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Biomedical subjects

H Nakajima

Publications and source records attributed to H Nakajima.

At least 73 records · Page 4Linked to original sources
PubMed2001

Kinetic analysis of binding between Shiga toxin and receptor glycolipid Gb3Cer by surface plasmon resonance.

Shiga toxin (Stx) binds to the receptor glycolipid Gb3Cer on the cell surface and is responsible for hemolytic uremic syndrome. Stx has two isoforms, Stx1 and Stx2, and in clinical settings Stx2 is known to cause more severe symptoms, although the differences between the mechanisms of action of Stx1 and Stx2 are as yet unknown. In this study, the binding modes of these two isoforms to the receptor were investigated with a surface plasmon resonance analyzer to compare differences by real time receptor binding analysis. A sensor chip having a lipophilically modified dextran matrix or quasicrystalline hydrophobic layer was used to immobilize an amphipathic lipid layer that mimics the plasma membrane surface. Dose responsiveness was observed with both isoforms when either the toxin concentration or the Gb3Cer concentration was increased. In addition, this assay was shown to be specific, because neither Stx1 nor Stx2 bound to GM3, but both bound weakly to Gb4Cer. It was also shown that a number of fitting models can be used to analyze the sensorgrams obtained with different concentrations of the toxins, and the "bivalent analyte" model was found to best fit the interaction between Stxs and Gb3Cer. This shows that the interaction between Stxs and Gb3Cer in the lipid bilayer has a multivalent effect. The presence of cholesterol in the lipid bilayer significantly enhanced the binding of Stxs to Gb3Cer, although kinetics were unaffected. The association and dissociation rate constants of Stx1 were larger than those of Stx2: Stx2 binds to the receptor more slowly than Stx1 but, once bound, is difficult to dissociate. The data described herein clearly demonstrate differences between the binding properties of Stx1 and Stx2 and may facilitate understanding of the differences in clinical manifestations caused by these toxins.

Bacterial Toxins↗
PubMed2001

Endothelial nitric oxide synthase limits left ventricular remodeling after myocardial infarction in mice.

Background- To investigate the role of endothelial nitric oxide synthase (NOS3) in left ventricular (LV) remodeling after myocardial infarction (MI), the impact of left anterior descending coronary artery ligation on LV size and function was compared in 2- to 4-month-old wild-type (WT) and NOS3-deficient mice (NOS3(-/-)). Methods and Results- Two days after MI, both strains of mice had a similar LV size, fractional shortening, and ejection fraction by echocardiography. Twenty-eight days after MI, both strains had dilated LVs with decreased fractional shortening and lower ejection fractions. Although the infarcted fraction of the LV was similar in both strains, LV end-diastolic internal diameter, end-diastolic volume, and mass were greater, but fractional shortening, ejection fraction, and the maximum rate of developed LV pressure (dP/dt(max)) were lower in NOS3(-/-) than in WT mice. Impairment of diastolic function, as measured by the time constant of isovolumic relaxation (tau) and the maximum rate of LV pressure decay (dP/dt(min)), was more marked in NOS3(-/-) than in WT mice. Mortality after MI was greater in NOS3(-/-) than in WT mice. Long-term administration of hydralazine normalized blood pressure in NOS3(-/-) mice, but it did not prevent the LV dilatation, impaired systolic and diastolic function, and increased LV mass that followed MI. In WT mice, capillary density and myocyte width in the nonischemic portion of the LV did not differ before and 28 days after MI, whereas in NOS3(-/-) mice, capillary density decreased and myocyte width increased after MI, whether or not hydralazine was administered. Conclusions- These results suggest that the presence of NOS3 limits LV dysfunction and remodeling in a murine model of MI by an afterload-independent mechanism, in part by decreasing myocyte hypertrophy in the remote myocardium.

Animals↗
PubMed2001

Anatomical study of the venous drainage architecture of the scapular skin and subcutaneous tissue.

Venous anatomy of the skin and subcutaneous adipofascial tissue in the scapular region was examined in 14 specimens of 12 fresh cadavers that had been injected systemically with contrast medium. Three-dimensional analysis was performed by radiographing the specimens stereoscopically and splitting them into the skin and subcutaneous adipofascial tissue layers. From the architecture, most of the venous blood that had perfused the dermis was considered to pool in a polygonal venous network, located in the skin layer; to flow chiefly through some large communicating veins; and to enter the scapular, parascapular, or circumflex scapular veins. Most of the venous blood that had perfused the subcutaneous adipofascial tissue was considered to enter the scapular or parascapular veins directly.

