[Show abstract][Hide abstract] ABSTRACT: Receptor usage by viral interleukin-6 (vIL-6), a virokine encoded by Kaposi sarcoma- associated herpesvirus, is an issue of controversy. Recently, the crystal structure of vIL-6 identified vIL-6 sites II and III as directly binding to glycoprotein (gp)130, the common signal transducer for the IL-6 family of cytokines. Site I of vIL-6, however, comprising the outward helical face of vIL-6, where human IL-6 (hIL-6) would interact with the specific alpha-chain IL-6 receptor (IL-6R), is accessible and not occupied by gp130. This study examined whether this unused vIL-6 surface is available for IL-6R binding. By enzyme-linked immunosorbent assay, vIL-6 bound to soluble gp130 (sgp130) but not to soluble IL-6R (sIL-6R). Using plasmon surface resonance, vIL-6 bound to sgp130 with a dissociation constant of 2.5 microM, corresponding to 1000-fold lower affinity than that of hIL-6/sIL-6R complex for gp130. sIL-6R neither bound to vIL-6 nor affected vIL-6 binding to gp130. In bioassays, vIL-6 activity was neutralized by 4 monoclonal antibodies (mAbs) recognizing a domain within vIL-6 site I, mapped to the C-terminal part of the AB-loop and the beginning of helix B. The homologous region in hIL-6 participates in site I binding to IL-6R. In addition, binding of vIL-6 to sgp130 was interfered with specifically by the 4 neutralizing anti-vIL-6 mAbs. Based on the vIL-6 crystal structure, the vIL-6 neutralizing mAbs map outside the binding interface to gp130, suggesting that they either produce allosteric changes or block necessary conformational changes in vIL-6 preceding its binding to gp130. These results document that vIL-6 does not bind IL-6R and suggest that conformational change may be critical to vIL-6 function.
[Show abstract][Hide abstract] ABSTRACT: In this study, we examined the effects of nonimmunological factors on cadaver kidney transplant.
Fifty-three cadaver kidney transplant recipients were studied. They were divided into two groups, Group I ( donor age<60, n=38 ) and Group II ( donor age > or = 60, n=15). These patients were studied to determine whether donor age and recipient Body Mass Index (BMI) affected transplant outcome.
The 3 and 5-year overall graft / patient survival was 80.6 / 100% and 72.7 97.3%, respectively. The best S-Cr after transplantation was significantly (p<0.05) lower in Group I compared to Group II. The 1, 5 and 8-year graft survival was significantly (p<0.05) better in Group I. In the low BMI patients of Group II, 5-year graft survival was significantly (p<0.01) better than high BMI patients.
An aged cadaver donor was a risk factor for decreased posttransplant renal functions and lower graft survival. However, if a recipient with a low BMI can be selected, the outcome of cadaver kidney transplants from aged donors may be improved.
[Show abstract][Hide abstract] ABSTRACT: We studied the voluntary contractile activity of the ischiocavernosus muscle (ICM) in 21 sexually potent and 97 erectile dysfunction (ED) subjects using a spring balance. A strap was placed around the coronal grove of the glans penis and tensioned with the spring balance. Subjects were asked and encouraged to contract the ICM against the spring balance. We evaluated the length of stroke, duration of contraction, and maximum contractile force. The length of stroke, duration of contraction, and maximum contractile force showed statistically significant differences between potent and ED subjects. Diagnosed psychogenic ED and arteriogenic ED showed higher contractile activity than cavernous ED and neurogenic ED. Our results corresponded to those of previous studies that have urged consideration of the role of the ICM during the process of erection in animal experiments and in human electrophysiological studies. The spring balance evaluation is a useful, inexpensive method for evaluating the ICM.
International Journal of Impotence Research 11/2001; 13(5):294-7. · 1.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Xenopus telomerase catalytic component gene, xTERT (Xenopus telomerase reverse transcriptase), has been cloned. The production of xTERT recombinant protein together with the proposed Xenopus telomerase RNA (xTR) (Chen et al., 2000. Cell 100, 503-514) in a rabbit reticulocyte lysate system led to the reconstitution of active telomerase, indicating that both products are functional telomerase components. Both xTERT expression and telomerase activity are high from the early to the late blastula stage. However, they are decreased at the gastrula stage and thereafter, suggesting that the xTERT expression level is the primary mechanism for regulating telomerase activity in Xenopus development. This is the first report of a non-mammalian vertebrate TERT gene. Sequence comparison of xTERT with human and mouse TERTs has uncovered four regions conserved in the amino-terminal halves of vertebrate TERT proteins, the functions of which will be discussed herein.
