Biomedical subjects

T Kamata

Publications and source records attributed to T Kamata.

At least 19 recordsLinked to original sources

PubMed2006

Inhibition of NADPH oxidase 4 activates apoptosis via the AKT/apoptosis signal-regulating kinase 1 pathway in pancreatic cancer PANC-1 cells.

Pancreatic adenocarcinoma is an aggressive human malignancy and is characterized by resistance to apoptosis. Recently, NADPH oxidase (Nox) 4-mediated generation of intracellular reactive oxygen species (ROS) was proposed to confer antiapoptotic activity and thus a growth advantage to pancreatic cancer cells. The signaling mechanism by which Nox4 transmits cell survival signals remains unclear. Here, we show that both a flavoprotein inhibitor, diphenylene iodonium (DPI), and small interfering RNAs designed to target Nox4 mRNA (siNox4RNAs) inhibited superoxide production in PANC-1 pancreatic cancer cells, and depletion of ROS by DPI or siNox4RNAs induced apoptosis. Parallely, DPI treatment and siNox4RNA transfection blocked activation of the cell survival kinase AKT by attenuating phosphorylation of AKT. Furthermore, AKT phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) on Ser-83 was reduced by DPI and siNox4RNAs. When ASK1Ser83Ala (an AKT phosphorylation-defective ASK1 mutant) was introduced into PANC-1 cells, this mutant alone induced apoptosis. But, addition of DPI or co-transfection of siNox4RNA had no additive effect, indicating that the mutant can substitute for these reagents in apoptosis induction. Taken together, these findings suggest that ROS generated by Nox4, at least in part, transmit cell survival signals through the AKT-ASK1 pathway in pancreatic cancer cells and their depletion leads to apoptosis.

Adenocarcinoma↗

PubMed2006

Metabolism of the recently encountered designer drug, methylone, in humans and rats.

The urinary metabolites of methylone in humans and rats were investigated by analysing urine specimens from its abuser and after administrating to rats with gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS), using authentic standards. The time-course excretion profiles of methylone and its three metabolites in rats were further investigated after a single intraperitoneal dosing of 5 mg kg-1 methylone hydrochloride. Two major metabolic pathways were revealed for both humans and rats as follows: (1) side-chain degradation by N-demethylation to the corresponding primary amine methylenedioxycathinone (MDC), partly conjugated; and (2) demethylenation followed by O-methylation of either a 3- or 4-OH group on the benzene ring to produce 4-hydroxy-3-methoxymethcathinone (HMMC) or 3-hydroxy-4-methoxymethcathinone (3-OH-4-MeO-MC), respectively, mostly conjugated. Of these metabolites, HMMC was the most abundant in humans and rats. The cumulative amount of urinary HMMC excreted within the first 48 h in rats was approximately 26% of the dose, and the amount of the parent methylone was not more than 3%. These results demonstrate that the analysis of HMMC will be indispensable for proof of the use of methylone in forensic urinalysis.

Adult↗

PubMed2006

Megakaryocytes derived from human embryonic stem cells: a genetically tractable system to study megakaryocytopoiesis and integrin function.

