Construction of recombinant feline herpesvirus type 1 expressing Toxoplasma gondii surface antigen 1.
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Alphaherpesvirinae↗
Biomedical subjects
N Yokoyama
Publications and source records attributed to N Yokoyama.
Inhibition of Src by direct interaction with protein phosphatase 2A.
In this study, we report that Src kinase is inhibited by protein phosphatase 2A (PP2A), a serine/threonine phosphatase. We carried out experiments in vitro using purified PP2A (AC dimer) and full-length v-Src or truncated forms of v-Src. The inhibition of v-Src by PP2A is concentration- and time-dependent. Addition of okadaic acid, a PP2A phosphatase inhibitor, abolished the PP2A-dependent inhibition of v-Src. When experiments were carried out at 4 degrees C under conditions where PP2A activity is inhibited, Src activity was unaffected by the presence of PP2A, suggesting that PP2A binding alone is insufficient to block Src activity. These results imply that PP2A activity is essential for inhibition of v-Src. We also demonstrate that PP2A binds to the catalytic and the regulatory domains of v-Src.
Amino Acid Sequence↗
Protein phosphatase 2A interacts with the Src kinase substrate p130(CAS).
In this study, we report that the Src substrate Cas (p130 Crk-associated substrate) associates with protein phosphatase 2A (PP2A), a serine/threonine phosphatase. We investigated this interaction in cells expressing a temperature-sensitive mutant form of v-Src. v-Src activation (by shifting cells from the nonpermissive to the permissive temperature) led to an increase in the tyrosine phosphorylation of v-Src and Cas, as well as in the association between v-Src and Cas. v-Src has previously been shown to bind to PP2A and to phosphorylate the catalytic subunit of PP2A, resulting in inhibition of phosphatase activity. We found that the association between v-Src and PP2A decreased as cells were shifted to the permissive temperature. In contrast, the levels of PP2A that co-immunoprecipitated with Cas increased when v-Src was activated. We obtained similar results in pull-down experiments with immobilized Microcystin, a PP2A inhibitor. Serine/threonine phosphorylation of Cas has previously been shown to occur in a cell cycle regulated matter. Treatment of NIH3T3 cells with okadaic acid, a PP2A inhibitor, augments the serine/threonine phosphorylation of Cas that occurs at mitosis. Furthermore, PP2A dephosphorylates serine residues on Cas in vitro. Taken together, our results suggest that PP2A may be involved in the cell cycle-specific dephosphorylation of Cas.
3T3 Cells↗
Characterization of acyl-CoA-binding protein (ACBP) in the pheromone gland of the silkworm, Bombyx mori.
Various fatty acyl-CoAs are involved as intermediates or precursors of sex pheromone components in the biosynthetic pathway of the pheromones in many lepidopteran insects. We have purified a 10-kDa protein from the cytosolic fraction of Bombyx mori pheromone glands by using affinity chromatography with a palmitoyl-CoA-agarose column and reversed-phase HPLC. Amino acid sequence analysis of the fragment peptides obtained from the purified protein, and a homology search, revealed that this protein was a member of acyl-CoA-binding proteins (ACBPs). MALDI-TOF mass spectral analysis of the purified protein and cloning of the gene from a pheromone gland cDNA library confirmed B. mori ACBP to be a 90 amino acid protein with 78.9% identity to that of Manduca sexta ACBP. The secondary structure of the recombinant B. mori ACBP was determined by NMR spectroscopy. Northern blot analysis demonstrated that B. mori ACBP was predominantly expressed in the pheromone gland and the corresponding transcript was expressed from the day before adult eclosion. Present results suggest that ACBP plays a significant role in the production of sex pheromones regulated by the neurohormone, pheromone biosynthesis activating neuropeptide (PBAN).
Acyl Coenzyme A↗
Novel lectin-like proteins on the surface of human monocytic leukemia cell line THP-1 cells that recognize oxidized cells.