[Show abstract][Hide abstract] ABSTRACT: Activation of phospholipase C-gamma2 (PLCgamma2) is the critical step in B cell antigen receptor (BCR)-coupled calcium signaling. Although genetic dissection experiments on B cells have demonstrated that Bruton's tyrosine kinase (Btk) and Syk are required for activating PLCgamma2, the exact activation mechanism of PLCgamma2 by these kinases has not been established. We identify the tyrosine residues 753, 759, 1197, and 1217 in rat PLCgamma2 as Btk-dependent phosphorylation sites by using an in vitro kinase assay. To evaluate the role of these tyrosine residues in phosphorylation-dependent activation of PLCgamma2, PLCgamma2-deficient DT40 cells were reconstituted with a series of mutant PLCgamma2s in which the phenylalanine was substituted for tyrosine. Substitution of all four tyrosine residues almost completely eliminated the BCR-induced PLCgamma2 phosphorylation, indicating that these residues include the major phosphorylation sites upon BCR engagement. Cells expressing PLCgamma2 with a single substitution exhibited some extent of reduction in calcium mobilization, whereas those expressing quadruple mutant PLCgamma2 showed greatly reduced calcium response. These findings indicate that the phosphorylations of the tyrosine residues 753, 759, 1197, and 1217, which have been identified as Btk-dependent phosphorylation sites in vitro, coordinately contribute to BCR-induced activation of PLCgamma2.
Journal of Biological Chemistry 11/2001; 276(42):38595-601. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cellular responsiveness to human interleukin 6 (hIL6) requires the expression of two receptor molecules: IL6-specific receptor (CD126'IL6R') and a nonspecific signal-transducing molecule (CD130'gp130'). Regulation of responsiveness to hIL6 is generally controlled by CD126'IL6R' expression. A viral homologue of hIL6 (vIL6) is encoded by human herpesvirus-8 and has biologic activity similar to hIL6 on a number of cell lines. vIL6 differs from hIL6 in its receptor utilization, requiring only CD130'gp130'. Total human B cells isolated from peripheral blood, which are predominantly CD126'IL6R'-negative, as well as sorted CD126'IL6R'-negative B cells, could be stimulated by recombinant vIL6, but not by hIL6, as indicated by induction of IL6-like signaling (STAT3 phosphorylation). This suggests that the ability of vIL6 to stimulate B cells expressing little or no CD126'IL6R' allows it to act on a larger pool of target B cells, compared to human IL6.
[Show abstract][Hide abstract] ABSTRACT: Inflammatory cytokines such as interleukin 1 (IL-1), IL-6, and tumor necrosis factor-alpha are produced in great quantities in inflamed rheumatoid joints. However, little is known about the pathogenic significance of each cytokine in the proliferative synovitis and destruction of bone and joint. We investigated the role of cytokine receptor signals transduced into cells at the foci of rheumatoid inflammation.
Synovial fluid (SF) cells from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) were examined for the activation of a group of cytokine receptor signaling molecules, signal transducers and activators of transcription (STAT).
DNA binding of STAT1 in SF cells was observed in 8 out of 14 patients with RA, but in none of the 10 patients with OA studied, and this was prevented by preincubation of these cells with neutralizing anti-IL-6 antibody. IL-6 activated both STAT1 and STAT3 in normal peripheral blood (PB) leukocytes, and preferentially STAT1 in rheumatoid SF cells. Moreover, STAT1 activation in rheumatoid SF cells appeared to be continuous, in contrast to the transient activation in normal PB leukocytes.
STAT1 and STAT3 are differentially regulated in response to IL-6 in different cell types. The continuous STAT1 activation may be of pathogenic significance in the progression and persistence of RA.
The Journal of Rheumatology 10/2001; 28(9):1952-9. · 3.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Checkpoint controls ensure chromosomal integrity through the cell cycle. Chk1 and Cds1/Chk2 are effector kinases in the G(2)-phase checkpoint activated by damaged or unreplicated DNA, and they prevent entry into M-phase through inhibition of cyclin B-Cdc2 kinase activation. However, little is known about how the effector kinases are regulated when the checkpoint is attenuated. Recent studies indicate that Chk1 is also involved in the physiological G(2)-phase arrest of immature Xenopus oocytes via direct phosphorylation and inhibition of Cdc25C, the activator of cyclin B-Cdc2 kinase. Bearing in mind the overlapping functions of Chk1 and Cds1, here we have studied the involvement of Xenopus Cds1 (XCds1) in the G(2)/M-phase transition of immature oocytes and the regulation of its activity during this period. Protein levels of XCds1 remained constant throughout oocyte maturation and early embryonic development. The levels of XCds1 kinase activity were high in immature oocytes and decreased at the meiotic G(2)/M-phase transition. Consistently, when overexpressed in immature oocytes, wild-type, but not kinase-deficient, XCds1 significantly delayed entry into M-phase after progesterone treatment. The inactivation of XCds1 depended on the activation of cyclin B-Cdc2 kinase, but not MAP kinase. Although XCds1 was not directly inactivated by cyclin B-Cdc2 kinase in vitro, XCds1 was inactivated by overexpression of cyclin B, which induces the activation of cyclin B-Cdc2 kinase without progesterone. Thus, the present study is the first indication of Cds1 activity in cells that are physiologically arrested at G(2)-phase, and of its downregulation at entry into M-phase.
