Biomedical subjects

K Nagashima

Publications and source records attributed to K Nagashima.

At least 37 records · Page 2Linked to original sources

PubMed2001

MM-1, a c-Myc-binding protein, is a candidate for a tumor suppressor in leukemia/lymphoma and tongue cancer.

The c-myc oncogene product (c-Myc) is a transcription factor that dimerizes with Max and recognizes the E-box sequence, and it plays key functions in cell proliferation, differentiation, and apoptosis. We previously showed that MM-1 bound to myc box II within the transactivation domain of c-Myc and repressed the E-box-dependent transcriptional activity of c-Myc. Here we report that MM-1 showed features of a tumor suppressor. In an EST data base search for cDNAs homologous to MM-1, we found a frequent substitution of amino acid 157 of MM-1, from alanine to arginine (A157R), and the substitution was observed more in tumor cells than in normal cells. A survey of the A157R mutation of MM-1 in 57 cultured cancer cells and 90 tissues from cancer patients showed that the A157R was present in about 50-60% of leukemia/lymphoma cells and in more than 75% of squamous cell carcinoma of tongue cancer. Although both the A157R and the wild-type MM-1 bound to c-Myc, only A157R lost the activities to repress both the E-box-dependent transcriptional activity of c-Myc and the myc/ras cooperative transforming activity in rat 3Y1 cells. Furthermore, the wild-type MM-1, but not A157R, arrested the growth of 3Y1 cells. The human MM-1 gene was mapped at chromosome 12q12-12q13, where many chromosome abnormalities in cancer cells have been reported. The results suggest that MM-1 is a novel candidate for a tumor suppressor that controls the transcriptional activity of c-Myc.

3T3 Cells↗

PubMed2001

Broad distribution of the JC virus receptor contrasts with a marked cellular restriction of virus replication.

To investigate the early events of JC virus (JCV) infection, including attachment, penetration, transport to the nuclei, and replication of the virus, we analyzed the susceptibility of 15 different cell lines to infection using a semiquantitative polymerase chain reaction (PCR) assay, in situ hybridization, laser scanning confocal microscopy, and a viral replication assay. The cell lines examined were human permissive and nonpermissive cells as well as cells of monkey and mouse origin. JCV entry into the nuclei of the all cell lines was observed within 10 min after inoculation, demonstrating that the virus receptor is widely distributed among mammalian cells. Inhibition of viral entry by an anti-JCV VP1 antibody and sialidase treatment to remove sialic acid residues, which are considered a candidate for the JCV receptor, suggested that VP1 may interact with the cellular surface sialic acid. In addition, chlorpromazine, a clathrin-dependent pathway inhibitor, significantly suppressed entry of JCV into nuclei. In spite of the broad spectrum of cells susceptible to JCV entry, replication of the virus occurred exclusively in human neuroblastoma cell lines. These results suggest that whereas JCV can enter a wide variety of cell types and localize to the nuclei, cell-specific intranuclear mechanisms are required for virus replication.

HeLa Cells↗

PubMed2001

Requirement for C3G-dependent Rap1 activation for cell adhesion and embryogenesis.

C3G is a guanine nucleotide exchange factor (GEF) for Rap1, and is activated via Crk adaptor protein. To understand the physiological role of C3G, we generated C3G knockout mice. C3G(-/-) homozygous mice died before embryonic day 7.5. The lethality was rescued by the expression of the human C3G transgene, which could be excised upon the expression of Cre recombinase. From the embryo of this mouse, we prepared fibroblast cell lines, MEF-hC3G. Expression of Cre abolished the expression of C3G in MEF-hC3G and inhibited cell adhesion-induced activation of Rap1. The Cre-expressing MEF-hC3G showed impaired cell adhesion, delayed cell spreading and accelerated cell migration. The accelerated cell migration was suppressed by the expression of active Rap1, Rap2 and R-Ras. Expression of Epac and CalDAG-GEFI, GEFs for Rap1, also suppressed the accelerated migration of the C3G-deficient cells. This observation indicated that Rap1 activation was sufficient to complement the C3G deficiency. In conclusion, C3G-dependent activation of Rap1 is required for adhesion and spreading of embryonic fibroblasts and for the early embryogenesis of the mouse.