Presence of lectin-like receptors on the membranes of human monocytic leukemia cell line THP-1 cells for clustered sialylated poly-N-acetyllactosaminyl sugar chains on the membranes of oxidized erythrocytes and T-lympoid cells was investigated. Membranes of THP-1 cells differentiated into macrophages were solubilized, and the membrane proteins obtained by affinity chromatographies using lactoferrin-Sepharose and band 3-Sepharose were purified by successive DE column chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins of 50, 60, and 80 kDa with specificity to bind to sialylated poly-N-acetyllactosaminyl sugar chains were detected in the chromatographic fractions. A 50-kDa protein was isolated in a pure form. N-Terminal amino acid sequence of the protein was Lys-Gln-Lys-Val-Ala-Gly-Lys-Gln-Pro-Val-, which has not been found in the N-terminal regions of the hitherto known proteins. The antibody, raised against the chemially synthesized peptide composed of the N-terminal amino acid sequence, bound to 50-, 60-, and 80-kDa proteins as analyzed by immunoblotting and immunoprecipitation, indicating that these proteins had the same N-terminal amino acid sequence. The results demonstrate that THP-1 cells have novel 50-, 60-, and 80-kDa lectin-like proteins with the same N-terminal amino acid sequence on the cell surface which would bind to clustered sialylated poly-N-acetyllactosaminyl sugar chains generated on oxidized erythrocytes and T-lymphoid cells.
Amino Acid Sequence↗
Liver metastasis in gastric cancer with particular reference to lymphatic advancement.
BACKGROUND: We have previously reported that, in models of mesenteric lymph vessel obstruction in rats, we observed lymphaticovenous communication. This suggested that cancer cells metastasized to the liver by a lymphatic route. In the present study, we investigated the relationship between liver metastasis and lymphatic involvement in gastric carcinoma by examining resected specimens. METHODS: Twenty gastric cancer patients who had synchronous liver metastasis and 17 who developed metachronous liver metastasis after gastrectomy, performed between 1985 and 1997, were included in this study. They were compared with 44 advanced gastric cancer patients who had neither synchronous nor subsequent liver metastasis, and who survived with a disease-free course for more than 5 years. We compared the patients' clinicopathological features; in particular, we investigated extranodal invasion in the resected lymph nodes. This invasion was classified according to the pattern of extranodal cancer invasion, with or without rupture of the lymph node capsule. RESULTS: Liver metastasis was more frequent in patients with extranodal invasion than in those without extranodal invasion (P < 0.002). Multivariate analysis revealed that the correlation between extranodal invasion and liver metastasis was significant (P < 0.024); the odds ratio was 4.412. Metastasis to the lymph nodes was the next most significant risk for liver metastasis. CONCLUSION: We consider that the lymphatic system is closely related to the establishment of liver metastasis; in particular, extranodal invasion is a significant risk factor for liver metastasis in patients with gastric cancer.
Aged↗
Further development of a recombinant feline herpesvirus type 1 expressing the Gag protein of feline immunodeficiency virus.
We have previously reported the construction of a recombinant feline herpesvirus type 1 (FHV-1), designated C7301ddlTK-gag, expressing the Gag precursor protein of feline immunodeficiency virus (FIV). In this study, we report the construction of a further recombinant FHV-1 (ddlTK(gBp)-gag) which carries an FHV-1 gB promoter sequence upstream of the FIV gag gene of C7301ddlTK-gag. Strong expression of the FIV Gag protein by ddlTK(gBp)-gag was confirmed by immunoblot analyses and enzyme-linked immunosorbent assays. Although C7301ddlTK-gag and ddlTK(gBp)-gag failed to induce anti-FIV Gag antibodies in cats, we confirmed the infectivity and stability of these recombinants in cats.
Animals↗
Angiotensin-converting enzyme inhibition improves defective angiogenesis in the ischemic limb of spontaneously hypertensive rats.
