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Biomedical subjects

N Suzuki

Publications and source records attributed to N Suzuki.

At least 91 records · Page 5Linked to original sources
PubMed2001

Serodiagnosis of Toxoplasma gondii infection in cats by enzyme-linked immunosorbent assay using recombinant SAG1.

The gene encoding surface antigen 1 (SAG1, P30) of Toxoplasma gondii (T. gondii) was cloned into the plasmid pGEX-4T-3 and subsequently expressed in Escherichia coli (E. coli) as a glutathione-S-transferase (GST) fusion protein. The recombinant SAG1 (rSAG1) was refolded using 8M urea solution followed by dialysis and thereafter evaluated in an enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of toxoplasmosis. The test sera were adsorbed with GST to block non-specific reactivity to the GST-SAG1 fusion protein. The ELISA with rSAG1 was able to differentiate very clearly between sera from cats or mice experimentally infected with T. gondii and sera from normal cats or mice. The ELISA detected no cross-reactivity with sera from mice experimentally infected with the closely related parasite Neospora caninum (N. caninum). Some 193 cat sera were tested for antibodies to T. gondii, out of which 40 (20.7%) reacted positively by ELISA with the rSAG1 while another 79.3% cats reacted negative to the assay. Both positive and negative sera were confirmed by Western blot analysis. The results of ELISA were in agreement with those of a commercially available latex agglutination test (LAT) kit, although the former had higher titers than the latter.

Animals↗
PubMed2001

Interaction of heterotrimeric G13 protein with an A-kinase-anchoring protein 110 (AKAP110) mediates cAMP-independent PKA activation.

Heterotrimeric G proteins and protein kinase A (PKA) are two important transmitters that transfer signals from a wide variety of cell surface receptors to generate physiological responses. The established mechanism of PKA activation involves the activation of the Gs-cAMP pathway. Binding of cAMP to the regulatory subunit of PKA (rPKA) leads to a release and subsequent activation of a catalytic subunit of PKA (cPKA). Here, we report a novel mechanism of PKA stimulation that does not require cAMP. Using yeast two-hybrid screening, we found that the alpha subunit of G13 protein interacted with a member of the PKA-anchoring protein family, AKAP110. Using in vitro binding and coimmunoprecipitation assays, we have shown that only activated G alpha 13 binds to AKAP110, suggesting a potential role for AKAP110 as a G alpha subunit effector protein. Importantly, G alpha 13, AKAP110, rPKA, and cPKA can form a complex, as shown by coimmunoprecipitation. By characterizing the functional significance of the G alpha 13-AKAP110 interaction, we have found that G alpha 13 induced release of the cPKA from the AKAP110-rPKA complex, resulting in a cAMP-independent PKA activation. Finally, AKAP110 significantly potentiated G alpha 13-induced activation of PKA. Thus, AKAP110 provides a link between heterotrimeric G proteins and cAMP-independent activation of PKA.

A Kinase Anchor Proteins↗
PubMed2001

Induction of interleukin-1beta and interleukin-6 mRNA by low doses of ionizing radiation in macrophages.

We have previously reported the antimetastatic effects and augmentation of immune responses, which would be a mechanism of the antimetastatic effects, of 0.1 to 0.2 Gy total body irradiation. To elucidate the cellular mechanisms of the augmentation of immune response, we investigated the effects of low-dose irradiation on gene expression of interleukin-1beta (IL-1beta) and IL-6 using mouse peritoneal macrophages in vitro. Absolute mRNA quantification was carried out using competitive polymerase chain reaction. Gene expression of IL-1beta and IL-6 was increased 1 to 2 hr after 2.0 Gy irradiation and then decreased to below the basal expression level 4 hr after irradiation. Irradiation with 0.1 Gy increased IL-6 expression 2 hr after irradiation, but it did not affect IL-1beta expression. Downregulation of IL-1beta and IL-6 observed 4 hr after 2.0 Gy irradiation was not observed with 0.1 Gy irradiation. The protein kinase C (PKC) inhibitor H7 and the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor wortmannin inhibited induction of IL-1beta and IL-6 expression, which suggests that radiation-induced IL-1beta and IL-6 expression is achieved by PKC- and PI3-kinase-mediated signaling.

