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Biomedical subjects

I Kitajima

Publications and source records attributed to I Kitajima.

At least 19 recordsLinked to original sources
PubMed2001

Simultaneous measurement of anandamide and 2-arachidonoylglycerol by polymyxin B-selective adsorption and subsequent high-performance liquid chromatography analysis: increase in endogenous cannabinoids in the sera of patients with endotoxic shock.

Anandamide (ANA) and 2-arachidonoylglycerol (2-AG), two endogenous cannabinoids, can be generated by activated macrophages and platelets, respectively, in the context of endotoxic shock, and are proposed to play a crucial role in the induction of the shock-related hypotension. Taking advantage of our recently discovered function of polymyxin B (PMB) binding to ANA and 2-AG, we developed a new method for measuring ANA and 2-AG by applying PMB-immobilized beads to selectively adsorb them in biological fluids, instead of organic solvent extraction. The eluate from beads can be directly fractionated by reverse-phase high-performance liquid chromatography (HPLC), and the fractionations corresponding to authentic ANA and 2-AG are collected and derivatized with fluorogenic reagent and subsequently quantified by HPLC with fluorometric detection. The calibration graphs of ANA and 2-AG were linear over a range of 1 to 500 pmol/ml. The limits of detection for ANA and 2-AG were 20 and 50 fmol, respectively. Intraassay precision was 2.24-4.25 and 3.47-5.44%, and interassay was 4.05-6.14 and 4.92-7.28% for ANA and 2-AG, respectively. Using this method, we first determined a 4-fold and 3-fold higher level of ANA and 2-AG, respectively, in the sera of patients with endotoxic shock than in normal serum. This finding should help in elucidating the role of the endogenous cannabinoids in the hypotension of human endotoxic shock. This method is rapid, sensitive, and reliable for simultaneously quantifying ANA and 2-AG in biological fluids, and has potential for clinical usage.

Adsorption↗
PubMed2001

Phosphoinositide-3 kinase-PKB/Akt pathway activation is involved in fibroblast Rat-1 transformation by human T-cell leukemia virus type I tax.

Activated phosphoinositide 3-kinase (PI3K) and its downstream target Akt are essential for the fibroblast transformation induced by many viral products. Tax, encoded by human T-cell leukemia virus type I (HTLV-I), has been demonstrated to induce the transformation of rat fibroblast Rat-1 cell through NF-kappaB activation. By stable transfection of Rat-1 cells with expressing constructs of Tax and its mutant M47, which is defective in HTLV-I LTR transactivation, we selected their transformed clones, which have characteristics of NF-kappaB activation and colony formation beyond the cell monolayer (a malignant phenotype). However, these two characteristics in the transformed clones of Tax and M47 disappear after these cells have been treated with wortmannin, a specific inhibitor of PI3K. Further, increased activity of the PI3K/Akt is observed in the transformed clones of Tax and M47 as compared to the clones of empty vector Neo and the M148, which is defective in NF-kappaB activation and cell transformation. Increased activity of PI5K is present in the transformed clones of both Tax and M47 and in the M148 clone as compared to that in the Neo cell. It is known that the efficiency of Tax-induced cell transformation is not high; a minority of Tax-expressing clones show transformation, although the majority of Tax-expressing clones show activated NF-kappaB. A Tax-expressing, nontransformed clone after transfection with an active form of the catalytic subunit of PI3K, p110alpha, becomes transformed. Consistent with these results, a Tax highly-expressing human T-cell line MT2 exhibits both higher polyphosphoinositide turnover and higher activities of PI3K and PI5K than those of Jurkat or MT1 and HTLV-I-negative and a Tax-unexpressing cell line, respectively. These results demonstrate that the activation of the PI3K/Akt signaling pathway, excepting for the NF-kappaB, is also required for the cell transformation induced by Tax.

Androstadienes↗
PubMed2001

A possible role for leptin in normo- or hypoparathyroid uremic bone in postmenopausal dialysis women.

Leptin has been proposed to be a key molecule involved in energy regulation. Based on the generally acknowledged concept that a heavier person has a higher bone density, leptin is thought to be potentially involved in bone metabolism. Serum leptin, various bone markers, and bone mineral density (BMD) were studied in 51 dialysis patients (26 men and 25 women). The serum concentrations of leptin in dialysis patients ranged from 0.7 to 10.4 ng/ml (mean 3.2 +/- 2.1 ng/ml) for males and from 1.4 to 44.6 ng/ml (mean 11.8 +/- 10.4 ng/ml) for females. There was a good correlation between the body mass index (BMI) and leptin concentration in both male and female patients (P < 0.05). Serum leptin levels also correlated well with the age-adjusted z-score for BMD (P < 0.02) and were inversely correlated with levels of the carboxy-terminal propeptide of type I procollagen (P < 0.05) in females patients, but not in male patients. In conclusion, these results suggest an actual contribution of serum leptin in maintaining bone density in postmenopausal female dialysis patients.