OBJECTIVES: Natural angiogenesis has been shown to be impaired in spontaneously hypertensive rats (SHR). The purpose of this study was to determine whether pathological angiogenesis in the setting of tissue ischemia is also impaired in SHR, and to what extent it is modified by angiotensin-converting enzyme (ACE) inhibition. METHODS: Ischemia was induced in the hindlimb of SHR by excision of the femoral artery, after which the animals were randomly assigned to receive low-dose perindopril (sub-antihypertensive, 0.2 mg/kg/day), high-dose perindopril (antihypertensive, 2.0 mg/kg/day), or vehicle for 3 weeks. Wistar-Kyoto rats (WKY) with femoral artery excision served as a control group. RESULTS: Tissue ACE activity in SHR was significantly increased compared to WKY (49.4+/-6.2 vs. 34.0+/-14.2 IU/mg, P<0.01). Administration of perindopril significantly reduced ACE activity in SHR (low dose: 12.4+/-2.3; high dose: 11.0+/-2.1 IU/mg, P<0.005). Angiogenesis of the ischemic limb muscles was significantly impaired at 4 weeks in SHR versus WKY as indicated by the lower capillary density in the former (364.5+/-43.0 vs. 463.8+/-63.0/mm(2), P<0.05) as well as the reduced hindlimb perfusion assessed by laser Doppler imaging (0.86+/-0.08 vs. 1.03+/-0.09, P<0.05). Administration of perindopril significantly augmented both the capillary density (low dose: 494.3+/-69.8; high dose: 543.9+/-76.9/mm(2), P<0.005) and the limb perfusion (low dose: 1.06+/-0.15; high dose: 1.05+/-0.12, P<0.05) of the ischemic limb in SHR. CONCLUSIONS: These findings indicate that pathological angiogenesis in the setting of tissue ischemia is impaired in SHR compared with WKY, and that this impairment can be reversed by ACE inhibition. The angiogenic properties of an ACE inhibitor may benefit patients with essential hypertension presenting with lower limb vascular insufficiency.
Analysis of Variance↗
Characterisation of the gene encoding a protective antigen from Babesia microti identified it as eta subunit of chaperonin containing T-complex protein 1.
Passive immunisations with a monoclonal antibody termed 1-5H showed a partial but significant inhibition of parasitaemia against Babesia microti challenge infection. By immunoscreening with 1-5H, a clone (termed p58 gene) was obtained from a cDNA expression library of B. microti and the complete nucleotide sequence was determined. A protein homology search showed significant amino acid identities to the eta subunit of the chaperonin containing T-complex protein 1 (CCT) of human (59%), mouse (58%) and Plasmodium falciparum (62%). Genomic analyses indicated that the p58 gene is present as a single copy gene and contains a total of approximately 400-bp introns in the genome of B. microti. The mAb 1-5H recognised a 58-kDa protein of B. microti and was found to cross-react with a 60-kDa protein of Babesia rodhaini. These results suggest the possibility that the p58 protein is the CCT eta subunit of B. microti and functions as a chaperonin.
Amino Acid Sequence↗
Increased leukocyte activity as a predictor for flow-limiting coronary lesions in patients with angina pectoris.
Myocardial ischemia initiates an acute inflammatory response, in which polymorphonuclear leukocytes (PMNs) are major participants. We investigated whether increased PMN activity in the peripheral blood is a marker for high-grade coronary artery stenosis in patients with angina pectoris. PMN activity was examined in 45 patients by measuring the luminol-dependent chemiluminescence (CL) response of the peripheral blood upon addition of phorbol myristate acetate (PMA). The CL of patients with angiographic filling delay was 1.6-fold higher than in patients without delay (480+/-54 vs. 302+/-33 counts/PMN, P<0.01). The CL of patients with a filling delay of the left anterior descending artery (LAD) was 1.7-fold higher than in patients without an LAD filling delay (537+/-79 vs. 317+/-27 counts/PMN, P<0.01). With the CL cut-off at 400 counts/PMN, the sensitivity and specificity for detecting an angiographic filling delay of the LAD were 69 and 75%, respectively. These data indicate that PMN activity in the peripheral blood is increased with the presence of a flow-limiting coronary lesion showing an angiographic filling delay. Measuring PMN activity may be a useful approach to assess indications for and timing of angiography and/or adjunctive invasive therapies in patients with angina pectoris.