[Show abstract][Hide abstract] ABSTRACT: Mice with cardiac-specific overexpression of signal transducer and activator of transcription 3 (STAT3) are resistant to doxorubicin-induced damage. The STAT3 signal may be involved in the detoxification of reactive oxygen species (ROS).
The effects of leukemia inhibitory factor (LIF) or adenovirus-mediated transfection of constitutively activated STAT3 (caSTAT3) on the intracellular ROS formation induced by hypoxia/reoxygenation (H/R) were examined using rat neonatal cardiomyocytes. Either LIF treatment or caSTAT3 significantly suppressed the increase of H/R-induced ROS evaluated by 2',7'-dichlorofluorescin diacetate fluorescence. To assess whether ROS are really involved in H/R-induced cardiomyocyte injury, the amount of creatine phosphokinase in cultured medium was examined. Both LIF treatment and caSTAT3 significantly decreased H/R-induced creatine phosphokinase release. These results indicate that the gp130/STAT3 signal protects H/R-induced cardiomyocyte injury by scavenging ROS generation. To investigate the mechanism of scavenging ROS, the effects of LIF on the induction of antioxidant enzymes were examined. LIF treatment significantly increased the expression of manganese superoxide dismutase (MnSOD) mRNA, whereas the expression of the catalase and glutathione peroxidase genes were unaffected. This induction of MnSOD mRNA expression was completely blocked by adenovirus-mediated transfection of dominant-negative STAT3. Moreover, caSTAT3 augmented MnSOD mRNA and its enzyme activity. In addition, the antisense oligodeoxyribonucleotide to MnSOD significantly inhibited both LIF and caSTAT3-mediated protective effects.
The activation of STAT3 induces a protective effect on H/R-induced cardiomyocyte damage, mainly by inducting MnSOD. The STAT3-mediated signal is proposed as a therapeutical target of ROS-induced cardiomyocyte injury.
[Show abstract][Hide abstract] ABSTRACT: Environmental factors essential for the first stages of B lymphopoiesis remain elusive. Here, we report that immediately after commitment to B lineage, precursors become dependent on a chemokine SDF-1 and its receptor CXCR4 using mutant and radiation chimeric mice. In bone marrow, generation of the earliest identifiable B cell precursor populations requires CXCR4. In fetal liver, we identified Lin(-)CD19(-)c-kit(+)IL-7Ralpha(+)AA4.1(+), the earliest unipotent B cell precursor population, and found that its development was severely affected in SDF-1(-/-) embryos but not in IL-7(-/-) embryos. Lin(-) T cell progenitors appeared normal in SDF-1(-/-) embryos. Moreover, SDF-1 exhibited specific biologic activities on the earliest B cell precursors. SDF-1 provides the first example of a cytokine responsible for the earliest B lineage stages.
[Show abstract][Hide abstract] ABSTRACT: Bone morphogenetic protein (BMP)-2 has been shown to induce ectopic expression of cardiac transcription factors and beating cardiomyocytes in non-precardiac mesodermal cells, suggesting that BMP-2 is an inductive signaling molecule that participates in cardiac development. However, direct evidence of the effects of BMP-2 on cardiac myocytes has not been reported. To examine the role of BMP-2 and its receptors, we studied the ability of BMP-2 to promote survival of isolated neonatal rat cardiac myocytes. BMP receptors IA, IB, and II and activin receptor I were found to be expressed in myocytes, and BMP-2 phosphorylated Smad1 and p38 MAPK. Interestingly, BMP-2 promoted survival and inhibited apoptosis of serum-deprived myocytes, although it did not strongly induce hypertrophic growth. To explore the mechanisms for this protective effect, an adenovirus-based vector system was used. Similar to BMP-2, Smad1 promoted survival that was repressed by Smad6. Moreover, BMP-2 and Smad1 enhanced the expression of the anti-apoptotic molecule Bcl-x(L). Antisense oligonucleotides to bcl-x(L) attenuated the survival effected by BMP-2. Overall, our findings suggest that BMP-2 prevents apoptosis of myocytes by induction of Bcl-x(L) via a Smad1 pathway and might be a novel survival factor without any hypertrophic effect on myocytes.
