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Biomedical subjects

M Sato

Publications and source records attributed to M Sato.

At least 163 records · Page 9Linked to original sources
PubMed2001

Imaging of conformational changes of proteins with a new environment-sensitive fluorescent probe designed for site-specific labeling of recombinant proteins in live cells.

We demonstrate herein a new method for imaging conformational changes of proteins in live cells using a new synthetic environment-sensitive fluorescent probe, 9-amino-6,8-bis(1,3,2-dithioarsolan-2-yl)-5H-benzo[a]phenoxazin-5-one. This fluorescent probe can be attached to recombinant proteins containing four cysteine residues at the i, i + 1, i + 4, and i + 5 positions of an alpha-helix. The specific binding of the fluorescent probe to this 4Cys motif enables fluorescent labeling inside cells by its extracellular administration. The high sensitivity of the fluorophore to its environment enables monitoring of the conformational changes of the proteins in live cells as changes in its fluorescence intensity. The present method was applied to calmodulin (CaM), a Ca2+-binding protein that was well-known to expose hydrophobic domains, depending on the Ca2+ concentration. A recombinant CaM fused at its C-terminal with a helical peptide containing a 4Cys motif was labeled with the fluorescent probe inside live cells. The fluorescence intensity changed reversibly depending on the intracellular Ca2+ concentration, which reflected the conformational change of the recombinant CaM in the live cells.

Amino Acid Sequence↗
PubMed2001

Proliferative activity of intratumoral CD8(+) T-lymphocytes as a prognostic factor in human renal cell carcinoma: clinicopathologic demonstration of antitumor immunity.

Tumor-infiltrating lymphocytes, particularly CD8(+) T cells, could be a manifestation of antitumor immunity. We clinicopathologically analyzed the biological significance of tumor-infiltrating lymphocytes in 221 patients with renal cell carcinoma without preoperative treatments. More abundant infiltration of tumor tissue not only by CD8(+) but also CD4(+) T cells was associated with shorter survival of the patients, because of the positive correlation between the number of lymphocytes and representative tumor grade factors. This suggests that immune cell reactions are more pronounced as the tumor grade/biological malignancy progresses, probably because of increased antigenicity of tumor cells. We next analyzed the proliferative activity of CD8(+) T cells that infiltrated in tumor cell nests, which could also reflect antitumor immunity. Higher labeling index of Ki-67, a proliferation-associated antigen, among CD8(+) T cells in contact to tumor cells was associated with a longer survival by both uni- and multivariate analyses. Our data in human renal cell carcinoma suggest that infiltration of tumor tissue by T cells itself does not denote the efficacy of antitumor immunity because of its dependence on the biological malignancy of tumor cells, but infiltration of tumor tissue by CD8(+) T cells bearing more pronounced proliferative activity could reflect effective antitumor immunity. This concept would be important for future immunotherapy of human cancer.

Adult↗
PubMed2001

Genomic organization of the Shc-related phosphotyrosine adapters and characterization of the full-length Sck/ShcB: specific association of p68-Sck/ShcB with pp135.

The Shc gene family is an emerging family, containing at least three members designated Shc/ShcA, Sck/Sli/ShcB, N-Shc/Rai/ShcC in mammals. In this study, we determined the genomic organization of the mouse Shc family. Coding regions of ShcA, B, and C each comprised 12 exons, spanned approximately 6, 20, and 65 kb, and located on chromosome 3, 10, and 13, respectively. Based on this genome analysis, we determined the full-length structure of mouse Sck/ShcB as a 68-kD protein. We found that the 68-kD full-length Sck/ShcB was more efficiently phosphorylated upon EGF treatment than the previously-analyzed CH2-deleted form. We also found that Sck specifically interacted with a 135-kD phosphoprotein (pp135) through its SH2 domain following membrane depolarization. The Sck-pp135 interaction was reduced by Src kinase inhibitors. These results suggest that Sck, but not N-Shc nor Shc, transmit signals in conjunction with pp135 following Src activation and/or calcium entry in the cell.

Adaptor Proteins, Signal Transducing↗
PubMed2001

The fission yeast meiotic regulator Mei2p undergoes nucleocytoplasmic shuttling.

