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Biomedical subjects

M Ikeda

Publications and source records attributed to M Ikeda.

At least 649 records · Page 36Linked to original sources

Structural characteristics of the pedicle and its role in screw stability.

STUDY DESIGN: Cross-sectional regional bone mineral density of the pedicle was measured by peripheral quantitative computed tomography. Biomechanical tests were performed to clarify the role of the pedicle in screw stability. OBJECTIVES: To identify the structural characteristics of the pedicle that supports pedicle screw stability and the differences in these characteristics between normal and osteoporotic vertebrae. SUMMARY OF BACKGROUND DATA: The pedicle screw is an essential component of many systems used to align the spine. The contribution of the pedicle to screw stability, however, has not been fully investigated. METHODS: Trabecular, subcortical, and cortical bone mineral density and the area of the pedicle were measured by peripheral quantitative computed tomography. Bone mineral density also was recalculated in four circumferential layers. These parameters were compared between normal and osteoporotic individuals. The relative contribution of the pedicle to screw stability was evaluated by caudocephalad and pull-out loading in a vertebra with or without its body. RESULTS: Inner trabecular, middle subcortical, and outer cortical bone mineral density and cortical bone area in the pedicle were significantly lower in osteoporotic vertebrae than those in normal vertebrae. In the pedicle, bone mineral density increased close to the outer layer. Bone mineral density not as thick even in the outer layer in osteoporotic subjects. Approximately 80% of the caudocephalad stiffness and 60% of the pullout strength of the pedicle screw depended on the pedicle rather than on the vertebral body. CONCLUSION: Screw stability depends on the structural characteristics of the pedicle. The pedicle was denser in the subcortical bone, in which the threads of the screw engage, than in trabecular bone. In osteoporosis, bone mineral density was not as dense even in the outer layer, and the cortex was thinner than normal. A larger screw would not enhance screw stability and may break the thin cortex in osteoporotic vertebrae.

Absorptiometry, Photon↗

DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2.

DNA-damaging agents signal to p53 through as yet unidentified posttranscriptional mechanisms. Here we show that phosphorylation of human p53 at serine 15 occurs after DNA damage and that this leads to reduced interaction of p53 with its negative regulator, the oncoprotein MDM2, in vivo and in vitro. Furthermore, using purified DNA-dependent protein kinase (DNA-PK), we demonstrate that phosphorylation of p53 at serines 15 and 37 impairs the ability of MDM2 to inhibit p53-dependent transactivation. We present evidence that these effects are most likely due to a conformational change induced upon phosphorylation of p53. Our studies provide a plausible mechanism by which the induction of p53 can be modulated by DNA-PK (or other protein kinases with similar specificity) in response to DNA damage.

Binding Sites↗

Determination of lead and cadmium in food and blood by inductively coupled plasma mass spectrometry: a comparison with graphite furnace atomic absorption spectrometry.

To compare inductively coupled plasma mass spectrometry (ICP-MS) and graphite furnace atomic absorption spectometry (GF-AAS) as the method for determining lead and cadmium in the human diet and blood, 418 diet homogenate samples and the same number of blood samples were collected from Chinese and Japanese women and were analyzed by the two methods. The results showed that our ICP-MS method is precise and accurate, being comparable to the GF-AAS method established previously. The ICP-MS method is simple and fast spending only one-tenth of the time necessary for GF-AAS and allows simultaneous analyses of lead and cadmium with low detection limits. When applied to actual sample analysis, however, ICP-MS results tend to be 10-20% lower than GF-AAS results in the analysis of lead in the diet and blood and cadmium in blood. This is possibly due to some interference in ICP-MS and matrix of samples. As the ICP-MS results could be mathematically corrected to be equivalent to the GF-AAS results, we conclude that this ICP-MS method can be used as a routine analytical method for the determination of lead and cadmium in human diet and blood samples.

Blood Chemical Analysis↗

The full agonistic effect of recombinant 20 kDa human growth hormone (hGH) on CHO cells stably transfected with hGH receptor cDNA.