BACKGROUND: The platelet fibrinogen receptor, a heterodimer consisting of integrin subunits alpha(IIb) and beta(3), is required for platelet aggregation, spreading, and hemostasis. Platelet agonists such as thrombin and adenosine diphosphate (ADP) lead to the activation of alpha(IIb)beta(3), thereby enhancing its affinity and avidity for binding fibrinogen (inside-out signaling). Furthermore, fibrinogen binding to alpha(IIb)beta(3) triggers cytoskeletal changes and granule release (outside-in signaling). AIM: Genetic approaches to characterize the molecular pathways involved in alpha(IIb)beta(3) signaling are not possible with anucleate blood platelets. Therefore, we have established an OP9 stromal cell co-culture system to generate megakaryocytes from human embryonic stem cells (hESCs). RESULTS: alpha(IIb)beta(3) activation, measured by soluble fibrinogen binding to hESC-derived megakaryocytes, /GPIbalpha(+) cells, is readily detectable following stimulation with known platelet agonists. Dose-response curves for peptide agonists specific for the two platelet thrombin receptors, protease-activated receptor 1 (PAR1) and PAR4, show a relative responsiveness that mirrors that of human platelets, and sub-maximal ADP responses are augmented by epinephrine. Moreover, hESC-derived megakaryocytes undergo lamellipodia formation, actin filament assembly, and vinculin localization at focal adhesions when plated on a fibrinogen-coated surface, characteristic of alpha(IIb)beta(3) outside-in signaling. Undifferentiated hESCs genetically modified by lentiviral infection can be cloned and maintained in an undifferentiated state and then differentiated into megakaryocytes capable of alpha(IIb)beta(3) activation. CONCLUSION: Using hESCs, we have developed a renewable source of human megakaryocytes, and a genetically tractable system for studying megakaryocytopoiesis and alpha(IIb)beta(3) signaling in the native cellular environment.

Cell Line↗

PubMed2005

Isolation, identification and excretion profile of the principal urinary metabolite of the recently banned designer drug 1-(3-trifluoromethylphenyl)piperazine (TFMPP) in rats.

The metabolism of 1-(3-trifluoromethylphenyl)piperazine (TFMPP), a recently banned designer drug, in rats was studied by analysing its urinary metabolites. p-Hydroxy-TFMPP (p-OH-TFMPP) was isolated and identified as the main metabolite by using nuclear magnetic resonance spectroscopy, gas chromatography-mass spectrometry and high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS). The time-course excretion profiles of TFMPP and p-OH-TFMPP in rats were investigated following a single intraperitoneal dosing of 5 mg kg(-1) TFMPP by using an optimized analytical procedure that combined solid-phase extraction and LC-ESI MS techniques. The cumulative amount of p-OH-TFMPP excreted within the first 48 h reached approximately 64% of the dose, of which 70% was the glucuronide conjugated form. The cumulative amount of parent TFMPP excreted was less than 0.7% of the dose. The results suggest that p-OH-TFMPP would be the most relevant metabolite to be detected for TFMPP exposure in the forensic and clinical analysis of human urine.

Animals↗

PubMed2005

Acute inflammatory sensorimotor polyradiculoneuropathy associated with immune thrombocytopenic purpura.

Although acute inflammatory polyneuropathy (AIP) and immune thrombocytopenic purpura (ITP) are both believed to be immune-mediated disorders, only a few cases have been reported in which these two diseases co-existed. We describe a case of a 67-year-old patient who developed quadriparesis, ophthalmoplegia and severe sensory impairment along with thrombocytopenia. Detailed examinations, including the measurement of anti-ganglioside antibodies and anti-glycoprotein-IIb-IIIa-IgG-producing B-cells, revealed that he developed AIP and ITP. By reviewing past similar reports, we noticed that AIP associated with ITP tends to manifest severe sensory impairment and is often preceded by upper respiratory tract infection, but not by gastrointestinal infection.

Acute Disease↗

PubMed2002

Effect of Seihai-to, a Kampo medicine, in relapsing aspiration pneumonia--an open-label pilot study.