Schizosaccharomyces pombe Mei2p is an RNA-binding protein that switches the cell cycle from mitotic to meiotic. Mei2p forms a unique dot in the nucleus prior to meiosis I, aided by a non-coding RNA molecule termed meiRNA. Here we show that Mei2p intrinsically undergoes nucleocytoplasmic shuttling. Artificial acceleration of nuclear migration of Mei2p advances nuclear dot formation, but meiRNA does not appear to promote the dot formation by modulating the migration rate of Mei2p into the nucleus. Rather, this RNA is likely to facilitate the assembly of Mei2p into a dot structure and trap the protein as such in the nucleus.

Active Transport, Cell Nucleus↗
PubMed2001

Reduction of ion thermal diffusivity associated with the transition of the radial electric field in neutral-beam-heated plasmas in the large helical device.

Recent large helical device experiments revealed that the transition from ion root to electron root occurred for the first time in neutral-beam-heated discharges, where no nonthermal electrons exist. The measured values of the radial electric field were found to be in qualitative agreement with those estimated by neoclassical theory. A clear reduction of ion thermal diffusivity was observed after the mode transition from ion root to electron root as predicted by neoclassical theory when the neoclassical ion loss is more dominant than the anomalous ion loss.

Journal Article↗
PubMed2001

Intrabursal transfer of spermatozoa (ITS): a new route for artificial insemination of mice.

Artificial insemination (AI) by direct injection of epididymal spermatozoa into the reproductive tract of females is simpler and more convenient than in vitro fertilization (IVF) and subsequent transfer of fertilized eggs to recipient oviducts for simultaneous acquisition of a large number of pups. Introduction of epididymal spermatozoa into oviducts via the oviductal wall or via vaginal and intrauterine routes is currently the most commonly used method for AI in mice. In this study, we explored another route for AI of the mouse and found that transfer of spermatozoa into a space near the infundibulum between the ovary and ovarian bursa enables in vivo fertilization of ovulated oocytes at the ampulla. When 1 microL of a sperm suspension containing 1 x 10(4) spermatozoa freshly isolated from B6C3F1 males was intrabursally injected into superovulated B6C3F1 females on E (embryonic day) 0.4 (10:00 AM), 5 of 7 females yielded 2-cell embryos with rates of efficiency ranging from 4 to 21% (11% on average), which were much lower than those (91% on average) for embryos obtained by natural mating. All the 2-cell embryos derived from injection of sperm developed in vitro to hatched blastocysts. Similar results were obtained from injection of 1 microL of sperm suspension containing 1 x 10(3) spermatozoa, although in vivo fertilizing ability was slightly improved (28% on average). When 1 microL of sperm suspension containing 1 x 10(4) spermatozoa was injected intrabursally into superovulated females that had been mated with vasectomized males, 6 of 10 mice (60%) yielded 19 normal mid-gestational fetuses with an average litter size of 3.2, which was much lower than that (14.5) for embryos obtained by natural mating. Although the present findings appear to be preliminary, this technique, based on the intrabursal transfer of spermatozoa, will be of practical use for AI in mice, particularly for transgenic and mutant mice that are often difficult to breed.

Animals↗
PubMed2001

Split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing.

We describe a new method for detecting protein-protein interactions in intact mammalian cells; the approach is based on protein splicing-induced complementation of rationally designed fragments of firefly luciferase. The protein splicing is a posttranslational protein modification through which inteins (internal proteins) are excised out from a precursor fusion protein, ligating the flanking exteins (external proteins) into a contiguous polypeptide. As the intein, naturally split DnaE from Synechocystis sp. PCC6803 was used: The N- and C-terminal DnaE, each fused respectively to N- and C-terminal fragments of split luciferase, were connected to proteins of interest. In this approach, protein-protein interactions trigger the folding of DnaE intein, wherein the protein splicing occurs and thereby the extein of ligated luciferase recovers its enzymatic activity. To test the applicability of this split luciferase complementation, we used insulin-induced interaction between known binding partners, phosphorylated insulin receptor substrate 1 (IRS-1) and its target N-terminal SH2 domain of PI 3-kinase. Enzymatic luciferase activity triggered by insulin served to monitor the interaction between IRS-1 and the SH2 domain in an insulin dose-dependent manner, of which amount was assessed by the luminescent intensity. This provides a convenient method to study phosphorylation of any protein or interactions of integral membrane proteins, a class of molecules that has been difficult to study by existing biochemical or genetic methods. High-throughput drug screening and quantitative analysis for a specific pathway in tyrosine phosphorylation of IRS-1 in insulin signaling are also made possible in this system.