Animals↗

PubMed2001

SCA17, a novel autosomal dominant cerebellar ataxia caused by an expanded polyglutamine in TATA-binding protein.

Genetic etiologies of at least 20% of autosomal dominant cerebellar ataxias (ADCAs) have yet to be clarified. We identified a novel spinocerebellar ataxia (SCA) form in four Japanese pedigrees which is caused by an abnormal CAG expansion in the TATA-binding protein (TBP) gene, a general transcription initiation factor. Consequently, it has been added to the group of polyglutamine diseases. This abnormal expansion of glutamine tracts in TBP bears 47--55 repeats, whereas the normal repeat number ranges from 29 to 42. Immunocytochemical examination of a postmortem brain which carried 48 CAG repeats detected neuronal intranuclear inclusion bodies that stained with anti-ubiquitin antibody, anti-TBP antibody and with the 1C2 antibody that recognizes specifically expanded pathological polyglutamine tracts. We therefore propose that this new disease be called SCA17 (TBP disease).

Adult↗

PubMed2001

Regulation of Ca2+ channel expression at the cell surface by the small G-protein kir/Gem.

Voltage-dependent calcium (Ca2+) channels are involved in many specialized cellular functions, and are controlled by intracellular signals such as heterotrimeric G-proteins, protein kinases and calmodulin (CaM). However, the direct role of small G-proteins in the regulation of Ca2+ channels is unclear. We report here that the GTP-bound form of kir/Gem, identified originally as a Ras-related small G-protein that binds CaM, inhibits high-voltage-activated Ca2+ channel activities by interacting directly with the beta-subunit. The reduced channel activities are due to a decrease in alpha1-subunit expression at the plasma membrane. The binding of Ca2+/CaM to kir/Gem is required for this inhibitory effect by promoting the cytoplasmic localization of kir/Gem. Inhibition of L-type Ca2+ channels by kir/Gem prevents Ca2+-triggered exocytosis in hormone-secreting cells. We propose that the small G-protein kir/Gem, interacting with beta-subunits, regulates Ca2+ channel expression at the cell surface.

Animals↗

PubMed2001

Protective effect of agiotensin II type I receptor antagonist, CV-11974, on ischemia and reperfusion injury of the liver.

BACKGROUND: Microcirculatory disturbance has been shown to play a critical role in hepatic ischemia and reperfusion (I/R) injury. Angiotensin II (AngII) is one of the most potent endogenous vasoconstrictors. Angiotensin II type I (AT1) receptor antagonist has been reported to have protective effects on I/R injury of the heart and kidney. However, effect on hepatic I/R injury has not been determined. In this study, we investigate our hypothesis that AT1 receptor antagonist, CV-11974, attenuates hepatic I/R injury. METHODS: Twelve beagle dogs underwent a 2-hr total hepatic vascular exclusion with veno-venous bypass. CV-11974 was given to animals at a dose of 0.002 mg/ kg/min for 5 min followed by 0.001 mg/kg/min for 25 min via portal vein before ischemia (group II, n=6). Nontreated animals were used as the control (group I, n=6). Animal survival, hemodynamics, hepatic tissue blood flow (HTBF), liver function, platelet count, renin activity, and AngII concentration of hepatic vein, energy metabolism, and histopathology were analyzed. RESULTS: Two-week survival was 33% in group I, in contrast, 100% in group II. Mean arterial blood pressure during early reperfusion was maintained, and HTBF after reperfusion was significantly higher in group II. Treatment attenuated liver enzyme release and decrease of platelet count, increased renin and AngII, suppressed ATP degradation during ischemia and enhanced ATP resynthesis after reperfusion. Neutrophil infiltration and histopathological damages were lessened in group II. CONCLUSIONS: Our data demonstrated that the local renin-angiotensin system might play a role in hepatic microcirculation. AT1 receptor blockade with CV-11974 attenuated hepatic microcirculatory disturbance and ameliorated I/R injury.