Journal of Biological Chemistry 09/2001; 276(33):31133-41. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Research indicates that aged heart-beating cadaveric donors cause greater risk factors in kidney transplantation. The influence of age on the outcome of non-heart-beating (NHB) cadaveric renal transplantations has not yet been clarified. From July 1986 to May 1999, 63 patients who received cadaveric renal transplantation at Osaka City University Hospital and Osaka City General Hospital were divided into two groups according to their age. Renal function and graft-survival rates of the two groups were compared. The mean values of nadir donor serum creatinine were significantly worse (P < 0.05) in the aged donor group. In the aged donor group the percentage of immediately functioning grafts was lower and the percentage of non-functioning grafts was higher. During the first 10 years post-transplant, graft survival in the aged donor group was significantly lower than that in the younger donor group. We conclude that cadaveric renal transplantation from NHB aged donors can be to the detriment of renal function and graft survival rates compared to transplantation from younger donors.
International Journal of Urology 08/2001; 8(8):S68-70. · 1.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cernitin pollen-extract (Cernilton, CN) is a preparation made from eight kinds of pollen and has been used for various prostatic diseases in Japan and Europe. We reported previously that CN possessed a recovery action on the sex-hormone-induced nonbacterial prostatitis in rats. To clarify the possible mechanism of action of CN, we investigated the effects of CN on inflammatory cytokines (IL-1 beta, IL-6 and TNF-alpha) in the same model. Aged Wistar rats were castrated and injected 17 beta-estradiol (0.25 mg/kg/day, s.c.) for 30 days. CN (630 and 1,260 mg/kg, p.o.) or testosterone (2.5 mg/kg, s.c.) was administered for the last 14 days of the treatment of 17 beta-estradiol. In control rats, prostatic IL-6 and TNF-alpha contents were increased approximately 2-3 fold, and acinar glandular inflammation and stromal proliferation were found histopathologically, as compared with those of intact rats. On the other hand, CN decreased the increased contents of cytokines in a dose-dependent manner. The histopathological changes mentioned above were restored in rats treated with 1,260 mg/kg. Testosterone also ameliorated them significantly. These results indicate that CN has an anti-inflammatory action, and that the inhibitory effect of CN on the prostatic inflammatory cytokine is an important factor in its action.
[Show abstract][Hide abstract] ABSTRACT: We reported previously that neurofilaments (NFs) of aged rats were highly packed in the axon and contained a smaller amount of NF-M as compared with those of young rats (Uchida et al.  J. Neurosci. Res. 58:337-348). We studied NFs of the mutant mouse, named Klotho, which displays prematurely symptoms resembling human aging. The transport of axonal cytoskeletal proteins, including NFs, tubulin and actin, was decreased at the leading portion of the peak of transported proteins in Klotho during the process of premature aging. The nearest neighbor inter-NF distance in Klotho axons (35-39 nm) was shorter than that of the wild-type mouse (48-49 nm), indicating the packing of NFs in Klotho. The ratio of NF-M to NF-L was slightly decreased in cytoskeletons from the spinal cords of Klotho. These changes are similar, though not identical, to those observed in aged rats, and are the first evidence of age-related changes in the neurons of Klotho.
Journal of Neuroscience Research 06/2001; 64(4):364-70. · 2.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We show that when telencephalic neural progenitors are briefly exposed to bone morphogenetic protein 2 (BMP2) in culture, their developmental fate is changed from neuronal cells to astrocytic cells. BMP2 significantly reduced the number of cells expressing microtubule-associated protein 2, a neuronal marker, and cells expressing nestin, a marker for undifferentiated neural precursors, but BMP2 increased the number of cells expressing S100-beta, an astrocytic marker. In telencephalic neuroepithelial cells, BMP2 up-regulated the expression of negative helix-loop-helix (HLH) factors Id1, Id3, and Hes-5 (where Hes is homologue of hairy and Enhancer of Split) that inhibited the transcriptional activity of neurogenic HLH transcription factors Mash1 and neurogenin. Ectopic expression of either Id1 or Id3 (where Id is inhibitor of differentiation) inhibited neurogenesis of neuroepithelial cells, suggesting an important role for these HLH proteins in the BMP2-mediated changes in the neurogenic fate of these cells. Because gliogenesis in the brain and spinal cord, derived from implanted neural stem cells or induced by injury, is responsible for much of the failure of neuronal regeneration, this work may lead to a therapeutic strategy to minimize this problem.