Animals↗
PubMed2001

NMDA receptor stimulation and brain-derived neurotrophic factor upregulate homer 1a mRNA via the mitogen-activated protein kinase cascade in cultured cerebellar granule cells.

In three alternative splice variants of Homer 1 transcripts, Homer 1a mRNA has been shown to be upregulated selectively and rapidly by neural stimulation and represents a member of the immediate early gene (IEG) family. We investigated the mechanism underlying Homer 1a mRNA induction in cerebellar granule cell culture. All Homer 1 variants were expressed in cultured granule cells as analyzed by RNA blotting and immunochemical characterization. Glutamate stimulation of granule cells selectively upregulated Homer 1a mRNA via NMDA receptor-mediated influx of extracellular calcium. The induction of Homer 1a mRNA was much slower (peaked at 4 hr) and sustained longer than that of the typical IEG c-fos mRNA. Actinomycin D and cycloheximide experiments have revealed that, despite the presence of the mRNA-destabilizing AU-rich motif, transcriptional activation is a main determinant for selective Homer 1a mRNA induction. Inhibitor analysis as well as immunochemical characterization has indicated that the MEK (MAPK/ERK kinase)-ERK (extracellular signal-regulated kinase) cascade plays an indispensable role in glutamate-stimulated induction of Homer 1a mRNA. Consistent with this observation, brain-derived neurotrophic factor, which is known to activate the ERK cascade, similarly upregulated Homer 1a mRNA. These results demonstrate that MAPK (mitogen-activated protein kinase) is a key mediator that links distinct extracellular stimuli to the transcriptional activation of Homer 1a mRNA.

Animals↗
PubMed2001

Gene induction pathways mediated by distinct IRFs during viral infection.

During viral infection, interferon-alpha/beta (IFN-alpha/beta) and many IFN-inducible genes are induced to elicit antiviral responses of the host. Using cells with a gene disruption(s) for the IRF family of transcription factors, we provide evidence that these genes, containing similar IRF-binding cis-elements, are classified into distinct groups, based on the gene induction pathway(s). The IFN-beta gene induction is dependent on either IRF-3 or IRF-7, whereas induction of the IFN-alpha gene family is IRF-7-dependent. On the other hand, ISG15, ISG54 and IP-10 are induced by either IRF-3 or IFN stimulated gene factor 3 (ISGF3). We also show that another group of genes is totally dependent on ISGF3. Thus, during viral infection, a given gene responds either directly to a virus or virus-induced IFN-alpha/beta or both through distinct pathways. The differential utilization of these induction pathways for these genes during viral infection may reflect their distinct functional roles in the efficient antiviral response.

2',5'-Oligoadenylate Synthetase↗
PubMed2001

Purification, crystallization and preliminary X-ray diffraction analysis of the yeast Sec12Deltap protein, a guanine nucleotide-exchange factor involved in vesicle transport.

Sec12 is a guanine nucleotide-exchange factor (GEF) of the GTP-binding protein Sar1. Its GEF activity on Sar1 makes it a key element in vesicle budding from the endoplasmic reticulum to the Golgi apparatus in yeast. Sec12 is an integral membrane glycoprotein of 70 kDa. A 38.5 kDa N-cytoplasmic domain (Sec12Deltap) has been expressed in Saccharomyces cerevisiae and in Escherichia coli, purified to homogeneity and crystallized. Two crystal forms were obtained. Crystal form I belongs to space group P6(2)/P6(4), with unit-cell parameters a = b = 191.7, c = 53.3 A, gamma = 120 degrees, and diffracts to 2.6 A resolution. Crystal form II belongs to space group P1, with unit-cell parameters a = 52.6, b = 53.0, c = 116.8 A, alpha = 98.0, beta = 97.4, gamma = 93.4 degrees, and diffracts to 2.0 A resolution.