Androstadienes↗
PubMed2001

Laparoscopic resection of a functional paraganglioma in the organ of Zuckerkandl.

We describe the successful laparoscopic resection of a functional paraganglioma in the organ of Zuckerkandl. A 47-year-old man with hypertension and diabetes mellitus was found to have an abdominal mass beside the aorta. The tumor was diagnosed as a functional paraganglioma by diagnostic imaging and biochemical tests. We then performed a transperitoneal laparoscopic resection for removal. After freeing the left ureter, resecting the inferior mesenteric artery, and dividing the small blood vessels, the tumor was isolated and found to be preserved in its capsule. It was retrieved in a bag through an enlarged incision. The operation time was 450 min and blood loss was 410 ml. The postoperative course was uneventful and there has been no local recurrence or distant metastasis during the 18-month follow-up period. Laparoscopic resection of functional extraadrenal paragangliomas is technically feasible and safe if adequate pre- and intraoperative medical management and a careful, steady surgical technique are used.

Abdominal Neoplasms↗
PubMed2001

Cell transplantation for the treatment of acute myocardial infarction using vascular endothelial growth factor-expressing skeletal myoblasts.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a promising reagent for inducing myocardial angiogenesis. Skeletal myoblast transplantation has been shown to improve cardiac function in chronic heart failure models by regenerating muscle. We hypothesized that transplantation of VEGF-expressing myoblasts could effectively treat acute myocardial infarction by providing VEGF-induced cardioprotection through vasodilatation in the early phase, followed by angiogenesis effects in salvaging ischemic host myocardium combined with the functional benefits of newly formed, skeletal myoblast-derived muscle in the later phase. METHODS AND RESULTS: Primary rat skeletal myoblasts were transfected with the human VEGF(165) gene using hemagglutinating virus of Japan-liposome with >95% transfection efficiency. Four million of these myoblasts (VEGF group), control-transfected myoblasts (control group), or medium only (medium group) was injected into syngeneic rat hearts 1 hour after left coronary artery occlusion. Myocardial VEGF-expression increased for 2 weeks in the VEGF group, resulting in enhanced angiogenesis without the formation of tumors. Grafted myoblasts had differentiated into multinucleated myotubes within host myocardium. Infarct size (33.3+/-1.4%, 38.1+/-1.4%, and 43.7+/-1.6% for VEGF, control, and medium groups, respectively; P=0.0005) was significantly reduced with VEGF treatment, and cardiac function improved in the VEGF group (maximum dP/dt: 4072.0+/-93.6, 3772.5+/-101.1, and 3482.5+/-90.6 mm Hg/s in the 3 groups, respectively; P=0.0011; minimum dP/dt: -504.2+/-68.5, -2311.3+/-57.0, and -2124.0+/-57.9 mm Hg/s, respectively; P=0.0008). CONCLUSIONS: This combined strategy of cell transplantation with gene therapy could be of importance for the treatment of acute myocardial infarction.

Animals↗
PubMed2001

Overexpression of interleukin-1 receptor antagonist provides cardioprotection against ischemia-reperfusion injury associated with reduction in apoptosis.