Angiotensin II↗

PubMed2001

Analysis of transforming activity of human synovial sarcoma-associated chimeric protein SYT-SSX1 bound to chromatin remodeling factor hBRM/hSNF2 alpha.

Human synovial sarcoma has been shown to exclusively harbor the chromosomal translocation t(X;18) that produces the chimeric gene SYT-SSX. However, the role of SYT-SSX in cellular transformation remains unclear. In this study, we have established 3Y1 rat fibroblast cell lines that constitutively express SYT, SSX1, and SYT-SSX1 and found that SYT-SSX1 promoted growth rate in culture, anchorage-independent growth in soft agar, and tumor formation in nude mice. Deletion of the N-terminal 181 amino acids of SYT-SSX1 caused loss of its transforming activity. Furthermore, association of SYT-SSX1 with the chromatin remodeling factor hBRM/hSNF2 alpha, which regulates transcription, was demonstrated in both SYT-SSX1-expressing 3Y1 cells and in the human synovial sarcoma cell line HS-SY-II. The binding region between the two molecules was shown to reside within the N-terminal 181 amino acids stretch (aa 1--181) of SYT-SSX1 and 50 amino acids (aa 156--205) of hBRM/hSNF2 alpha and we found that the overexpression of this binding region of hBRM/hSNF2 alpha significantly suppressed the anchorage-independent growth of SYT-SSX1-expressing 3Y1 cells. To analyze the transcriptional regulation by SYT-SSX1, we established conditional expression system of SYT-SSX1 and examined the gene expression profiles. The down-regulation of potential tumor suppressor DCC was observed among 1,176 genes analyzed by microarray analysis, and semi-quantitative reverse transcription--PCR confirmed this finding. These data clearly demonstrate transforming activity of human oncogene SYT-SSX1 and also involvement of chromatin remodeling factor hBRM/hSNF2 alpha in human cancer.

Agar↗

PubMed2001

Comparison of the timing of acute blood-brain barrier breakdown to rabbit immunoglobulin G in the cerebellum and spinal cord of mice with experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model for human multiple sclerosis (MS). Similar to MS patients, EAE animals can exhibit chronic or relapsing, remitting paralysis; leukocyte infiltration of the central nervous system (CNS); and breakdown of the blood-brain barrier (BBB), allowing access to serum components. EAE pathology in rodents is generally thought to progress from the spinal cord to the more rostral brain. This common notion is based on numerous reports on the severity and progression of cellular infiltration and BBB breakdown during the course of disease. We studied opening of the BBB in EAE mice immunized to the proteolipid protein (PLP) peptide, PLP 139-151, with or without the use of pertussis toxin. Peripherally injected rabbit immunoglobulin G showed significant penetration through a compromised BBB in EAE mice and was observed throughout the parenchyma as well as intracellularly in multiple neuronal types. Results demonstrate the novel finding that the cerebellar BBB is dramatically and briefly comprised, even before breakdown of the BBB in the thoracolumbar spinal cord and prior to symptomatic disease. The demonstration of susceptibility in the cerebellum provides an important target for studying the factors predisposing certain CNS regions to autoimmune-related compromise of the BBB, such as MS.

Animals↗

PubMed2001

Relationship between thallium-201 myocardial SPECT and findings of endomyocardial biopsy specimens in dilated cardiomyopathy.

The purpose of this study was to clarify which myocardial histological findings associated with dilated cardiomyopathy (DCM) are reflected in quantitative 201Tl myocardial SPECT. We obtained studied SPECT images from 21 patients with DCM 10 minutes and 2 hours after they received an injection of 111 MBq 201Tl at rest. We calculated the percent coefficient of variation of myocardial 201Tl counts [%CV(TI)], the washout rate (WR), standard deviation of WR [SD(WR)], extent score (ES) and severity score (SS). We used image analysis to measure % fibrosis, % myocytes, the ratio of fibrous tissue to myocyte tissue (F/My), myocyte size and standard deviation of myocyte size [SD(My)] in left ventricular endomyocardial biopsy specimens. The %CV(Tl) was correlated with % fibrosis and F/My. The ES and SS also correlated with F/My. The correlation between SD(WR) and SD(My) was significant. The present findings suggest that %CV(Tl), ES and SS of rest 201Tl SPECT reflect myocardial fibrosis and that the standard deviation of washout reflects the distribution of myocyte size.