Cadaver↗
PubMed2001

Role of CD4(+) CD25(+) regulatory T cells in T helper 2 cell-mediated allergic inflammation in the airways.

It has recently been shown that CD4(+) CD25(+) T cells are immunoregulatory T cells that prevent CD4(+) T cell-mediated organ-specific autoimmune diseases. To determine whether CD4(+) CD25(+) T cells downregulate Th2 cell-mediated allergic inflammation in the airways, we studied antigen-induced eosinophil recruitment in the airways in BALB/c Rag-2(-)(/-) mice transferred with CD4(+) CD25(+) T cell-depleted or unfractionated T cells from ovalbumin-specific TCR transgenic mice. Antigen-induced eosinophil recruitment into the airways was significantly decreased in the mice transferred with CD4(+) CD25(+) T cell-depleted splenocytes as compared with those transferred with unfractionated splenocytes. On the other hand, the depletion of CD4(+) CD25(+) T cells increased antigen-induced neutrophil and T cell recruitment in the airways of the mice. The depletion of CD4(+) CD25(+) T cells also decreased antigen-induced IL-4 and IL-5 production in the airways of the mice. Finally, the depletion of CD4(+) CD25(+) T cells prevented antigen-induced Th2 cell differentiation in vitro but increased the differentiation of Th1 cells. These results indicate that CD4(+) CD25(+) T cells modulate the Th1 and Th2 cell balance toward Th2 cells and thus upregulate Th2 cell-mediated allergic inflammation in the airways.

Animals↗
PubMed2001

Granulocyte colony-stimulating factor regulates myeloid differentiation through CCAAT/enhancer-binding protein epsilon.

Granulocyte colony-stimulating factor (G-CSF) is a major cytokine that regulates proliferation and differentiation of myeloid cells, although the underlying mechanisms by which G-CSF controls myeloid differentiation are largely unknown. Differentiation of hematopoietic cells is regulated by lineage-specific transcription factors, and gene-targeting studies previously revealed the critical roles of CCAAT/enhancer-binding protein (C/EBP) alpha and C/EBP epsilon, respectively, in the early and mid-late stages of granulocyte differentiation. The expression of C/EBP epsilon in 32Dcl3 cells and FDCP1 cells expressing mutant G-CSF receptors was examined and it was found that G-CSF up-regulates C/EBP epsilon. The signal for this expression required the region containing the first tyrosine residue of G-CSF receptor. Dominant-negative signal transducers and activators of transcription 3 blocked G-CSF--induced granulocytic differentiation in 32D cells but did not block induction of C/EBP epsilon, indicating that these proteins work in different pathways. It was also found that overexpression of C/EBP epsilon greatly facilitated granulocytic differentiation by G-CSF and, surprisingly, that expression of C/EBP epsilon alone was sufficient to make cells differentiate into morphologically and functionally mature granulocytes. Overexpression of c-myc inhibits differentiation of hematopoietic cells, but the molecular mechanisms of this inhibition are not fully understood. In 32Dcl3 cells overexpressing c-myc that do not differentiate by means of G-CSF, induction of C/EBP epsilon is completely abrogated. Ectopic expression of C/EBP epsilon in these cells induced features of differentiation, including changes in nuclear morphologic characteristics and the appearance of granules. These data show that C/EBP epsilon constitutes a rate-limiting step in G-CSF-regulated granulocyte differentiation and that c-myc antagonizes G-CSF-induced myeloid differentiation, at least partly by suppressing induction of C/EBP epsilon. (Blood. 2001;98:897-905)

Animals↗
PubMed2001

Lecithinized Cu, Zn-superoxide dismutase limits the infarct size following ischemia-reperfusion injury in rat hearts in vivo.

Covalent binding of 4 molecules of phosphatidylcholine palmitoyl to human recombinant superoxide dismutase (SOD) results in a compound (lecithinized SOD) that has a longer half-life and greater affinity to the cell membrane than unmodified SOD. We investigated whether lecithinized SOD played a protective role against myocardial ischemia-reperfusion injuries in rats. Rats underwent 45 min of myocardial ischemia by occluding the left coronary artery followed by 120 min of reperfusion. They were randomly assigned to receive either lecithinized SOD, polyethylene glycol conjugated SOD (PEG-SOD), unmodified SOD, free lecithin derivative, or PBS intravenously at 5 min prior to reperfusion. Myocardial infarct area assessed by TTC staining was smaller in lecithinized SOD group than PEG-SOD, unmodified SOD, free lecithin derivative or control group. Blood pressure and heart rate was similar in each group. ELISA demonstrated SOD level in the heart was significantly high in lecithinized SOD group, especially in the heart of ischemia at risk. Although serum SOD level of PEG-SOD was as high as lecithinized SOD, SOD level of the heart was low. These data suggested lecithinized SOD had a protective effect in myocardial ischemia-reperfusion injuries through its increased bioavailability.