Adult↗
PubMed2001

Fibrin monomer could be a useful predictor of pulmonary embolism after total hip arthroplasty: preliminary report.

We examined 17 total hip arthroplasty patients in order to develop a method for the predictive diagnosis of pulmonary embolism (PE) after joint arthroplasty. Scintigraphy revealed the presence of PE in 4 patients. Prothrombin time (PT), activated partial thromboplastin time (aPTT), antithrombin III (ATIII), and thrombin-AT III complex (TAT) did not show significant differences between patients with and without PE. D-dimer 7 days after surgery showed significant differences between patients with and without PE. Fibrin monomer (FM) increased sharply after surgery, and it was significantly different between the patients with and without PE immediately after surgery and 2 days after surgery. Our findings suggest the importance of FM in the predictive diagnosis of pulmonary embolism after total hip arthroplasty, and 40 microg/ml or higher levels with our measurement method could represent a high-risk condition.

Aged↗
PubMed2001

Study of cell-material interaction by estimating NF-kappaB activation in HeLa S3 cells adhered onto hydrophilic substrates.

In order to interpret how cells recognize biomaterials, nucleic factor-kappa B (NF-kappaB) activation in the attached HeLa S3 cells on various substrates was evaluated. As substrates, materials of hydrophilic nature (cellulose, poly(acrylamide)-grafted poly(ethylene) (PAAm-g-PE), and lipids films) were used. The contemporary assay method for NF-kappaB was modified to fit our system. As a result, NF-kappaB activation varied depending on the substrates. The NF-kappaB outcome was induced significantly in the HeLa S3 cells that had adhered onto the lipid films in a short time. On the other hand, high levels of NF-kappaB induction were observed in the HeLa cells adhered to the celluose and PAAm-g-PE after a 24 h incubation period. The induction of NF-kappaB by cell-material interaction is discussed from the point of view of biocompatibility.

Cell Adhesion↗
PubMed2001

Vascular endothelial growth factor (VEGF) is one of the cytokines causative and predictive of hepatic veno-occlusive disease (VOD) in stem cell transplantation.

Hepatic veno-occlusive disease (VOD) is one of the most serious complications in patients receiving stem cell transplantation (SCT). However, the cause of VOD remained to be elucidated. Vascular endothelial growth factor (VEGF) has been reported to have various physiological effects including neovascularization and acceleration of vasopermeability. Because we postulated that VEGF could be one of the causative factors in VOD after SCT, serum VEGF levels were measured by ELISA in 50 patients receiving SCT. Six of the patients showed typical manifestations of VOD and four of them died due to VOD. The mean maximum serum VEGF level in the six patients with VOD was markedly increased compared to that in the patients without VOD (P < 0.001) and in normal controls (P < 0.001). Moreover, the mean maximum serum VEGF level in patients with VOD before conditioning chemoradiotherapy for SCT was also high compared to patients without VOD (P = 0.0012) in the same period. Similarly, serum VEGF levels were significantly higher in patients whose plasma protein C activities decreased below 40% (P < 0.001). During the clinical course of VOD after SCT, the increase of serum VEGF synchronized fairly well with the development of VOD. Since VEGF causes the expression of tissue factor on circulating monocyte/macrophages and results in hypercoagulability, our observation suggests that in the patients with VOD who showed high serum VEGF it might account for the development of VOD. Furthermore, this observation may indicate a novel therapeutic strategy for prevention of VOD.

Adolescent↗
PubMed2001

One-portal technique of endoscopic fasciotomy: Chronic compartment syndrome of the lower leg.

Many athletes complain of exercise-induced pain in the lower leg that can be caused by inflammatory diseases, peripheral nervous system disease, fatigue fracture, shin splint, and chronic compartment syndrome (CCS). CCS is the most typical exercise-induced condition and it often requires surgical decompression of the several compartments. There are already many techniques reported in the literature. Recently, an endoscopic technique for CCS was reported with which excellent results were achieved. We have modified it and developed a new technique for treating CCS of the lower leg. We report a case of CCS of the lower leg treated with 1-portal endoscopic fasciotomy. The technique helps to decrease damage to soft tissue and patients will immediately return to normal activities of daily living.

Adolescent↗
PubMed2001

Upregulation of toll-like receptor 2 gene expression in macrophage response to peptidoglycan and high concentration of lipopolysaccharide is involved in NF-kappa b activation.