Proceedings of the National Academy of Sciences 06/2001; 98(10):5868-73. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In order to determine the modality of prophylactic intravesical instillation of pirarubicin (THP = tetrahydropyranyladriamycin) following transurethral resection (TUR) of superficial bladder cancer, a prospective randomized study was performed. A total of 79 patients were randomized into "2-hour instillation" (A), "5-min instillation" (B) and "control" (C) groups. Prophylactic efficacy and side effects were analyzed in each group. In groups A and B, 20 mg of THP was first dissolved in 10 ml of distilled water, adjusted to 40 ml with saline and was administered intravesically once a week for 10 weeks, starting from 1 week after TUR. The recurrence-free rate was calculated in 65 evaluable patients. The one-year recurrence-free rate was 70.2% in group A, 62.8% in group B and 52.1% in group C. The one-year recurrence-free rate was significantly higher in group A than in group C. Adverse effects were observed in 21.4% of the patients in group A and 40.7% in group B. There was no significant difference in the occurrence rate of side effects between these two groups. Taking the prophylactic efficacy and side effects into consideration, "2-hour instillation" seemed to be better than "5-min instillation".
[Show abstract][Hide abstract] ABSTRACT: The authors evaluted the efficacy of vaccination with murine renal cell carcinoma (Renca) secreting the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene and interleukin-6 (IL-6) gene for the treatment of Renca tumor. Murine GM-CSF and murine IL-6 genes were introduced and expressed in Renca cells (Renca-GM-CSF and Renca-IL-6). For a prevaccination study, wild-type Renca cells were injected subcutaneously into Balb/c mice that had been vaccinated three times with inactivated wild-type Renca, Renca-GM-CSF, Renca-IL-6, or a mixture of Renca-GM-CSF and Renca-IL-6 cells 7, 14, and 21 days before this tumor inoculation. For vaccination experiments, Renca tumor-bearing (8 to 10 mm) mice were injected subcutaneously weekly for 3 weeks with inactivated wild-type Renca cells, or either one or a combination of Renca-GM-CSF and Renca-IL-6. A nonvaccinated control was included in all experiments. The animals were monitored for survival and tumor development for 8 weeks. Mice inoculated with wild-type Renca alone died from the tumor within 35 days. Renca-IL-6 grew slower than wild-type Renca (p < 0.05). No tumor was produced by Renca-GM-CSF. Prevaccination with the combination of Renca-GM-CSF and Renca-IL-6 prevented subsequently inoculated wild-type Renca from forming tumors, and prevaccination with either one of them, compared with prevaccination with wild-type Renca, retarded tumor growth and prolonged survival time. Tumor-bearing mice vaccinated with wild-type Renca died within 42 days. Vaccination with Renca-GM-CSF or Renca-IL-6 alone prolonged the survival time, but only Renca-GM-CSF drastically reduced the tumor size. Vaccination with the combination of them achieved complete remission. Neither of the cytokine-secreting cells enhanced the expression of MHC class I or II molecules. Autologous tumor cell vaccine secreting GM-CSF is effective in preventing and treating established tumors. Its efficacy is enhanced by the cosecretion of IL-6.
[Show abstract][Hide abstract] ABSTRACT: Interleukin-18 (IL-18), a proinflammatory cytokine, leads to IFN-gamma production by NK or T cells, induces Th1 differentiation and suppresses IgE synthesis by B cells when acting on responding cells together with IL-12. IL-18 also exhibits biological activities related to allergic inflammation such as histamine or IL-4 release from basophils and accumulation of eosinophils in localized lesions in allergic model mice. In this study, Reverse transcription (RT)-PCR analysis revealed that IL-18 receptor alpha chain mRNA was expressed in both freshly prepared eosinophils and two eosinophilic cell lines (YY-1 and EoL-1 cells). Flow cytometry and RT-PCR analyses revealed that the treatment of YY-1 cells with n-butyric acid promoted cell maturation and caused an enhancement of IL-18 receptor alpha chain expression. IL-18 had little effect on the survival of peripheral eosinophils, but it dose-dependently augmented IL-8 synthesis by YY-1 cells. In addition, IL-18-mediated up-regulation of IL-8 expression in eosinophils from a patient suffering from hyper-eosinophilic syndrome was confirmed. Our findings using peripheral blood eosinophils and eosinophilic cell line suggest the functional importance of IL-18 in the induction of IL-8 and a potential proinflammatory role in allergy.
European Journal of Immunology 05/2001; 31(4):1010-6. · 4.97 Impact Factor