Two published case reports described palliation of disease after Seihai-to therapy for refractory aspiration pneumonia caused by recurrent laryngeal nerve paralysis and cerebrovascular disease. We undertook an open-label trial in patients with relapsing aspiration pneumonia. Fifteen patients with relapsing aspiration pneumonia were randomly divided into conventional therapy group (n = 8) or Seihai-to group (n = 7). In Seihai-to group, patients were treated with Seihai-to in addition to conventional therapy (Western medicines). Frequency of feverish days and antibiotics-use, CRP value and chest CT or X-ray findings were compared between the two groups during the study period of 16 weeks. In the Seihai-to group, the latency of swallowing reflex was measured in 6 patients before and after administration of Seihai-to. The mean values of fever index, CRP value and antibiotics-use in the Seihai-to group were decreased significantly, compared with those of the conventional therapy group. However, the latency of the swallowing reflex after 4 weeks of treatment was not significantly changed (p = 0.249), compared with the latency before administration of Seihai-to. No adverse reaction was observed in either group. Seihai-to was effective in reducing relapse of aspiration pneumonia in this small group. Seihai-to might not improve the swallowing reflex, but might instead improve a defense mechanism or excessive inflammation caused by pneumonia in the lower airway. Further evaluation of Seihai-to therapy for patients with aspiration pneumonia in a larger population is warranted.

Aged↗

PubMed2002

Urinary excretion of selegiline N-oxide, a new indicator for selegiline administration in man.

1. The metabolism of selegiline (SG) has been studied by investigating the time-course of urinary excretion of SG and its metabolites using high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS) in combination with solid-phase extraction. 2. The excretion profiles of SG and its four major metabolites, selegiline-N-oxide (SGO), N-desmethylselegiline (DM-SG), methamphetamine (MA) and amphetamine (AP), were investigated in six healthy volunteers after oral administrations of SG hydrochloride in a single dose of 2.5 or 7.5mg, and a repeat twice-daily dose of 5.0 mg day(-1) (for 3 days). 3. The cumulative amount of SGO excreted within approximately the first 8-12h was comparable with MA, and the amount in the first 72 h was 2.0-7.8 times larger (2.8-13.2% of the dose) than that of DM-SG. 4. These results demonstrate that SGO can be used in place of DM-SG, which is known to be a main specific metabolite of SG, as a new indicator for the discrimination of SG use compared with MA abuse.

Adrenergic Uptake Inhibitors↗

PubMed2002

Complete response of Sister Mary Joseph Nodule from gastric adenocarcinoma treated with combination chemotherapy of low-dose S-1 and cisplatin.

A case of an unresected, advanced gastric cancer with Sister Mary Joseph nodule was presented. It was treated with new combination chemotherapy of low-dose S-1 and cisplatin producing complete response of periumbilical metastasis. Few treatments are efficacious for umbilical invasion of peritoneal dissemination. A complete response for Sister Mary Joseph nodule from gastric adenocarcinoma has not been ever reported.

Adenocarcinoma↗

PubMed2001

The Sos1-Rac1 signaling. Possible involvement of a vacuolar H(+)-ATPase E subunit.

We have purified and identified a 32-kDa protein interacting with the Dbl oncogene homology domain of mSos1(Sos-DH) from rat brains by glutathione S-transferase-Sos-DH affinity chromatography. Peptide sequencing revealed that the protein is identical to a positive regulatory E subunit (V-ATPase E) of a vacuolar H(+)-ATPase, which is responsible for acidification of endosome and alkalinization of intracellular pH. The interaction between V-ATPase E and Sos-DH was confirmed by yeast two-hybrid assay. A coimmunoprecipitation assay demonstrated that a V-ATPase E protein physiologically bound to mSos1, and the protein was colocalized with mSos1 in the cytoplasm, as determined by immunohistochemistry. mSos1 was found in the early endosome fraction together with V-ATPase E and Rac1, suggesting the functional involvement of mSos1/V-ATPase E complexes in the Rac1 activity at endosomes. Overexpression of V-ATPase E in COS cells enhanced the ability of mSos1 to promote the guanine nucleotide exchange activity for Rac1 and stimulated the kinase activity of Jun kinase, a downstream target of Rac1. Thus, the data indicate that V-ATPase E may participate in the regulation of the mSos1-dependent Rac1 signaling pathway involved in growth factor receptor-mediated cell growth control.

3T3 Cells↗

PubMed2001

Amino acid residues in the alpha IIb subunit that are critical for ligand binding to integrin alpha IIbbeta 3 are clustered in the beta-propeller model.