Angina Pectoris↗
Forward signaling mediated by ephrin-B3 prevents contralateral corticospinal axons from recrossing the spinal cord midline.
To investigate Eph-ephrin bidirectional signaling, a series of mutations were generated in the ephrin-B3 locus. The absence of both forward and reverse signaling resulted in mice with mirror movements as typified by a hopping locomotion. The corticospinal tract was defective as axons failed to respect the midline boundary of the spinal cord and bilaterally innervated both contralateral and ipsilateral motor neuron populations. A second mutation that expresses a truncated ephrin-B3 protein lacking its cytoplasmic domain did not lead to hopping, indicating that reverse signaling is not required for corticospinal innervation. Ephrin-B3 is concentrated at the spinal cord midline, while one of its receptors, EphA4, is expressed in postnatal corticospinal neurons as their fibers pathfind down the contralateral spinal cord. Our data indicate ephrin-B3 functions as a midline-anchored repellent to stimulate forward signaling in EphA4-expressing axons.
Alleles↗
Simultaneous detection of colorectal carcinoma liver and lung metastases does not warrant resection.
BACKGROUND: Recent evidence suggests that metastasectomy is efficacious for selected patients with hepatic and pulmonary metastases from a colorectal primary. The aim of this study was to identify a subgroup of patients who best benefit from hepatic and pulmonary metastasectomy among those with colorectal carcinoma metastases. STUDY DESIGN: We analyzed retrospectively a total of 136 patients who underwent resection of hepatic or pulmonary metastases of colorectal origin at Niigata University Medical Hospital between 1982 and 2000. The median follow-up period was 94 months. Eighty-four patients underwent hepatectomy alone, 25 underwent pulmonary resection alone, and 27 underwent both hepatic and pulmonary resection. The 27 patients undergoing hepatic and pulmonary resection were divided into two groups: 17 patients with sequentially detected hepatic and pulmonary metastases and 10 patients with simultaneously detected metastases. Survival time was determined from the date of initial metastasectomy. Differences in cumulative survival were evaluated using the log-rank test. Sixteen factors were assessed for their influence on the survival of the 27 patients undergoing resection of hepatic and pulmonary metastases; univariate and multivariate analyses were used in this evaluation. RESULTS: Patient survival after hepatic and pulmonary resection was comparable with that after hepatectomy alone (p = 0.536) and that after pulmonary resection alone (p = 0.294). Among the 27 patients undergoing hepatic and pulmonary resection, the outcomes after resection were significantly better in patients with sequentially detected metastases (cumulative 5-year survival of 44%) than in those with simultaneously detected ones (cumulative 5-year survival of 0%) (p < 0.001). On multivariate analysis sequential detection of hepatic and pulmonary metastases was the strongest independent favorable prognostic factor (p <0.001). CONCLUSIONS: Patients with sequentially detected hepatic and pulmonary metastases from a colorectal primary are good candidates for aggressive metastasectomy. Simultaneous detection of these metastases does not warrant resection.
Adult↗
Early gallbladder carcinoma does not warrant radical resection.
BACKGROUND: This study was designed to address whether gallbladder cancer invading the muscle layer (stage pT(1b)) is a local disease and whether radical resection is necessary. METHODS: A retrospective analysis of 25 patients with pT(1b) gallbladder tumours, 13 of whom underwent simple cholecystectomy and 12 radical resection with regional lymph node dissection, was performed. A total of 147 regional lymph nodes was examined for metastasis. The median follow-up time was 95 months. RESULTS: No patient had blood vessel or perineural invasion on histology. Lymphatic vessel invasion was seen in one patient. Both overt metastasis and micrometastases were absent in all lymph nodes examined. Overall 10-year survival was 87 per cent. The outcome after simple cholecystectomy was comparable to that after radical resection (P = 0.16). Two patients who underwent radical resection died from tumour relapse in distant sites. CONCLUSION: Most pT(1b) gallbladder carcinomas spread only locally. Additional radical resection is not necessary when the depth of invasion of gallbladder carcinoma is limited to the muscle layer after simple cholecystectomy.