Toll-like receptors 2 and 4 (TLR2 and TLR4) have been found to transduce signals of peptidoglycan (PGN) and lipopolysaccharide (LPS), respectively, for NF-kappa B activation. However, little is known about the expression and regulation of the TLR2 gene in monocytes/macrophages in response to the two typical bacterial products. We show in the present study that both PGN and a high concentration of LPS increase TLR2 gene expression in macrophage-like cells, 1 alpha,25-dihydroxyvitamin D(3)-differentiated human HL60 and mouse RAW264.7 cells, and human monocytes in a dose- and time-dependent manner. Actinomycin D and pyrrolidine dithiocarbamate inhibition of gene transcription and NF-kappa B activation, respectively, blocks LPS- and PGN-elevated TLR2 mRNA in monocytic cells. The LPS-induced increase in TLR2 mRNA in monocytic cells is abolished by polymyxin B pretreatment and is observed in peripheral blood mononuclear cells from pigs subjected to endotoxic shock. Further, high concentrations of LPS and synthetic lipid A increase TLR2 mRNA expression in peritoneal macrophages from both TLR4-deficient C3H/HeJ mice and normal C3H/HeN mice, a process that constitutes induction of TLR4-independent TLR2 expression. These findings demonstrate that TLR2 gene expression is upregulated in macrophage responses to PGN and to high concentrations of LPS in vitro and in vivo and correlates with NF-kappa B activation.

Animals↗
PubMed2001

Expression of transduced HSP70 gene protects chondrocytes from stress.

OBJECTIVE: To investigate the efficacy of adenovirus vector mediated transduction of heat shock protein 70 (HSP70) gene to human chondrocyte-like cell (HCS-2/8) against heat stress. METHODS: Two adenovirus vectors that contain wild-type (AxSHEwt) or mutant-type (AxSHEmt) HSP70 gene, and that are regulated by SRalpha promoter, were constructed. The mutant-type lacks the area that expresses stress durability. One of the 2 adenovirus vectors was added to the cultures of human chondrocyte-like cells (HCS-2/8). Heat stress (48 degrees C) was applied to the transduced cells for 2 h, and the efficacy of adenovirus vector mediated transduction of HSP70 gene against heat stress in the chondrocytes was investigated using alamar blue assay and MTT assay. RESULTS: Absorbance levels at 48 degrees C were 300.3 +/- 51.9 and 1.173 +/- 0.011 in the controls, 278.5 + 33.8 and 1.217 +/- 0.018 in the AxSHEmt transduced cells, and 349 +/- 14.7 and 1.371 +/- 0.033 in the AxSHEwt transduced cells. The level in the AxSHEwt transduced cells was significantly higher than in the other 2 groups (p < 0.05). With 37 degrees C treatment, no significant difference was observed. CONCLUSION: Chondrocytes to which HSP70 gene was transduced had a significantly higher metabolic activity and viability under heat stress.

Adenoviridae↗
PubMed2000

PEA3 and AP-1 are required for constitutive IL-8 gene expression in hepatoma cells.

Interleukin-8 (IL-8) mRNA was constitutively expressed in human hepatoma cell line, HepG2 and in human hepatocellular carcinoma (HCC), which often form hypervascular tumors. The sequence 5'-AGGAAG-3' at -137 to -132 bp of IL-8 promoter was shown to be polyomavirus enhancer A binding protein-3 (PEA3) binding site, which can cooperate with activator protein-1 (AP-1). Both PEA3 and AP-1 are essential for constitutive IL-8 expression in HepG2 cells, determined by promoter assays. Moreover, PEA3 and IL-8 proteins coexisted in HCC tissues, but not in uninvolved liver tissues. It is possible PEA3 may have important roles in tumor progression and in angiogenesis in HCC.

Base Sequence↗
PubMed2000

Type I muscle atrophy caused by microgravity-induced decrease of myocyte enhancer factor 2C (MEF2C) protein expression.

To investigate the molecular mechanisms of muscle atrophy under microgravity, the paraspinal muscles of rats after 14 days spaceflight and those of ground-based controls were examined. In the microgravitational environment, expressions of 42 genes changed, and the expressions of heat shock protein 70 and t complex polypeptide 1 increased. In Northern blotting, myocyte-specific enhancer binding factor 2C (MEF2C) and MEF2C-related genes including aldolase A and muscle ankyrin decreased. After 9 days ground recovery, expression of MEF2C increased and it was located mainly on the satellite cells in the muscle regeneration state. MEF2C could be a key transcriptional factor for skeletal muscle atrophy and regeneration under microgravity.

Animals↗
PubMed2000

Anandamide induces apoptosis of PC-12 cells: involvement of superoxide and caspase-3.