Animals↗
PubMed2001

Ligand-switching intermediates for the CO-sensing transcriptional activator CooA measured by pulse radiolysis.

CooA is a heme-containing and CO-sensing transcriptional activator whose activity is regulated by CO. The protoheme that acts as a CO sensor in CooA shows unique properties for its coordination structure. The Cys75 axial ligand of the ferric heme is replaced by His77 upon the reduction of the heme iron and vice versa. In this work, the ligand-switching process induced by the reduction of the heme was investigated by the technique of pulse radiolysis. Hydrated electron reduced the heme iron in ferric CooA within 1 micros to form the first intermediate with the Soret peak at 440 nm, suggesting that a six-coordinate ferrous heme with a thiolate axial ligand was formed initially. The first intermediate was converted into the second intermediate with the time constant of 40 micros (k = 2.5 x 10(4) x s(-1)). In the second intermediate, the thiolate from Cys75 was thought to be protonated and/or the Fe-S bond was thought to be elongated. The second intermediate was converted into the final reduced form with the time constant of 2.9 ms (k = 3.5 x 10(2) x s(-1)) for wild-type CooA. The ligand exchange between Cys75 and His77 took place during the conversion of the second intermediate into the final reduced form.

Bacterial Proteins↗
PubMed2001

Lecithinized copper, zinc-superoxide dismutase ameliorates ischemia-induced myocardial damage.

We have reported that lecithin-conjugated recombinant human Cu, Zn-superoxide dismutase (lecithinized SOD) has greater pharmacological potency than unmodified SOD through an increase in cell membrane affinity and half-life in plasma. Recently, ischemia or hypoxia alone has been suggested to result in increased superoxide anions, which lead to apoptosis in cardiomyocytes. We tested the effect of lecithinized SOD in reducing the infarct size following prolonged myocardial ischemia without reperfusion. Rats were subjected to a 24-h left coronary occlusion. Lecithinized SOD, unmodified SOD, free lecithin derivative or PBS was administered intravenously 30 min before coronary occlusion. SOD concentration of the heart, measured by ELISA, was higher in the lecithinized SOD-treated group than in the other groups 24 h after administration. The infarct area ratio of the heart, assessed by TTC staining, in the lecithinized SOD-treated group was significantly smaller than those of the other groups. Both TUNEL-positive cardiomyocytes and DNA laddering were attenuated in the ischemic area of the heart treated with lecithinized SOD. Single bolus administration of lecithinized SOD had a cardioprotective effect against ischemia without reperfusion in the rat model of acute myocardial infarction, possibly due to its sustained high tissue concentration.

Animals↗
PubMed2001

Estrogen protects neuronal cells from amyloid beta-induced apoptotic cell death.

Accumulating studies have shown that estrogen replacement therapy reduces the risk of Alzheimer's disease. In this study, we clarified that 17beta-estradiol (E2) significantly rescues PC12 neuronal cells from amyloid beta protein (Abeta)-induced cell death. We found that the amino acid residues of 25 to 35 (Abeta25-35) were more cytotoxic than the full length protein (Abeta1-40) and these residues induced DNA fragmentation typical for apopto- sis. In addition, E2 was confirmed to inhibit calcium influx and cytochrome c release induced by Abeta25-35. Since these sequential events cause apoptosis, the protective effect of E2 may be exerted not by the direct interaction with Abeta, but by the blockade of the mitochondrial apoptotic pathway induced by Abeta.

Alzheimer Disease↗
PubMed2001

Winter seasonality and rotavirus diarrhoea in adults.

We investigated the aetiological role of group A rotavirus in adults with acute diarrhoea in a 4-year prospective study. Of 683 patients with acute diarrhoea, 97 (14%) shed rotavirus as a sole agent, whereas six (5%) of 115 patients without diarrhoea shed rotavirus. Half of patients with rotavirus diarrhoea required admission to hospital. Unlike rotavirus diarrhoea in children, the occurrence of rotavirus-positive cases did not show a significant winter seasonality. Rotavirus infection should be included in the differential diagnosis of diarrhoeal diseases in adults.

Adult↗
PubMed2001

The distal region and receptor tyrosines of the Epo receptor are non-essential for in vivo erythropoiesis.