Adult↗
Fas/FasL mediated apoptosis of thyrocytes in Graves' disease.
We examined in the present study the possible involvement of Fas and its ligand (FasL) in the process of Graves' disease. Immunohistochemical analysis showed that few normal thyrocytes expressed Fas but many thyrocytes in Graves' disease expressed this molecule. The percentage of FasL-positive thyrocytes in Graves' thyroids was, however, less than in normal thyroids. Several apoptotic thyrocytes and infiltrating mononuclear cells (MNCs) were detected scattered throughout Graves' thyroid tissues and abundant proliferating cell nuclear antigen (PCNA)-positive thyrocytes were present. Apoptotic cells, as well as PCNA-positive cells, were scarcely detectable in normal thyroid glands, however. In vitro treatment of thyrocytes by IL-1beta a cytokine found to be expressed in Graves' thyroid glands, increased Fas but reduced FasL expression. IL-1beta-stimulated thyrocytes became sensitive to apoptosis by anti-Fas IgM monoclonal antibody (mAb). Activated T cells, which strongly expressed FasL, showed cytotoxic activity toward IL-1beta-stimulated thyrocytes but not toward unstimulated thyrocytes. This cytotoxic activity involved the Fas/FasL pathway. Importantly, unstimulated thyrocytes could kill activated, but not resting, T cells. IL-1beta-stimulated thyrocytes, with down-regulated FasL expression, could not efficiently kill activated T cells. The cytotoxic activity of unstimulated thyrocytes toward activated T cells was inhibited by anti-FasL mAb. Interestingly, unstimulated thyrocytes induced apoptosis in IL-1beta-stimulated thyrocytes but not in unstimulated thyrocytes. These interactions were also blocked by anti-FasL mAb. Our results suggest that the apoptotic cell death of both thyrocytes and infiltrating MNCs found in Graves' thyroid glands is regulated by IL-1beta through Fas/FasL interactions.
Apoptosis↗
Clinical significance of lymph node micrometastasis in gallbladder carcinoma.
BACKGROUND: This retrospective study was intended to define the clinical significance of lymph node micrometastasis in gallbladder carcinoma. METHODS: A total of 1136 regional lymph nodes taken from 63 consecutive patients undergoing radical resection were examined histologically. Micrometastasis was defined as a metastasis missed on routine histologic examination with hematoxylin-and-eosin but detected by immunohistochemical examination with an antibody against cytokeratins 8 and 18. RESULTS: None of 9 patients (0%) with pT1 disease and 19 of 54 patients (35%) with pT2-4 disease had nodal micrometastases. Univariate analysis identified nodal micrometastasis, type of radical resection, M classification, pT classification, perineural invasion, pTNM stage, timing of radical resection, lymphatic vessel invasion, and pN classification as significant variables. Multivariate analysis revealed that nodal micrometastasis (P =.0003) and type of radical resection (P=.0044) were independent prognostic factors. Nodal micrometastasis affected survival adversely, despite the absence (P=.0002) or presence (P <.0001) of overt nodal metastasis. Nodal micrometastasis correlated significantly with invasive characteristics: lymphatic vessel invasion, perineural invasion, and distant metastasis. CONCLUSIONS: Lymph node micrometastasis is the strongest independent predictor of worse survival regardless of the overt nodal status and may indicate aggressive tumor biology among patients undergoing curative resection for gallbladder carcinoma.
Adult↗
Involvement of protein phosphatase 2A in the interleukin-3-stimulated Jak2-Stat5 signaling pathway.