BACKGROUND: Interleukin-1 (IL-1) plays a role in mediating acute inflammation during ischemia-reperfusion (I/R) injury in the heart, which leads to both necrosis and apoptosis of cardiomyocytes. IL-1 receptor antagonist (IL-1ra) is known to inhibit the effects of IL-1alpha and IL-1beta, resulting in attenuated inflammatory injury, and to protect cells from IL-1beta-induced apoptosis in vitro. We hypothesized that IL-1ra overexpression would provide cardioprotection by reducing inflammation-mediated myocardial damage including apoptosis after I/R injury in vivo. METHODS AND RESULTS: Rat hearts were transfected with human secreted-type IL-1ra gene by intracoronary infusion of Hemagglutinating Virus of Japan liposome and were heterotopically transplanted. IL-1ra overexpression in these hearts was confirmed by enzyme immunoassay and immunohistochemistry. Myocardial tolerance of the transplanted heart was evaluated with the use of a novel system in which the heart, existing within the recipient's abdomen, was given 30 minutes of ischemia by left coronary artery occlusion and 24 hours of reperfusion. Consequently, infarct size was decreased in IL-1ra-transfected hearts compared with control-transfected ones (26.9+/-3.2% versus 46.2+/-3.0%, P=0.001), corresponding to lower myocardial myeloperoxidase activity (2.20+/-0.69 versus 6.82+/-1.19 U/g wet wt, P<0.001) and decreased neutrophil infiltration in histological study. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and DNA-laddering studies demonstrated that cardiomyocyte apoptosis was attenuated in IL-1ra-transfected hearts (21.4+/-3.3 versus 41.4+/-3.4%, P=0.002), correlating with reduced post I/R upregulation of Bax, Bak, and caspase-3. CONCLUSIONS: IL-1ra introduced by gene transfection protected myocardium from I/R injury by attenuating the inflammatory response, which was associated with decreased apoptosis. This suggests a potentially important role of IL-1/IL-1ra in myocardial I/R injury and the value of IL-1ra-gene therapy for myocardial preservation.

Animals↗
PubMed2001

Intracoronary infusion of skeletal myoblasts improves cardiac function in doxorubicin-induced heart failure.

BACKGROUND: Skeletal myoblast transplantation is promising for the treatment of end-stage heart failure. Direct intramyocardial injection is useful for local cell delivery but may not be effective in global dissemination of cells into the heart, which would be advantageous in treating generalized cardiac dysfunction as in dilated cardiomyopathy. We hypothesized that intracoronary infusion of myoblasts would disseminate cells more effectively, leading to functional improvement in global heart failure. METHODS AND RESULTS: Heart failure was induced by the intraperitoneal administration of doxorubicin (total dose 15 mg/kg) in rat. One million primary skeletal myoblasts were then infused via the coronary arteries of an excised, failing doxorubicin-treated heart. After incubation under increased intracoronary pressure, the hearts were subsequently transplanted into syngeneic recipients. For the control group, doxorubicin-treated hearts were infused with medium only and transplanted. Four weeks after transplantation, Langendorff perfusion demonstrated that both maximum dP/dt (2797.6+/-103.3 versus 2326.9+/-133.1 mm Hg/s, P=0.01) and minimum dP/dt (-2067.4+/-88.1 versus -1718.8+/-91.3 mm Hg/s, P=0.02) were improved in myoblast-transplanted hearts compared with medium-infused hearts. This was associated with a sharper slope of the left ventricular developed pressure-volume curve and a reduced slope of the end-diastolic pressure-volume relation in the myoblast-transplanted hearts. Immunohistochemistry for skeletal myosin heavy chain showed that globally disseminated myoblasts had survived and differentiated into multinucleated myotubes that had aligned with the cardiac fiber axis within host myocardium. No significant myocardial infarction was observed. CONCLUSIONS: We demonstrated the feasibility and efficiency of skeletal myoblast transplantation via the intracoronary route as a promising strategy for improving cardiac function in global heart failure.

Animals↗
PubMed2001

Levels of vascular endothelial growth factor are elevated in patients with obstructive sleep apnea--hypopnea syndrome.

To better understand how humans adapt to hypoxia, the levels of hemoglobin (Hb), serum erythropoietin (Epo), and vascular endothelial growth factor (VEGF) were measured in 106 patients with severe obstructive sleep apnea-hypopnea syndrome. The results indicated that temporal hypoxic stimulation increases Hb. Furthermore, a minor increase in Epo and a substantial increase in VEGF were found. The induction in patients with severe sleep apnea was greater than that reported in other types of hypoxia. (Blood. 2001;98:1255-1257)

Case-Control Studies↗
PubMed2001

A novel secreted tumor antigen with a glycosylphosphatidylinositol-anchored structure ubiquitously expressed in human cancers.