The erythropoietin receptor (EpoR) is required for the proliferation and survival of committed erythroid lineage cells. Previous studies have utilized receptor mutations to show the requirement for the distal half of the cytoplasmic domain of the EpoR and receptor tyrosines for activation of signaling pathways potentially critical to Epo function. To extend these studies to in vivo erythropoiesis, we have created two mutant strains of mice. One strain (H) contains a truncation of the distal half of the cytoplasmic domain, while the second strain (HM) contains the same truncation as well as the mutation of the residual tyrosine (Y(343)) to a phenylalanine. Strikingly, both strains of mice are viable, with only slight alterations in constitutive erythropoiesis or in in vitro assays of red cell lineage function. Challenging H mutant mice with continuous injections of Epo results in an erythrocytosis that is not seen in HM mice. The results demonstrate that neither the distal region nor receptor tyrosines are essential for in vivo EpoR function, but contribute to receptor function in a subtle manner.

Animals↗
PubMed2001

Cloning and characterization of Aspergillus nidulans vpsA gene which is involved in vacuolar biogenesis.

In Saccharomyces cerevisiae, vacuoles play very important roles in pH and osmotic regulation, protein degradation and storage of amino acids, small ions as well as polyphosphates. In filamentous fungi, however, little is known about vacuolar functions at a molecular level. In this paper, we report the isolation of the vpsA gene from the filamentous fungus Aspergillus nidulans as a homologue of the VPS1 gene of S. cerevisiae which encodes a dynamin-related protein. The vpsA gene encodes a polypeptide consisting of 696 amino acids that is nearly 60% homologous to the S. cerevisiae Vps1. Similar to Vps1, VpsA contains a highly conserved tripartite GTPase domain but lacks the pleckstrin homology domain and proline-rich region. The vpsA disruptant shows poor growth and contains highly fragmented vacuoles. These results suggest that A. nidulans VpsA functions in the vacuolar biogenesis.

Amino Acid Sequence↗
PubMed2001

Stat5a regulates T helper cell differentiation by several distinct mechanisms.

We have previously shown that CD4(+) T cell-mediated allergic inflammation is diminished in signal transducer and activator of transcription (Stat)5a-deficient (Stat5a(-/-)) mice. To determine whether Stat5a regulates T helper cell differentiation, we studied T helper (Th)1 and Th2 cell differentiation of Stat5a(-/-)CD4(+) T cells at single-cell levels. First, Th2 cell differentiation from antigen-stimulated splenocytes was significantly decreased in Stat5a(-/-) mice as compared with that in wild-type mice. Further, Th2 cell differentiation was also impaired in Stat5a(-/-) mice even when purified CD4(+) T cells were stimulated with anti-CD3 plus anti-CD28 antibodies in the presence of interleukin-4. Moreover, the retrovirus-mediated gene expression of Stat5a in Stat5a(-/-)CD4(+) T cells restored the Th2 cell differentiation at the similar levels to that in wild-type CD4(+) T cells. In addition, interleukin-4 normally phosphorylated Stat6 in CD4(+) T cells from Stat5a(-/-) mice. Second, the development of CD4(+)CD25(+) immunoregulatory T cells was impaired in Stat5a(-/-) mice, as indicated by a significant decrease in the number of CD4(+)CD25(+) T cells in Stat5a(-/-) mice. Furthermore, the depletion of CD4(+)CD25(+) T cells from wild-type splenocytes significantly decreased Th2 cell differentiation but increased Th1 cell differentiation, whereas the depletion of CD4(+)CD25(+) T cells from Stat5a(-/-) splenocytes had no significant effect on the Th1 and Th2 cell differentiation. Together, these results indicate that the intrinsic expression of Stat5a in CD4(+) T cells is required for Th2 cell differentiation and that Stat5a is involved in the development of CD4(+)CD25(+) immunoregulatory T cells that modulate T helper cell differentiation toward Th2 cells.

Animals↗
PubMed2001

Binding of CO at the Pro2 side is crucial for the activation of CO-sensing transcriptional activator CooA. (1)H NMR spectroscopic studies.