In this study, we report that the tyrosine kinase, Janus kinase 2 (Jak2), associates with the serine/threonine protein phosphatase 2A (PP2A) in 32Dcl3 myeloid progenitor cells. The association between Jak2 and PP2A transiently increases following interleukin-3 (IL-3) stimulation and activation of Jak2. The catalytic subunit of PP2A is tyrosine phosphorylated by Jak2 in vitro and in vivo, resulting in inhibition of phosphatase activity. PP2A also associates with Stat5 in 32Dcl3 cells in an IL-3-dependent manner. Pretreatment of 32Dcl3 cells with okadaic acid (OA), an inhibitor of PP2A, resulted in increased tyrosine phosphorylation and nuclear translocation of Stat5. Our results suggest that PP2A plays a negative regulatory role in regulating the IL-3 signaling pathway via formation of complexes with Jak2 and Stat5.
Animals↗
Gastric and intestinal phenotypic marker expression in gastric carcinomas and its prognostic significance: immunohistochemical analysis of 136 lesions.
OBJECTIVE: It is well known that both gastric and intestinal phenotypic cell markers are expressed in gastric carcinomas, irrespective of their histologic type. However, the clinicopathologic significance of these expressions has not yet been clarified. METHODS: We analyzed the correlations among gastric and intestinal phenotypic marker expression patterns of the tumor, clinicopathologic findings and the patient's outcome in 136 advanced gastric carcinomas. RESULTS: Phenotypic marker expression was immunohistochemically evaluated using the monoclonal antibodies 45M1 (anti-human gastric mucin; HGM), CLH5 (anti-MUC6), Ccp58 (anti-MUC2) and 56C6 (anti-CD10). All tumors were classified as gastric (G), gastric and intestinal mixed (GI), intestinal (I) or unclassified (UC) phenotype. Of the 136 gastric carcinomas, 50 (36.8%), 56 (41.2%), 21 (15.4%) and 9 (6.6%) were classified as G, GI, I and UC phenotype, respectively. The G-phenotype tumors were associated with a higher rate of undifferentiated-type and infiltrative histology as compared with the I-phenotype tumors (p < 0.05 and p < 0.001, respectively). Furthermore, both univariate and multivariate analysis of survival revealed the G-phenotype tumor to be associated with a significantly poorer outcome than the I-phenotype tumor (p < 0.05). CONCLUSION: Our present results indicate that the gastric and intestinal phenotypic marker expression pattern of tumors, determined by the combination of HGM, MUC6, MUC2 and CD10 expression, is prognostically useful for patients with gastric carcinoma.
Adenocarcinoma↗
Enhanced adhesion of oxidized mouse polymorphonuclear leukocytes to macrophages by a cell-surface sugar-dependent mechanism.
Mouse thioglycollate-induced peritoneal macrophages effectively, in the absence of serum, recognized mouse polymorphonuclear leukocytes (PMNs) mildly oxidized with diamide, superoxide (hypoxanthine/xanthine oxidase) or t-butyhydroperoxide, or modified with N-ethylmaleimide (NEM). The recognition reached a maximum when PMNs were treated wtih each of the reagents at relatively low concentrations, and the recognition was decreased on treatment with reagents at higher concentrations. Glutathione depletion in the diamide-oxidized PMNs may cause enhanced adhesion to macrophages. Sialylated sugar chains attached to a peptide chain in glycophorin A and sialylated poly-N-acetyllactosaminyl sugar chains in lactoferrin and band 3 glycoprotein effectively inhibited the increased adhesion of the diamide-oxidized PMNs. Enzymatic removal of sialyl residues and the degradation of poly-N-acetyllactosaminyl sugar chains by pretreatment of PMNs with neuraminidase or endo-beta-galactosidase, respectively, lost their increasing ability for macrophage adhesion after oxidation with diamide, superoxide or t-butylhydroperoxide. Clustered sialylated poly-N-acetyllactosaminyl sugar chains on the cell surface may be involved in the increased adhesion of the oxidized PMNs to macrophages.
Animals↗