In a search for novel genes expressed in human cancers, we identified one gene from an assembled expressed sequence tag database. Northern blot analysis revealed that the gene, termed alcan, was expressed in various types of human cancer cell lines and in the fetus, but not in normal tissues. The alcan gene is located on chromosome 6 and is encoded on a 246-amino-acid protein with weak homology to classical major histocompatibility complex class I. Its gene product, ALCAN, had hydrophobic amino acid clusters at both the N- and C-terminal regions and was predicted to be a glycosylphosphatidylinositol (GPI)-anchored membrane protein. Flow cytometric analysis revealed that ALCAN was detected on the surface of human cancer cells and on alcan-transfected CHO-K1 cells. ALCAN was also secreted from these cells, suggesting that some portion of the molecules was secreted by enzymatic cleavage by, for example, phospholipases. Mutational analysis of ALCAN suggested that the GPI-anchored position was the Ser(216) residue. These findings indicate that ALCAN may be a potential target for cancer diagnosis or therapy.

Amino Acid Sequence↗
PubMed2001

Variations in Prkdc encoding the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) and susceptibility to radiation-induced apoptosis and lymphomagenesis.

DNA double-strand breaks (DSBs) induced by ionizing radiation enforce cells to die, if unrepaired; while if misrepaired, DSBs may cause malignant transformation. The DSB repair system predominant in mammals requires DNA-dependent protein kinase (DNA-PK). Previously, we identified the apoptosis susceptibility gene Radiation-induced apoptosis 1 (Rapop1) on mouse chromosome 16. The STS/A (STS) allele at Rapop1 leads to decreased sensitivity to apoptosis in the BALB/cHeA (BALB/c) background. In the present study, we established Rapop1 congenic strains C.S-R1 and C.S-R1L, which contain the STS genome in a 0.45 cM interval critical for Rapop1 in common in the BALB/c background. Within the segment critical for Rapop1, Prkdc encoding the catalytic subunit of DNA-PK (DNA-PKcs) was assigned. Two variations T6,418C and G11,530A, which induce amino acid substitutions C2,140R downstream from the putative leucine zipper motif and V3,844M near the kinase domain, respectively, were found between BALB/c and STS for Prkdc. The majority of inbred strains such as C57BL/6J carried the STS allele at Prkdc; a few strains including 129/SvJ and C.B17 carried the BALB/c allele. DNA-PK activity as well as DNA-PKcs expression was profoundly diminished in BALB/c and 129/SvJ mice as compared with C57BL/6 and C.S-R1 mice. In the crosses (C.S-R1 x BALB/c)F(1) x 129/SvJ and (C.S-R1 x BALB/c)F(1) x C.B17, enhanced apoptosis occurred in the absence of the wild-type allele at Prkdc. C.S-R1 and C.S-R1L were both less sensitive to radiation lymphomagenesis than BALB/c. Our study provides strong evidence for Prkdc as a candidate for Rapop1 and a susceptibility gene for radiation lymphomagenesis as well.

Alleles↗
PubMed2001

Mechanism of lower genotoxicity of toremifene compared with tamoxifen.

An increased incidence of endometrial cancer has been reported in breast cancer patients taking tamoxifen (TAM) and in healthy women participating in the TAM chemoprevention trials. Because TAM-DNA adducts are mutagenic and detected in the endometrium of women treated with TAM, TAM adducts are suspected to initiate the development of endometrial cancer. Treatment with TAM has been known to promote hepatocarcinoma in rats, but toremifene (TOR), a chlorinated TAM analogue, did not. TAM adducts are primarily formed via sulfonation of the alpha-hydroxylated TAM metabolites. To explore the mechanism of the lower genotoxicity of TOR, the formation of DNA adducts induced by TOR metabolites was measured using (32)P-postlabeling/ high-performance liquid chromatography analysis and compared with that of TAM metabolites. When alpha-hydroxytoremifene was incubated with DNA, 3'-phosphoadenosine 5'-phosphosulfate, and either rat or human hydroxysteroid sulfotransferase, the formation of DNA adducts was two orders of magnitude lower than that of alpha-hydroxytamoxifen. alpha-hydroxytoremifene was a poor substrate for rat and human hydroxysteroid sulfotransferases. In addition, the reactivity of alpha-acetoxytoremifene, a model activated form of TOR, with DNA was much lower than that of alpha-acetoxytamoxifen. Thus, TOR is likely to have lower genotoxicity than TAM. TOR may be a safer alternative by avoiding the development of endometrial cancer.