Several distinct regions of the integrin alpha(IIb) subunit have been implicated in ligand binding. To localize the ligand binding sites in alpha(IIb), we swapped all 27 predicted loops with the corresponding sequences of alpha(4) or alpha(5). 19 of the 27 swapping mutations had no effect on binding to both fibrinogen and ligand-mimetic antibodies (e.g. LJ-CP3), suggesting that these regions do not contain major ligand binding sites. In contrast, swapping the remaining 8 predicted loops completely blocked ligand binding. Ala scanning mutagenesis of these critical predicted loops identified more than 30 discontinuous residues in repeats 2-4 and at the boundary between repeats 4 and 5 as critical for ligand binding. Interestingly, these residues are clustered in the predicted beta-propeller model, consistent with this model. Most of the critical residues are located at the edge of the upper face of the propeller, and several critical residues are located on the side of the propeller domain. None of the predicted loops in repeats 1, 6, and 7, and none of the four putative Ca(2+)-binding predicted loops on the lower surface of the beta-propeller were important for ligand binding. The results map an important ligand binding interface at the edge of the top and on the side of the beta-propeller toroid, centering on repeat 3.

Amino Acid Sequence↗

PubMed2001

Platelet integrin alphaIIbbeta3-ligand interactions: what can we learn from the structure?

Upon vascular injury, platelets initiate interaction with exposed subendothelial matrices through various receptors such as glycoprotein (GP) Ib/IX/V complex, alpha2beta1 integrin, and GPVI/FcRgamma. Although these interactions cannot sustain stable platelet thrombus formation by themselves, they ultimately lead to the activation of alphaIIbbeta3 integrin (GPIIb-IIIa complex [GPIIb-IIIa]), the most abundant receptor in platelets. The alphaIIbbeta3 integrin plays a central role in primary hemostasis by serving as a receptor for fibrinogen and von Willebrand factor (vWf). It establishes a stable interaction with vWf bound to the extracellular matrices and uses fibrinogen as a bridging molecule in platelet aggregate formation. The alphaIIbbeta3 integrin also plays an important role in the pathogenesis of thrombosis. Over the past decades, a tremendous amount of effort has been made to elucidate the ligand-binding mechanisms of alphaIIbbeta3, in part because of its clinical significance. Most of the studies have relied on biochemical analyses of purified alphaIIbbeta3 or recombinant proteins generated in vitro. With the lack of actual 3-dimensional structure, molecular modeling has provided a useful framework for interpreting such experimental data on structure-function correlation of integrin molecules. However, it has also generated disagreement between different models. The aim of this minireview is to summarize the past efforts as well as the recent accomplishments in elucidating the structure/function of alphaIIbbeta3. Finally, we will try to explain all those experimental data using the recently published crystal structure of the extracellular domains of the alphaVbeta3 heterodimeric complex.

Animals↗

PubMed2001

Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site.

The beta2 integrin leukocyte function antigen-1 (LFA-1) has an important role in the pathophysiology of inflammatory and autoimmune diseases. Here we report that statin compounds commonly used for the treatment of hypercholesterolemia selectively blocked LFA-1-mediated adhesion and costimulation of lymphocytes. This effect was unrelated to the statins' inhibition of 3-hydroxy-3-methylglutaryl coenzyme-A reductase; instead it occurred via binding to a novel allosteric site within LFA-1. Subsequent optimization of the statins for LFA-1 binding resulted in potent, selective and orally active LFA-1 inhibitors that suppress the inflammatory response in a murine model of peritonitis. Targeting of the statin-binding site of LFA-1 could be used to treat diseases such as psoriasis, rheumatoid arthritis, ischemia/reperfusion injury and transplant rejection.

Allosteric Site↗

PubMed2001

Biphasic alterations in gastrointestinal transit following endotoxaemia in mice.