The agonistic effect of the recombinant 20 kilodalton human GH (20K-hGH) with authentic primary structure was studied using Chinese hamster ovary (CHO) cells stably transfected with hGH receptor (hGHR) cDNA and was compared with that of 22K-hGH. The binding affinities (dissociation constants) of 20K- and 22K-hGH were identical (0.41 +/- 0.11 nM and 0.41 +/- 0.04 nM, respectively). In addition, the two hGHs possessed the same potencies in activating the rat serine protease inhibitor (Spi) 2.1 gene promoter. 20K-hGH was similarly internalized as 22K-hGH but its internalization rate was a little slower than that of 22K-hGH. We also found that proliferation of CHO-hGHR cells stimulated by serum was remarkably inhibited by both hGHs to the same degree. In conclusion, both hGH isoforms exhibited the same binding affinities for hGHR and were potent enough to induce some hGHR-mediated cellular events. These suggest that 20K-hGH exerts a full agonistic activity for hGHR.

Animals↗

Involvement of caspase-1 and caspase-3 in the production and processing of mature human interleukin 18 in monocytic THP.1 cells.

Recently, human interleukin 18 (hIL-18) cDNA was cloned, and the recombinant protein with a tentatively assigned NH2-terminal amino acid sequence was generated. However, natural hIL-18 has not yet been isolated, and its cellular processing is therefore still unclear. To clarify this, we purified natural hIL-18 from the cytosolic extract of monocytic THP.1 cells. Natural hIL-18 exhibited a molecular mass of 18.2 kDa, and the NH2-terminal amino acid was Tyr37. Biological activities of the purified protein were identical to those of recombinant hIL-18 with respect to the enhancement of natural killer cell cytotoxicity and interferon-gamma production by human peripheral blood mononuclear cells. We also found two precursor hIL-18 (prohIL-18)-processing activities in the cytosol of THP.1 cells. These activities were blocked separately by the caspase inhibitors Ac-YVAD-CHO and Ac-DEVD-CHO. Further analyses of the partially purified enzymes revealed that one is caspase-1, which cleaves prohIL-18 at the Asp36-Tyr37 site to generate the mature hIL-18, and the other is caspase-3, which cleaves both precursor and mature hIL-18 at Asp71-Ser72 and Asp76-Asn77 to generate biologically inactive products. These results suggest that the production and processing of natural hIL-18 are regulated by two processing enzymes, caspase-1 and caspase-3, in THP.1 cells.

Amino Acid Sequence↗

Interleukin 18 induces the sequential activation of natural killer cells and cytotoxic T lymphocytes to protect syngeneic mice from transplantation with Meth A sarcoma.

We have recently reported that interleukin 18 (IL-18) pretreatment induces immunologically mediated antitumor effects in BALB/c mice injected i.p. with syngeneic Meth A sarcoma. In this study, mice were pretreated with IL-18 before Meth A transplantation, and immunocompetency in pretreated or untreated tumor-bearing mice (TBM) 3, 9, and 15 days after transplantation was compared with that of normal mice. On day 3, pretreated TBM mitogen-stimulated spleen cells produced significantly decreased levels of IL-2 and IFN-gamma during 24-h culture. In contrast, IL-10 and granulocyte macrophage colony-stimulating factor productions were significantly enhanced in pretreated TBM cultures, and natural killer (NK) cell activity was also significantly augmented. Splenomegaly was also observed in the pretreated TBM on day 3, and the proliferating cells were identified as asialo GM1+ cells by flow cytometry. Cytotoxic activity of pretreated TBM spleen cells after a 5-day mixed lymphocyte-tumor cell culture did not differ from that of untreated TBM and normal mice on day 3 but was significantly enhanced on days 9 and 15 compared with that observed in normal mice and untreated TBM. Concurrently, the production of IL-2 and of IL-10 recovered and decreased, respectively, and NK activity dropped to normal levels. The effects of IL-18 on cytokine production and NK activity observed on day 3 treated TBM were also reproduced in normal mice. In conclusion, IL-18 seems to enhance the generation of NK activity early after tumor transplantation and simultaneously induces an increase and a decrease in the production of IL-10 and IL-2, respectively. As NK activity subsides to normal levels and IL-10 synthesis decreases, IL-2 synthesis is restored, and cytolytic cell activity is significantly enhanced. These results provide new insight into the immunologically mediated antitumor effects of IL-18.

Animals↗

Purification and characterization of the human interleukin-18 receptor.