Animals↗
PubMed2001

Origin and Co-localization of nitric oxide synthase, CGRP, PACAP, and VIP in the cerebral circulation of the rat.

The origin of perivascular nerve fibres storing nitric oxide synthase (NOS) and co-localisation with perivascular neuropeptides were examined in the rat middle cerebral artery (MCA) by retrograde tracing with True Blue (TB) in combination with immunocytochemistry. Application of TB to the proximal part of the middle cerebral artery labelled nerve cell bodies ipsilaterally in the trigeminal, sphenopalatine, otic, and superior cervical ganglia. A few labelled cell bodies were seen contralaterally, suggesting bilateral innervation. In the parasympathetic sphenopalatine and otic ganglia, numerous TB-labelled cell bodies contained neuronal NOS (C- and N-terminal), vasoactive intestinal peptide (VIP), and pituitary adenylate cyclase activating peptide (PACAP). In the trigeminal ganglion, almost all TB-labelled cell bodies contained calcitonin gene-related peptide (CGRP) but only a few cells contained NOS. In the superior cervical ganglion, the majority of the TB-labelled nerve cells contained neuropeptide Y (NPY) but none of them contained NOS. Removal of the ipsilateral sphenopalatine ganglion caused a slight reduction in the number of perivascular VIP-, PACAP-, and NOS-containing fibres after 3 days in the MCA while there was no difference at 2 and 4 weeks after the denervation as compared to control. This indicates that the parasympathetic VIP-, PACAP-, and NOS-immunoreactive nerve fibres in the rat MCA originate from several sources.

Animals↗
PubMed2001

Miscoding potential of the N2-ethyl-2'-deoxyguanosine DNA adduct by the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I.

Acetaldehyde, a major metabolite of ethanol, reacts with dG residues in DNA, resulting in the formation of the N(2)-ethyl-2'-deoxyguanosine (N(2)-Et-dG) adduct. This adduct has been detected in lymphocyte DNA of alcohol abusers. To explore the miscoding property of the N(2)-Et-dG DNA adduct, phosphoramidite chemical synthesis was used to prepare site-specifically modified oligodeoxynucleotides containing a single N(2)-Et-dG. These N(2)-Et-dG-modified oligodeoxynucleotides were used as templates for primer extension reactions catalyzed by the 3' --> 5' exonuclease-free (exo(-)) Klenow fragment of Escherichia coli DNA polymerase I. The primer extension was retarded one base prior to the N(2)-Et-dG lesion and opposite the lesion; however, when the enzyme was incubated for a longer time or with increased amounts of this enzyme, full extension occurred. Quantitative analysis of the fully extended products showed the preferential incorporation of dGMP and dCMP opposite the N(2)-Et-dG lesion, accompanied by a small amounts of dAMP and dTMP incorporation and one- and two-base deletions. Steady-state kinetic studies were also performed to determine the frequency of nucleotide insertion opposite the N(2)-Et-dG lesion and chain extension from the 3' terminus from the dN.N(2)-Et-dG (N is C, A, G, or T) pairs. These results indicate that the N(2)-Et-dG DNA adduct may generate G --> C transversions in living cells. Such a mutational spectrum has not been detected with other methylated dG adducts, including 8-methyl-2'-deoxyguanosine, O(6)-methyl-2'-deoxyguanosine, and N(2)-methyl-2'-deoxyguanosine. In addition, N(2)-ethyl-2'-deoxyguanosine triphosphate (N(2)-Et-dGTP) was efficiently incorporated opposite a template dC during DNA synthesis catalyzed by the exo(-) Klenow fragment. The utilization of N(2)-Et-dGTP was also determined by steady-state kinetic studies. N(2)-Et-dG DNA adducts are also formed by the incorporation of N(2)-Et-dGTP into DNA and may cause mutations, leading to the development of alcohol- and acetaldehyde-induced human cancers.