Crystallization↗
PubMed2001

Prognostic significance of frequent acidophilic nuclear inclusions in adenocarcinoma of the lung with immunohistochemical and ultrastructural studies.

BACKGROUND: Adenocarcinoma of the lung occasionally has acidophilic nuclear inclusions (ANIs). Some studies have reported that the incidence of ANIs was higher in well differentiated tumor types and have suggested that adenocarcinoma patients with ANIs might have a more favorable prognosis; however, to the authors' knowledge, statistically significant prognostic findings were not reported. The objective of the current study was to assess the prognostic significance of ANI in patients with pulmonary adenocarcinoma and, moreover, to characterize ANI immunohistochemically and ultrastructurally. METHODS: Surgically resected tumor specimens from 147 patients with primary pure adenocarcinoma of the lung were examined. Only obvious ANIs surrounded by a clear halo on hematoxylin and eosin-stained slides were counted; the authors classified cases with > or = 10 ANIs per 10 high-power fields (/10 HPF) as frequent-ANI cases, cases with < 10 ANIs/10 HPF as infrequent-ANI cases, and cases without ANIs as non-ANI cases in the current study. RESULTS: Nineteen frequent-ANI cases (12.9%) and 16 infrequent-ANI cases (10.9%) were found; the remaining 112 cases (76.2%) were considered to be non-ANI cases. The majority of ANIs immunohistochemically contained surfactant apoprotein and ultrastructurally corresponded to invagination of the inner nuclear membrane, showing a tubular or amorphous configuration. Frequent-ANI patients showed significantly better prognosis than the other two groups on both overall univariate analysis and univariate analysis limited to patients with International Union Against Cancer Stage I disease (P = 0.0096 and P = 0.0095, respectively). However, on the multivariate analysis only disease stage was shown to be a significant prognostic factor and frequent-ANI showed borderline significance (P = 0.0956). CONCLUSIONS: Frequent ANIs appear to be of limited value in clarifying the prognosis of patients with lung adenocarcinoma.

Adenocarcinoma↗
PubMed2001

2-Acylimino-3H-thiazoline derivatives: a novel template for platelet GPIIb/IIIa receptor antagonists.

In the course of our research for the low-molecular weight RGD peptide mimics, we have found that a rigid 2-acylimino-3H-thiazoline structure is suitable for the peptide backbone mimics. Introduction of amidinophenyl and beta-alanine moiety as arginine and aspartic acid side-chain surrogates to this backbone mimic resulted in a highly potent fibrinogen receptor antagonist 2-(4-amidinobenzoylimino)-3,4-dimethyl-N-(2-carboxyethyl)-3H-thiazoline-5-carboxamide (7c), namely PS-028 (Ki = 46.5 +/- 5.8 microM).

Animals↗
PubMed2001

New therapy complementary to transcatheter arterial embolization for hepatocellular carcinoma.

A new therapy complementary to transcatheter arterial embolization (TAE) in which anticancer agents are percutaneously injected into the tumor under ultrasound guidance (percutaneous injection chemotherapy combined with TAE; TAE-PICT) has been developed and found to be effective. A total of 68 hepatocellular carcinoma (HCC) nodules in 50 patients were treated with TAE-PICT, and the complications were evaluated. Among those with solitary HCC nodules < or = 5 cm in diameter, 17 were treated with TAE-PICT and 24 with only TAE in a prospective comparative study. No major complications were seen, and the anticancer drugs could still be found in the tumor 3 months after injection. A significantly higher cumulative survival rate (p= 0.03961) and significantly higher cumulative recurrence-free survival rate (p = 0.00647) were achieved in patients treated with TAE-PICT than in those treated with TAE alone.

Aged↗
PubMed2001

Carbon dioxide degassing by advective flow from Usu volcano, Japan.

Magmatic carbon dioxide (CO2) degassing has been documented before the 31 March 2000 eruption of Usu volcano, Hokkaido, Japan. Six months before the eruption, an increase in CO2 flux was detected on the summit caldera, from 120 (September 1998) to 340 metric tons per day (September 1999), followed by a sudden decrease to 39 metric tons per day in June 2000, 3 months after the eruption. The change in CO2 flux and seismic observations suggests that before the eruption, advective processes controlled gas migration toward the surface. The decrease in flux after the eruption at the summit caldera could be due to a rapid release of CO2 during the eruption from ascending dacitic dikes spreading away from the magma chamber beneath the caldera.