Interleukin (IL)-18 was identified as a molecule that induces IFN-gamma production and enhances NK cell cytotoxicity. In this paper, we report upon the purification and characterization of human IL-18 receptor (hIL-18R). We selected the Hodgkin's disease cell line, L428, as the most strongly hIL-18R-expressing cell line based on the results of binding assays. This binding was inhibited by IL-18 but not by IL-1beta. The dissociation constant (Kd) of 125I-IL-18 binding to L428 cells was about 18.5 nM, with 18,000 binding sites/cell. After immunizing mice with L428 cells and cloning, a single monoclonal antibody (mAb) against hIL-18R was obtained (mAb 117-10C). Sequentially, hIL-18R was purified from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-extracted L428 cells by wheat germ lectin-Sepharose 4B chromatography and mAb 117-10C-Sepharose chromatography. The internal amino acid sequences of hIL-18R all matched those of human IL-1 receptor-related protein (IL-1Rrp), the ligand of which was unknown to date. When expressed in COS-1 cells, the cDNA of IL-1Rrp conferred IL-18 binding properties on the cells and the capacity for signal transduction. From these results, we conclude that a functional IL-18 receptor component is IL-1Rrp.

Amino Acid Sequence↗

Decrease in the amount of focal adhesion kinase (p125(FAK)) in interleukin-1beta-stimulated human umbilical vein endothelial cells by binding of human monocytic cell lines.

Monocytes in the blood circulation migrate across endothelial cell monolayers lining the blood vessels and infiltrate into the underlying tissues in inflammation. However, little is known about the mechanisms by which leukocytes migrate across the endothelial barrier after binding and what molecules participate in the process. Addition of the human monocytic cell line THP-1 to interleukin-1beta (IL-1beta)-stimulated human umbilical vein endothelial cells (HUVEC) induced a decrease in the amount of focal adhesion kinase (p125(FAK)) protein, a tyrosine kinase localized at focal contacts and essential for cell attachment to the extracellular matrix, whereas little change was observed in the amount of other molecules associated with cell adhesion such as vascular cell adhesion molecule-1, alpha-catenin, and talin. A maximum decrease in the amount of p125(FAK) was observed 15-30 min after addition of THP-1 cells to HUVEC, after which the level of p125(FAK) gradually recovered. A reduction in the density of actin stress fibers in IL-1beta-activated HUVEC was observed in parallel with the decrease in p125(FAK). The p125(FAK) decrease was partially inhibited by preventing THP-1 binding to HUVEC using a mixture of antibodies to adhesion molecules. We suggest that the decrease in p125(FAK) triggered by binding of monocytes in inflammation facilitates the transendothelial migration of the monocytes by altering the adhesiveness of endothelial cells to the extracellular matrix.

Antibodies, Monoclonal↗

Interleukin-18 induces activation and association of p56(lck) and MAPK in a murine TH1 clone.

Interleukin-18 (IL-18) was identified as an inducer of interferon-gamma (IFN-gamma) production by stimulated T cells. In this study, we used an ovalbumin-responsive murine Th1 clone (OVA#4), in which DNA synthesis was reportedly enhanced after IL-18 treatment in the presence of a non-mitogenic TCR/CD3 stimulus, to examine signal transduction pathways. In the presence of the stimulus, IL-18 induced the appearance of tyrosine-phosphorylated proteins and herbimycin A inhibited DNA synthesis. It is suggested that protein tyrosine kinase (PTK) mediated signaling is induced by IL-18. Specifically, IL-18 induced phosphorylation of phosphorylates p56(lck) (LCK) and mitogen-activated protein kinase (MAPK). IL-18 alone induced the kinase activities of both LCK and MAPK, and the activities were increased by the TCR/CD3 stimulus. Simultaneously, IL-18 induced the association of LCK with MAPK and this was also increased by the TCR/CD3 stimulus. The activation of the LCK-MAPK pathway correlated with enhanced DNA synthesis in OVA#4 cells. These results suggest that the LCK-MAPK pathway is involved in IL-18 signaling and that IL-18 may play an important role in modification of TCR/CD3-mediated response.

Animals↗

Characterization of anti-human interleukin-18 (IL-18)/interferon-gamma-inducing factor (IGIF) monoclonal antibodies and their application in the measurement of human IL-18 by ELISA.