Biopsy↗

PubMed2001

Molecular characterization of a mitochondrial DNA segment from the Japanese scallop (Patinopecten yessoensis): demonstration of a region showing sequence polymorphism in the population.

A 1.3-kb mitochondrial DNA segment from the Japanese scallop Patinopecten yessoensis was cloned and sequenced. This segment contained the transfer RNA(Met) gene and partial sequences of 2 ribosomal RNA genes, together with 2 separate noncoding regions (designated NcR1 and NcR2). The NcR regions derived from 78 individuals cultured in Lake Saroma or Matsu Bay, were sequenced, and we found 15 loci with sequence alterations including 13 substitutions, 1 deletion, and 1 insertion (1 locus in NcR1, 14 loci in NcR2), and 17 haplotypes. Of the 17 haplotypes, 10 were found in the Saroma population only, 3 in the Mutsu population only, and 4 in both populations. The gene diversity and nucleotide diversity values were, respectively, 0.87 and 0.0069 for the Saroma population, 0.63 and 0.0040 for the Mutsu population, and 0.83 and 0.0203 overall. Thus the NcR segment was considered to have sufficient sequence variation for population genetic studies. The 16 variants of the NcR2 sequence were separated successfully by denaturing gradient gel electrophoresis, confirming the sequence variation in NcR2.

Journal Article↗

PubMed2001

Molecular analysis of mutant and wild-type tau deposited in the brain affected by the FTDP-17 R406W mutation.

Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) is a familial neurological disorder, characterized genetically by autosomal dominant inheritance, clinically by behavioral abnormalities and parkinsonism, and neuropathologically by tauopathy. Linkage analyses of affected families have led to identification of several exonic and intronic mutations in the tau gene. In this study, we analyzed molecular species of tau in the soluble and insoluble fractions of brain affected by the FTDP-17 R406W mutation. Protein chemical analysis and Western blotting using site-specific antibodies indicated that almost equal amounts of wild-type and mutant tau were present in the Sarkosyl-insoluble fraction of the R406W brain. Consistent with this, wild-type and mutant tau colocalized in neurofibrillary tangles in the frontal cortex and hippocampus of the R406W brain. In contrast to soluble R406W tau, which was less phosphorylated than soluble wild-type tau, the Sarkosyl-insoluble mutant tau was highly phosphorylated as well as the insoluble wild-type tau.

Aged↗

PubMed2001

Cyclin D1, p16 and retinoblastoma gene product expression as a predictor for prognosis in non-small cell lung cancer at stages I and II.

The association of the immunohistochemical expressions of cyclin D1, p16 and the retinoblastoma gene product (pRB) with the prognoses of 106 patients with non-small cell lung cancer (NSCLC) at stages I and II after a complete resection was investigated. We used antibodies recognizing nuclear and cytoplasmic cyclin D1, p16 and pRB. In 106 tumors, the positive rates of cyclin D1, p16 and pRB were 46, 54 and 48%, respectively. Cyclin D1-positive (cyclin D1(+)) patients had significantly poorer survival prognoses than cyclin D1-negative (cyclin D1(-)) patients (log-rank test, P=0.0002; Wilcoxon test, P=0.0005), whereas p16-positive (p16(+)) patients had significantly better prognoses than p16-negative (p16(-)) patients (log-rank test, P=0.0063; Wilcoxon test, P=0.0044). The survival period was over 65% for patients with cyclin D1(-)/p16(+) (n=34) at 120 months after surgery, whereas patients with cyclin D1(+)/p16(-) patients (n=22) had a 50% survival period at 49 months. The cumulative survival rate of cyclin D1(+)/p16(-) patients was significantly lower than that of cyclin D1(-)/p16(+) patients (log-rank test, P=0.0004; Wilcoxon test, P=0.0002). The pRB did not influence significantly the survival rate. Our results indicate that cyclin D1 and p16, especially a combination of cyclin D1 and p16, are very useful to predict the prognosis of patient with NSCLC after curative resection independent of pathological stages I and II.