Anandamide (arachidonoylethanolamide), an endogenous cannabinoid receptor ligand has been suggested to have physiological role in mammalian nervous system. However, little is known about the role of anandamide on neuronal cells. Here, we demonstrate that anandamide causes death of PC-12 cells, showing marked DNA condensation and fragmentation, appearance of cells at sub-G(0)/G(1) and redistribution of phosphatidyl serine, the hallmark features of apoptosis. Anandamide raised intracellular superoxide level and CPP32-like protease activity in PC-12 cells markedly. Furthermore, antioxidant N-acetyl cysteine prevented anandamide-induced superoxide anion formation and cell death, implying that intracellular superoxide is a novel mediator of anandamide-induced apoptosis of PC-12 cells.

Acetylcysteine↗
PubMed2000

Polymyxin B binds to anandamide and inhibits its cytotoxic effect.

Anandamide (ANA), an endogenous cannabinoid, can be generated by activated macrophages during endotoxin shock and is thought to be a paracrine contributor to hypotension. We discovered that ANA in saline/ethanol solution and in serum was efficiently adsorbed in a polymyxin B (PMB)-immobilized beads column and eluted with ethanol. We confirmed the direct binding of PMB to ANA by using surface plasmon resonance. The adsorption of ANA by PMB may abolish the diverse effects of ANA such as hypotension, immunosuppression, and cytotoxicity, and may suggest a new therapeutic strategy for endotoxin shock.

Adsorption↗
PubMed2000

Inhibition of caspase-3 activation by SB 203580, p38 mitogen-activated protein kinase inhibitor in nitric oxide-induced apoptosis of PC-12 cells.

Mitogen-activated protein kinase (MAPK) p38 plays pivotal role in cell proliferation, differentiation, and apoptosis when cysteine protease caspase induces apoptosis in different cell systems. SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1 H-imidazole) is widely used as a specific inhibitor of p38 MAPK, and prevents apoptosis induced by various agents. The effect of SB 203580 on nitric oxide(NO)- or peroxynitrite-induced cell death is not known. Western blotting results indicate that p38 MAPK was activated significantly in NO- or peroxynitrite-induced cell death in a time-dependent manner, and subsequently this cell death was markedly inhibited by SB 203580, as determined by fluorescence-activated cell sorting (FACS)-can analyzer. Furthermore, NO/peroxynitrite-induced caspase-3 activation was notably inhibited by SB 203580, however, phosphorylation of either p38 MAPK or p44/42 was not influenced by SB 203580. Thus, it is likely that SB 203580 prevents NO/peroxynitrite-induced cell death by inhibiting caspase-3 activation in PC-12 cells.

Animals↗
PubMed2000

VEGF is causative for pulmonary hypertension in a patient with Crow-Fukase (POEMS) syndrome.

We report a case of Crow-Fukase (POEMS) syndrome associated with pulmonary hypertension (PH). In this case, the concentration of vascular endothelial growth factor (VEGF) was extremely high in the serum, and the levels of IL-1beta, IL-6, TNF-alpha, and thiamine, which were thought in past reports to be mediators of PH in Crow-Fukase syndrome, were normal. After prednisolone therapy, PH disappeared with a dramatic decrease in serum VEGF. Our results suggest that VEGF is closely correlated with PH in Crow-Fukase syndrome.

Cytokines↗
PubMed2000

Apoptotic cell death in steroid induced osteonecrosis: an experimental study in rabbits.

OBJECTIVE: We investigated apoptosis, i.e., programmed cell death, in steroid induced osteonecrosis in a rabbit model. METHODS: Forty-four adult Japanese White rabbits were divided into 3 groups: Group A were untreated controls and had a subsequent 8 week no-treatment period; Group B received intramuscular injection of methylprednisolone 4 mg/kg once weekly for 4 weeks; and Group C received the same treatment and had a subsequent 8 week no-treatment period. At the end of each period, all animals were sacrificed and tissue samples were obtained from the femur and humerus for histopathologic examination. Terminal deoxynucleotidyl transferase (TdT) mediated deoxyuridine triphosphate (dUTP) biotin nick end labeling (TUNEL) was used to detect fragmented DNA known to be associated with apoptotic cell death. RESULTS: Group A rabbits did not develop osteonecrosis-like lesions (ONL) in the femur and humerus, and few TUNEL positive cells were observed in bone marrow cells. In Group B, ONL developed in 11/15 rabbits, and many TUNEL positive cells were found in the area surrounding ONL. In Group C, ONL were found in 6/10 rabbits, but only a few TUNEL positive cells were present around the lesion. CONCLUSION: These findings revealed that apoptosis occurs in the early stage of steroid induced osteonecrosis.

Animals↗