Interleukin-18 (IL-18)/interferon-gamma-inducing factor (IGIF) is a novel cytokine, which is a potent inducer of IFN-gamma production and plays an important role in Th1 responses. In order to develop a specific ELISA for the measurement of human IL-18, we established 13 anti-human IL-18 monoclonal antibodies and characterized them. 7 murine anti-human IL-18 mAbs and 6 rat anti-human IL-18 mAbs were obtained by fusion of splenocytes from mice or rats immunized with human IL-18, with SP2/0 myeloma cells. These antibodies were classified into 4 groups according to competitive binding ELISAs to the human IL-18 molecule. 1 murine mAb and all 6 rat mAbs neutralized IFN-gamma production induced by IL-18. A specific human IL-18 ELISA was developed using two neutralizing mAbs (#125-2H and #159-12B). This ELISA detects human IL-18 with a minimum detection limit of 10 pg/ml, but does not react with heat-denatured human IL-18. The ELISA does not show any cross-reactivity with other cytokines. Using this assay, human IL-18 was measurable in the plasma of leukemia patients. This ELISA would become a powerful tool for investigating the relationship between IL-18 and various diseases or analyzing the control mechanisms of IL-18 production from IL-18 producing cells.

Adult↗

Identification of mu-, m-calpains and calpastatin and capture of mu-calpain activation in endothelial cells.

The presence of the calpain-calpastatin system in human umbilical vein endothelial cells (HUVEC) was investigated by means of ion exchange chromatography, Western blot analysis, and Northern blot analysis. On DEAE anion exchange chromatography, calpain and calpastatin activities were eluted at approximately 0.30 M and 0.15-0.25 M NaCl, respectively. For half-maximal activity, the protease required 800 microM Ca2+, comparable to the Ca2+ requirement of m-calpain. By Western blot analysis, the large subunit of mu-calpain (80 kDa) was found to be eluted with calpastatin (110 kDa). Both the large subunit of m-calpain (80 kDa) and calpastatin were detected in the respective active fractions. By Northern blot analysis, mRNAs for large subunits of mu- and m-calpains were detected in single bands, each corresponding to approximately 3.5 Kb. Calpastatin mRNA was observed in two bands corresponding to approximately 3.8 and 2.6 Kb. Furthermore, the activation of mu-calpain in HUVEC by a calcium ionophore was examined, using an antibody specifically recognizing an autolytic intermediate form of mu-calpain large subunit (78 kDa). Both talin and filamin of HUVEC were proteolyzed in a calcium-dependent manner, and the reactions were inhibited by calpeptin, a cell-permeable calpain specific inhibitor. Proteolysis of the cytoskeleton was preceded by the appearance of the autolytic intermediate form of mu-calpain, while the fully autolyzed postautolysis form of mu-calpain (76 kDa) remained below detectable levels at all time points examined. These results indicate that the calpain-calpastatin system is present in human endothelial cells and that mu-calpain may be involved in endothelial cell function mediated by Ca2+ via the limited proteolysis of various proteins.

Antibody Specificity↗

Specific activation of retinoic acid receptors (RARs) and retinoid X receptors reveals a unique role for RARgamma in induction of differentiation and apoptosis of S91 melanoma cells.

Retinoic acid (RA) and 9-cis-RA induce growth arrest and differentiation of S91 melanoma cells. RA activates retinoic acid receptors (RARs), whereas 9-cis-RA activates both RARs and retinoid X receptors (RXRs). Both classes of receptors function as ligand-dependent transcription factors. S91 melanoma cells contain mRNA for RXRalpha, RXRbeta, RARalpha, RARgamma, and RARbeta in low levels. Among these, only RARbeta gene transcription is induced by retinoids. However, at present the individual role(s) for each RXR and RAR isoform in these processes is unclear. We assessed the function of all isoforms in the S91 melanoma model by using RXR and RAR isoform-specific retinoids to study their effects on cell growth, RARbeta expression, and differentiation. Activation of each of the endogenous RXR or RAR isoforms induces RARbeta gene expression, and blocks cellular proliferation. However, only the RARgamma-ligands cause additional differentiation toward a melanocytic phenotype, which coincides with substantial apoptosis well before morphological changes are apparent. Apoptosis is completely dependent on de novo protein synthesis but cannot be induced by changes in activities of AP-1, protein kinase C, and protein kinase A, nor can it be blocked by the presence of the antioxidant glutathione. These results argue against a specific role for RARbeta, but suggest that RARgamma has a critical role in a genetic switch between melanocytes and melanoma, and induction of ligand-dependent apoptosis.