Aged↗

PubMed2001

A mitochondrial encephalo-myo-neuropathy with a nucleotide position 3271 (T-C) point mutation in the mitochondrial DNA.

We report three members of a family, who exhibited a phenotype similar to 'myoclonus epilepsy with ragged-red fibers' but had a genotype usually associated with 'mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes'. The patients, a 48-year-old female, and her two sons, aged 21 and 19 respectively, presented with photo-reactive syncopal episodes, disturbances of gait and writing, dysarthria and finger tremor since the 3rd and 2nd decade of life, respectively, that were accompanied also by numbness and weakness of the extremities. Subsequently, cerebellar ataxia and myoclonus were also noted. Electromyography revealed both myogenic and neurogenic muscular changes, and nerve conduction studies demonstrated a sensory-motor neuropathy. Biopsy showed ragged-red fibers with strongly stained SDH-positive vessels in skeletal muscles, and a marked loss of myelinated fibers of the sural nerves. Mitochondrial (mt) DNA analyses of peripheral blood, muscles and nerves revealed that all members had a heteroplasmic np3271 (T-C) point mutation in the mitochondrial tRNA-Leu gene (UUR). This family is unique, in that all patients presented with a myoclonus epilepsy with ragged-red fibers-like phenotype and had a distinctive peripheral neuropathy, while the detected mtDNA 327l (T-C) mutation has been reported to date only in rare cases of mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes

Adult↗

PubMed2001

Alternative EWS-FLI1 fusion gene and MIC2 expression in peripheral and central primitive neuroectodermal tumors.

Primitive neuroectodermal tumors (PNET) occur either in the central nervous system (CNS; central PNET, cPNET) or in the peripheral sites (peripheral PNET, pPNET). Recent molecular approaches have been defining a new concept of PNET, that is, the pPNET including Ewing's sarcoma (ES) which expresses MIC2 glycoprotein and shows the specific chimeric gene of EWS-FLI1. The expression of MIC2 and the genetic rearrangement of EWS-FLI1 are considered to be highly specific to the pPNET/ES. This study examined the expression of MIC2 and EWS-FLI1 gene by means of immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) on various small round cell tumors originating in the CNS or non-CNS organs. All peripheral PNET tested expressed MIC2 and were positive for EWS-FLI1 (11/11). In contrast, all cPNET and other blastic CNS tumors were negative for MIC2: medulloblastoma (0/3), cerebral PNET (0/2), spinal PNET (0/2), glioblastoma (0/2), retinoblastoma (0/3), and pineoblastoma (0/2). These MIC2-negative tumors were also negative for the chimeric gene product of EWS-FLI1. Interestingly, one PNET originating in the intracranial dura mater was positive for both MIC2 and EWS-FLI1 fusion gene. The results indicate that cPNET lacks any genetic or protein markers, except for a meningeal PNET which falls into the same phenotypic spectrum of pPNET.

12E7 Antigen↗

PubMed2001

JC virus large T protein transforms rodent cells but is not involved in human medulloblastoma.

JC virus (JCV) together with Simian virus 40 (SV40) and BK virus (BKV), belong to the polyomavirus group and these viruses are neuro-oncogenic to rodents by expression of large T antigen (LT), which binds to cellular p53 and pRB thus reducing the anticancer potential of the cell. The function of LT has not been clarified because small t antigen (st) is transcribed from the same start codon as the overlapping reading frame of LT, and is translated as a different protein with the same N-terminal residues (1-81 amino acids) by a splice-site variant of mRNA. To elucidate the function of LT without st, we constructed plasmids that express LT only by deleting the splicing region including the C-terminus of st, and consequently stable cell lines were established that express only JCLT, SV40LT and BKLT. The growth rates of these cells were examined in colonies on soft agar and it was found that LT alone has a transforming capacity; the order of efficiency being SV40LT, BKLT and JCLT. In addition, to verify the involvement of JCV in human medulloblastoma, eight cases of medulloblastoma, six cases of frozen material and five cases of paraffin-embedded tissues which included three cases of frozen tissues, were examined. PCR assay, genomic Southern blotting, and in situ hybridization were applied to detect the JCV genome, and LT and st were examined by immunohistochemistry; the results were compared with JCV-infected tissues as a positive control. All methods failed to detect not only JCV genome but also LT protein in medulloblastoma and it was concluded that JCV LT has transforming activities in rodent cells, but is not related to human medulloblastoma.