Journal Article↗
PubMed2001

Hand-assisted laparoscopic total colorectal resection for familial adenomatous polyposis with coexisting rectal cancer.

When familial adenomatous polyposis (FAP) is diagnosed in a patient, prophylactic surgery must be performed whether colorectal cancer is present or not. Operations for FAP have been performed through a large median abdominal incision or an additional perineal incision, depending on the coexistence of rectal cancer. Recently, we reported a technique of laparoscopic rectal amputation without abdominal skin incision for patients with rectal cancer to minimize postoperative cardiac and respiratory complications [6]. In this article, we report a case of laparoscopically assisted proctocolectomy with ileostomy through a minimal abdominal and perineal skin incision performed by a hand-assisted procedure. The purpose of combining the perineal and laparoscopic approaches is to minimize the skin incision, while retaining a rate of cure and safety equivalent to those of conventional rectal amputation, by using the advantages of laparoscopic procedures, and to facilitate postoperative recovery and improve the quality of life for relatively young patients with FAP.

Adenomatous Polyposis Coli↗
PubMed2001

5-Fluorouracil suppression of NF-KappaB is mediated by the inhibition of IKappab kinase activity in human salivary gland cancer cells.

We have recently shown that 5-Fluorouracil (5-FU) suppresses the transcription factor NF-kappaB in human salivary gland cancer cells (cl-1) by mediating upregulation of IkappaB-alpha expression. However, the precise mechanism involved in this action has not yet been elucidated. IkappaB kinases (IKK-alpha and IKK-beta) are the key components of the IKK complex that mediates activation of NF-kappaB in response to external stimuli such as cytokines. In addition, NF-kappaB-inducing kinase (NIK) and mitogen-activated protein kinase kinase kinase 1 (MEKK-1), both of which are the upstream kinases for the IKKs, interact with and activate the IKKs. Thus, we investigated the molecular mechanisms involved in the suppression of NF-kappaB by 5-FU. Although 5-FU did not affect the expression levels of IKKs, NIK, or MEKK-1, IKK activity in cl-1 cells was suppressed at both 6 h and 12 h after treatment with 2 microgram/ml 5-FU. Moreover, when cells were treated with various concentrations of 5-FU for 12 h, the concentration of 2 microgram/ml efficiently inhibited the IKK activity as compared to 1, 5, or 10 microgram/ml. The expression of Fas-associated death domain-like interleukin 1-converting enzyme-inhibitory protein (FLIP), which acts as an inhibitor of an initiator caspase (caspase-8), was down-regulated by 5-FU treatment in cl-1 cells. Apoptosis, as evidenced by cleavage of poly(ADP-ribose) polymerase through the action of an executioner caspase (caspase-3), was also clearly observed. Thus, these results suggest that 5-FU induction of apoptosis in cl-1 cells may be mediated by suppression of NF-kappaB via inhibition of IKK activity.

Fluorouracil↗
PubMed2001

Application of a new analytical method using gas chromatography and gas chromatography-mass spectrometry for the azide ion to human blood and urine samples of an actual case.

We have established a practical and reliable method to identify and quantify the azide ion in human whole blood and human urine by transforming the ion into pentafluorobenzyl azide (PFBN3). PFBN3 was simply derived from a reaction of the ion with an excess amount of pentafluorobenzyl bromide (PFBBr). The excess amount of PFBBr was removed from the products by its reaction with sodium thiosulfate. PFBN3 in the sample was detected in high sensitivity by gas chromatography with nitrogen-phosphorus detector (GC-NPD) and gas chromatography-mass spectrometry (GC-MS). The lower detection limits of the ion by GC-NPD were 5 ng/ml for human whole blood sample and 0.5 ng/ml for human urine sample at S/N=3. On the other hand, they were 100 ng/ml for human whole blood sample and 10 ng/ml for human urine sample by the full-scan mode of GC-MS. The analytical method was applied to identification and quantification of the ion in the actual whole blood and urine samples of the victims in an actual criminal case.

Azides↗