Apoptosis↗

Expression, nuclear localization and interactions of human MCM/P1 proteins.

We report here the comparative analysis of human Mcm/P1 proteins (HsMcm2, -3, -5 and -7), including a characterization of their mutual interactions, cell cycle dependent expression and nuclear localization during the cell cycle and the quiescent state. The mRNA levels of these genes, which undergo cell cycle dependent oscillations with a peak at G1/S phase, may be regulated by E2F motifs, two of which were detected in the 5' upstream region of the HsMCM5 gene. In contrast, the protein levels of these Mcm proteins were found to remain rather constant during the HeLa cell cycle. However, their levels gradually increased in a variable manner as KD cells progressed from GO into the G1/S phase. In the GO stage, the amounts of HsMcm2 and -5 proteins were much lower than those of HsMcm7 and -3 proteins, suggesting that they are not present in stoichiometric amounts, and that only a proportion of these molecules actively participate in cell cycle regulation as part of Mcm/P1 complexes.

Amino Acid Sequence↗

Hepatitis G virus replication in human cultured cells displaying susceptibility to hepatitis C virus infection.

We recently developed a hepatitis C virus (HCV) replication system using two cultured human cell lines: MT-2C, a human T-cell leukemia virus type I-infected cloned T cell line, and PH5CH, a non-neoplastic human hepatocyte line. In the course of the study, we found evidence that replication of hepatitis G virus (HGV), which has recently been identified, was supported in both MT-2C and PH5CH cells. When these cells were inoculated with serum 1B-3 containing both HCV and HGV obtained from a blood donor, the 5'-noncoding (5'-NC) regions of HCV RNA and HGV RNA were detected by RT-nested PCR in both cell lines more than 30 days postinoculation, and the 3'-noncoding region of HGV RNA was also detected in the both cell lines more than 30 days postinoculation. Sequence analysis of the 5'-NC region of HGV RNA revealed the quasispecies nature of HGV, although the 5'-NC region was highly conserved among HGV isolates. This HGV-infected culture system will be useful for various biological and virological studies of HGV.

Base Sequence↗

Secretion of authentic 20-kDa human growth hormone (20K hGH) in Escherichia coli and properties of the purified product.

Using Bacillus amyloliquefaciens neutral protease gene (npr), we have constructed a secretion system of 20-kDA human growth hormone (20K hGH) in E. coli. The secretion-signal region from npr was modified inserting a fragment coding a 2Lys-5Leu cluster. In this system we found that co-expression of glutathione reductase remarkably increased accumulation level of 20K hGH in periplasm and confirmed that secreted 20K hGH was correctly processed. The recombinant 20K hGH was highly purified and subjected to analyses of physicochemical properties and biological activities which are still unclear and controversial due to difficulty in preparing the sample with authentic structure. The secreted recombinant product had authentic disulfide linkages and showed molecular weight of 20,270.5 +/- 3.7 (theoretical value, 20,269.9). The results suggest that the recombinant 20K hGH is a full agonist on rat growth promotion and lipolysis stimulation in isolated rat adipose tissues. In particular, the lipolysis-stimulating activity of 20K hGH was distinct as compared with that of 22K hGH under physiological concentration. Cell proliferation activity via prolactin-receptor in Nb-2 lymphoma was obviously low as compared with that of 22K hGH.

Amino Acid Sequence↗

Circadian rhythms in vitamin B12 content of the rat brain.

