) kinase plays a central role in these infection steps. CMK1 can complement appressorium formation of the Pmk1 MAP kinase mutant of Magnaporthe grisea. Deletion of CMK1 causes reduction of conidiation and complete lack of pathogenicity to the host plant. Surprisingly, in contrast to M. grisea pmk1 mutants, conidia of cmk1 mutants fail to germinate on both host plant and glass surfaces, demonstrating that the CMK1 MAP kinase regulates conidial germination. However, addition of yeast extract rescues germination, indicating the presence of a CMK1-independent pathway for regulation of conidial germination. Germinating conidia of cmk1 mutants fail to form appressoria and the mutants are unable to grow invasively in the host plant. This strongly suggests that MAP kinase signaling pathways have general significance for infection structure formation and pathogenic growth in phytopathogenic fungi. Furthermore, three melanin genes show no or slight expression in the cmk1 mutant when conidia fail to germinate, suggesting that CMK1 plays a role in gene expression required for appressorial melanization.

Amino Acid Sequence↗

PubMed2000

Results of breast-conserving therapy for early stage breast cancer: Kyoto University experiences.

This study evaluated the results of breast-conserving therapy (BCT). Nine hundred six patients who underwent BCT at our hospital between November 1987 and February 1998 were analyzed. The mean age was 48 years. According to the Union Internationale Contre le Cancer 1997 classification system, stages 0, I, IIA, IIB, IIIA, and IIIB were 37, 400, 344, 117, 7, and 1, respectively. Radiation therapy consisted of 50 Gy to the ipsilateral whole breast. Boost irradiation of 10 Gy was administered to 186 of 231 patients with close or positive margins. Nearly all patients received adjuvant chemohormonal therapy with tamoxifen and 5-fluorouracil or its derivatives for 2 years. The minimum and median follow-up periods were 18 and 52 months, respectively. The 5-year overall survival, cause-specific survival, local recurrence-free survival, and disease-free survival rates were 97.3%, 98.4%, 98.1%, and 91.5%, respectively. Local recurrence in preserved breast occurred in 20 patients 7 to 86 months after surgery. Multivariate analysis revealed that the most predictive factor for disease-free survival rates and distant failures was the number of pathologically positive lymph nodes (p < 0.0001), and that the factor for local failure was marginal status (p = 0.005). This study demonstrated that BCT was suitable for the treatment of early-stage breast cancer with its reasonable survival rates and acceptable toxicity.

Breast Neoplasms↗