3T3 Cells↗

PubMed2001

Adult T-cell lymphoma involving the leptomeninges associated with a spinal cord schwannoma.

Adult T-cell lymphoma (ATL-L) developing initially in the meninges is rare. An autopsy case of ATL-L with an acute onset of meningitis and generalized lymphadenopathy in association with a cervical cord schwannoma is reported here. A 78-year-old woman with sensori-motor weakness of both arms over a 1-year period, developed febrile episodes and drowsiness with neck stiffness. Lumbar puncture revealed an increased protein content (161 mg/dL) and increased cell count (463/3) consisting of 99% of lymphocytes which contained atypical lymphocytes with multilobulated nuclei ('flower cells'), which are characteristic of ATL-L. Viral titers were positive only for HTLV-I antibodies (serum X 640: CSF X 16). Biopsy of an enlarged retroperitoneal lymph node revealed malignant lymphoma of the T-cell type. Brain MRI was negative, whereas an intradural extramedullary mass was found at the C4 level. With a diagnosis of ATL-L stage IV, chemotherapy was commenced, which was effective in reducing the generalized lymphadenopathy as well as the cervical mass and restoring the CSF findings to normality. The cervical cord mass was verified to be a solitary schwannoma, and ATL-L involvement was found not only in the leptomeninges, but also within the cervical cord schwannoma.

Aged↗

PubMed2001

Primary extranodal marginal zone B-cell lymphoma of the mucosa-associated lymphoid tissue type in the CNS.

Extranodal marginal zone B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT) type has been reported in various internal organs. Here a case is reported of MALT lymphoma developing in the cerebellopontine (CP) angle in a patient with Sjogren syndrome, and the concept of MALT lymphoma of the CNS is introduced. Pathologically, the tumor showed inflammatory features of reactive lymphocytic infiltration with follicle formation, but there were slightly atypical lymphocytes and plasmacytes with B-cell markers. These cells invaded reactive follicles, showing follicular colonization, and showed aberrant expression indicating their neoplastic nature. A review of the literature revealed eight cases of MALT lymphoma originating from the dura mater and one from the CP angle. The average age of patients was 50 years (range 28-66 years), and all patients were female. The tumors were slow to develop and the patients were cured after surgical removal and/or additional therapies. It is proposed that MALT lymphoma should be considered as a differential diagnosis of inflammatory pseudotumor of the CNS.

Adult↗

PubMed2001

HHV8-negative primary effusion lymphoma of the peritoneal cavity presenting with a distinct immunohistochemical phenotype.

Primary effusion lymphoma (PEL) has been recognized as a body-cavity-based lymphoma that was originally reported to be associated with human herpes virus 8 (HHV8) infection, and was frequently found in human immunodeficiency virus-positive (HIV) patients. Here we describe an autopsy case of PEL of the peritoneal cavity in an immunocompetent patient. Cytological analysis of tumor cells within ascites revealed immunocytochemical features of keratin positivity and CD45 negativity. At autopsy, the presence of a massive volume of ascites as well as diffuse tumor cell infiltrates within the serosa of the intestine and mesenterium were observed. Tumor cells were morphologically similar to anaplastic large-cell lymphoma, but were immunohistochemically positive for keratin and epithelial membrane antigen (EMA). They also showed no reactivity to representative lymphocyte surface markers including CD45, in addition to being negative for CD30 and p80NPM/ALK. Molecular analysis of the tumor cells revealed monoclonality of the immunoglobulin heavy-chain gene rearrangement which demonstrated a lymphoma of the B-cell lineage. Furthermore, HHV8 was not detected by immunohistochemical analysis, PCR or nested PCR technique. Based on these results, we consider the present case to be an HHV8-negative PEL with keratin and EMA positivity.

Adult↗