The whole brain content of vitamin B12 (VB12) in the rat was assayed by the chemiluminescent immunoassay at 4 h intervals in 12:12 h light-dark cycles (LD) and five different circadian times (CTs) under constant dim illumination (dim LL). In LD-entrained rats, the content of VB12 exhibited a day-night rhythm with a peak 2 h after the dark onset time and a trough 2 h before the light onset time. In freerunning rats under dim LL, the content of VB12 also exhibited a circadian variation with a peak ca. 2 h after the activity onset time (CT14) and a trough ca. 12 h after the activity onset time (CT 0). These findings clearly indicate that the brain VB12 content decreased during the active phase and increased during the resting phase regardless of the lighting schedule. On the other hand, the drinking behavior, as an index of the intake activity, was observed less frequently in the light phase of LD cycles and the resting phase of dim LL. Since most of the digested VB12 is known to be continuously stored in the liver, it is demonstrated that the rhythm of the brain content of VB12 may be caused by brain consumption of VB12 during the active phase and the transportation from the peripheral storage during the resting phase, independent of intake activities.

Animals↗

Expression of p21 (waf1/cip1/sdi1), but not p53 protein, is a factor in the survival of patients with advanced gastric carcinoma.

BACKGROUND: The authors examined whether expression of p21 (waf1/cip1/sdi1) and p53 protein was related to survival, rates in patients with advanced gastric carcinoma. METHODS: The expression of p21 and p53 protein was analyzed by immunohistochemistry in 93 patients with advanced gastric carcinoma with serosal invasion and lymph node metastasis. All patients underwent curative surgery. The probability of survival was calculated by the Kaplan-Meier method and compared by the generalized Wilcoxon test. RESULTS: Various levels of p21 and p53 immunoreactivities in carcinoma cells were detected in 30 (32%) and 60 (65%), respectively, of 93 samples. There was no correlation between p21 and p53 expression. The 5-year survival rate of patients with p21 expression was 69.4%, which was significantly better than that of patients without p21 expression (38.1%; P < 0.05). However, p53 protein expression did not correlate with patient survival. CONCLUSIONS: Expression of p21 protein may be a better prognostic factor than p53 protein expression in patients with advanced gastric carcinoma.

Adenocarcinoma↗

An experimental study of a combination method using a pedicle screw and laminar hook for the osteoporotic spine.

STUDY DESIGN: Using human cadaver spines, the authors compared the effect of using a combination of pedicle screw and laminar hook on the same vertebra with that of using a pedicle screw alone in reference to bone mineral density of the vertebra under nondestructive cyclic loading. OBJECTIVES: To quantify stiffness obtained by pedicle screw alone and by the combination method and to clarify a relationship between stiffness obtained by each instrumentation method and bone mineral density of the vertebra. SUMMARY OF BACKGROUND DATA: The use of pedicle screws apparently improves the union rate of spinal fusion. Instrumentation failures sometimes occur, however, such as loosening or loss of correction of the spine, especially in patients with osteoporosis. Some augmentation method in instrumentation is necessary to overcome bone fragility in the osteoporotic spine. METHODS: Thirteen cadaver lumbar vertebrae were used for this study. Bone mineral density was measured by dual-energy X-ray absorptiometer. After separating each vertebrae, the pedicle screw was screwed into a vertebra. Five cycles of cephalocaudal loading were performed to the shank of the screw with a cross-head speed of 3 mm/min under a maximum load control of 29.4 N using an Instron type testing machine, and the stiffness obtained with the pedicle screw (Kj) was calculated from the load-deformation curve. Then, a laminar hook was set and connected to the screw via a rod. Mechanical testing was performed in the same way, and the stiffness obtained with the combination method (Kf) was determined. Kj and Kf were compared using the paired t test. The relationship between Kj, Kf, or the stiffness improvement ratio ([Kf-Kj]/Kj) by the combination method and bone mineral density was analyzed by linear regression analysis. RESULTS: Stiffness obtained by the combination method was significantly greater than that obtained by pedicle screw alone (89.8 +/- 35.0 N/mm by the combination method, 60.2 +/- 19.6 N/mm by pedicle screwing alone; P < 0.0001). Stiffness, whether obtained by pedicle screw alone or by the combination method, was positively correlated with bone mineral density (with pedicle screw alone, R2 = 0.614, P < 0.0001; with the combination method, R2 = 0.645, P < 0.0001). However, there was no significant correlation between stiffness improvement ratio and bone mineral density. CONCLUSION: Instrumentation stiffness obtained by the combination method was significantly greater than that obtained by the use of pedicle screw alone. There was no significant correlation between the improvement ratio by the combination method and bone mineral density. These results suggest that the combination method is valuable irrespective of the presence of spinal osteoporosis.

Aged↗