DNA Adducts↗
PubMed2001

Isolation and characterization of major glycoproteins of pigeon egg white: ubiquitous presence of unique N-glycans containing Galalpha1-4Gal.

Ovotransferrin (POT), two ovalbumins (POA(hi) and POA(lo)), and ovomucoid (POM) were isolated from pigeon egg white (PEW). Unlike their chicken egg white counterparts, PEW glycoproteins contain terminal Galalpha1-4Gal, as evidenced by GS-I lectin (specific for terminal alpha-Gal), anti-P(1) (Galalpha1-4Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glcbeta1-1Cer) monoclonal antibody, and P fimbriae on uropathogenic Escherichia coli (specific for Galalpha1-4Gal). Galalpha1-4Gal on PEW glycoproteins were found in N-glycans releasable by treatment with glycoamidase F. The respective contents of N-glycans in each glycoprotein were 3.5%, POT; 17%, POA(hi); and 31-37%, POM. POA(hi) has four N-glycosylation sites, in contrast to chicken ovalbumin, which has only one. High performance liquid chromatography analysis showed that N-glycans on POA(hi) were highly heterogeneous. Mass spectrometric analysis revealed that the major N-glycans were monosialylated tri-, tetra-, and penta-antennary oligosaccharides containing terminal Galalpha1-4Gal with or without bisecting N-acetylglucosamine. Oligosaccharide chains terminating in Galalpha1-4Gal are rare among N-glycans from the mammals and avians that have been studied, and our finding is the first predominant presence of (Galalpha1-4Gal)-terminated N-glycans.

Animals↗
PubMed2001

N-glycan structures from the major glycoproteins of pigeon egg white: predominance of terminal Galalpha(1)Gal.

N-Glycans from major glycoproteins of pigeon egg white (ovotransferrin, ovomucoid, and ovalbumins) were enzymatically released and were reductively aminated with 2-aminopyridine, separated, and structurally characterized by mass spectrometry and a three-dimensional mapping technique using three different columns of high performance liquid chromatography (HPLC) (Takahashi, N., Nakagawa, H., Fujikawa, K., Kawamura, Y., and Tomiya, N. (1995) Anal. Biochem. 226, 139-146). Twenty-five major N-glycan structures, all of them hitherto unknown, were identified as pyridylamino derivatives. Of these, 13 were neutral, 10 were monosialyl, and 2 were disialyl oligosaccharides. All N-glycans contain from one to four Galalpha(1,4)Galbeta(1,4) sequences at the nonreducing terminal positions and are devoid of fucose residues. N-Acetylneuraminic acids were alpha(2,6)-linked only to beta-galactose. The HPLC profiles of the N-glycans from four different glycoproteins were qualitatively very similar to each other, but not identical in the peak distributions. Monosialyl glycans were most abundant in all four glycoproteins, followed by neutral glycans. Disialyl glycans were lowest in ovotransferrin, and highest in ovomucoid. Triantennary structures with bisecting GlcNAc were predominant in ovotransferrin, and tetra-antennary (with and without bisecting GlcNAc-containing) structures were predominant in other glycoproteins. Penta-antennary structures (with a sialic acid and without bisecting GlcNAc residue) were also found in small quantities in all four glycoproteins. In contrast to the chicken egg white counterparts, which contain mostly high mannose and hybrid types, all N-glycan structures in the major pigeon egg white glycoproteins are complex type.

Aminopyridines↗