CooA is a heme-containing transcriptional activator that anaerobically binds to DNA at CO atmosphere. To obtain information on the conformational transition of CooA induced by CO binding to the heme, we assigned ring current-shifted (1)H NMR signals of CooA using two mutants whose axial ligands of the heme were replaced. In the absence of CO, the NMR spectral pattern of H77Y CooA, in which the axial histidine (His(77)) was replaced with tyrosine, was similar to that of wild-type CooA. In contrast, the spectra of CooADeltaN5, in which the NH(2) termini including the other axial ligand (Pro(2)) were deleted, were drastically modulated. We assigned three signals of wild-type CooA at -4.5, -3.6, and -2.8 ppm to delta(1)-, alpha-, and delta(2)-protons of Pro(2), respectively. The Pro(2) signals were undetectable in the upfield region of the spectrum of the CO-bound state, which confirms that CO displaces Pro(2). Interestingly, the Pro(2) signals were observed for CO-bound H77Y CooA, implying that CO binds to the trans position of Pro(2) in H77Y CooA. The abolished CO-dependent transcriptional activity of H77Y CooA is therefore the consequence of Pro(2) ligation. These observations are consistent with the view that the movement of the NH(2) terminus triggers the conformational transition to the DNA binding form.

Bacterial Proteins↗
PubMed2001

Activation of the caspase cascade during Stx1-induced apoptosis in Burkitt's lymphoma cells.

Shiga toxin 1 (Stx1) produced by Escherichia coli has been reported to induce apoptosis in many different cell types, including Burkitt's lymphoma (BL) cells. Since it has been established that the caspases play essential roles as the effector molecules in the apoptotic process in most cases, we examined the kinetics of caspase activation during the process of Stx1-mediated apoptosis of BL cells. Using Ramos BL cells that are highly sensitive to Stx1-mediated cytotoxicity, we observed that multiple caspases, including caspase-3, -7, and -8 were promptly activated following Stx1 treatment, as indicated by both the procaspase cleavages and enhancement of cleavage of the tetrapeptide substrates of the caspases. In addition, the inhibition assay revealed that caspase-8 is located upstream of both caspase-3 and -7, suggesting that Stx1-mediated apoptosis utilizes a similar caspase cascade to that involved in Fas-mediated apoptosis. Neither anti-Fas mAb nor TNF-alpha, however, affected the Stx1-mediated apoptosis of Ramos cells. Although the precise mechanism of Stx1-mediated activation of caspase-8 is still unclear, we have demonstrated that crosslinkage of CD77, a functional receptor for Stx1, with specific antibody is sufficient to induce activation of caspase-8. Our findings should provide new insight into the understanding of the molecular basis of Stx1-mediated cell injury.

Antibodies, Monoclonal↗
PubMed2001

Interaction between Smad anchor for receptor activation and Smad3 is not essential for TGF-beta/Smad3-mediated signaling.

Regulation of subcellular localization of Smad proteins is supposed to be critical for the effective initiation and maintenance of TGF-beta signaling. Recently, Smad anchor for receptor activation (SARA) has been identified as a Smad2 binding protein. SARA regulates the subcellular localization of Smad2 and is required for TGF-beta/Smad2-mediated signaling. In this study, we determined whether the interaction between SARA and Smad3 is essential for TGF-beta/Smad3-mediated signaling. We found that a mutant Smad3 (Smad3NS) that lacked the binding to SARA was phosphorylated by TGF-beta type I receptor at the similar level to that in wild-type Smad3 (Smad3WT). Smad3NS also formed complexes with Smad4 and translocalized into the nucleus. Moreover, Smad3NS and Smad3WT equally enhanced TGF-beta-induced transcription. Therefore, these findings indicate that, in contrast to SARA/Smad2 interaction, SARA/Smad3 interaction is not essential for TGF-beta/Smad3-mediated signaling.

Active Transport, Cell Nucleus↗
PubMed2001

Single-step method for purification of Shiga toxin-1 B subunit using receptor-mediated affinity chromatography by globotriaosylceramide-conjugated octyl sepharose CL-4B.

A new single-step purification method for Shiga toxin (Stx) was developed using receptor-mediated affinity chromatography, in which Gb3Cer (globotriaosylceramide) was conjugated to octyl Sepharose CL-4B as a carrier. This method achieves high yield and high purity in a small column on which Gb3Cer has been immobilized at high density. Using this affinity column, the Stx1 B subunit was purified with homogeneity by a one-step procedure from a crude extract of recombinant Stx1 B subunit-producing Escherichia coli. The purified Stx1 B subunit conserved a natural pentamer structure confirmed by gel filtration and sedimentation equilibrium analysis. Furthermore, the purified Stx1 B subunit was able to bind specifically to Gb3Cer expressed on Burkitt's lymphoma cells. This versatile purification method can be used to isolate various types of natural as well as recombinant Stx, facilitating fundamental studies of human diseases caused by this toxin.

Blotting, Western↗