Lipopolysaccharide (LPS)-induced alterations of gastrointestinal transit were studied in mice using activated charcoal. LPS (10 mg kg-1) induced biphasic alterations of intestinal transit. Increase (acceleration phase) and delay (lag phase) in gastrointestinal transit were observed at 90 and 480 min after LPS injection, respectively. LPS administration induced significant increases in tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta and nitrate levels in blood serum with maximal levels observed at 1.5, 4, and 8 h after LPS administration, respectively. The effects of recombinant human lzactoferrin (rhLF) on LPS- induced alteration of gastrointestinal transit, and production of TNF-alpha, IL-1beta and nitrate were also studied. Animals were pretreated with rhLF 24 hours before intraperitoneal administration of LPS. RhLF significantly increased gastrointestinal transit during the lag phase. In addition, rhLF decreased the level of TNF-alpha in endotoxaemic animals. The levels of IL-1beta and nitrate were not significantly changed by rhLF. In conclusion, the effect of LPS on gastrointestinal transit is biphasic and the mechanism controlling the second phase most likely depends on TNF-alpha production, while the first phase most likely does not depend on TNF-alpha. On the other hand, it may be regulated by IL-1beta and nitric oxide production.

Animals↗

PubMed2001

Coagulation process proceeds on cultured human mesangial cells via expression of factor V.

BACKGROUND: In a previous clinicopathological study, we observed mesangial factor V expression accompanied by the intact form of cross-linked fibrin deposition in the active type of IgA nephropathy. The conversion of prothrombin to thrombin by factor Xa is potently accelerated more than 104-fold by the presence of factor V, which is a membrane-bound cofactor. Another membrane-bound cofactor, tissue factor, is known to play an initiating role in the coagulation cascade and to be synthesized in mesangial cells (MCs) by the stimulation of tumor necrosis factor-alpha (TNF-alpha). However, the synthesis of factor V, which plays on the terminating stage of prothrombin activation, has not been reported previously in MCs by in vitro study. Our current study tested the coagulation process via expression of factor V by the stimulation of proinflammatory cytokine, TNF-alpha, in cultured human MCs. METHODS: To evaluate factor V protein expression, immunoperoxidase staining with densitometric evaluation and Western blot analysis were conducted after stimulation of TNF-alpha. To test factor V activity, stimulated MCs were incubated in combination with factor Xa, prothrombin, fibrinogen and factor XIII, and fibrin production on MCs was assessed after immunoperoxidase staining on the cell surface. In a blocking test using an antibody against factor V, suppression of fibrin production was evaluated to clarify the role of factor V activity. For the evaluation of factor V mRNA expression in cultured human MCs, in situ hybridization and Northern blot analysis were performed. RESULTS: Factor V protein expression in MCs after TNF-alpha stimulation increased both time- and dose-dependently. As a marker of factor V activity with exogenous factor Xa, fibrin production on TNF-alpha-stimulated MCs was increased in a time-dependent manner and was inhibited by the addition of anti-factor V antibody. Factor V mRNA was identified in MCs by in situ hybridization and showed an increase after stimulation with TNF-alpha on Northern blot analysis. CONCLUSIONS: Our data suggest that the coagulation process proceeds on MCs as the result of increased expression of endogenous factor V activity on its cell surface in cooperation with exogenous factor Xa.

Antibodies↗

PubMed2001

Some Lactobacillus L-lactate dehydrogenases exhibit comparable catalytic activities for pyruvate and oxaloacetate.

The nonallosteric and allosteric L-lactate dehydrogenases of Lactobacillus pentosus and L. casei, respectively, exhibited broad substrate specificities, giving virtually the same maximal reaction velocity and substrate K(m) values for pyruvate and oxaloacetate. Replacement of Pro101 with Asn reduced the activity of the L. pentosus enzyme toward these alternative substrates to a greater extent than the activity toward pyruvate.

Amino Acid Sequence↗

PubMed2001

Up-regulated TGF-beta mRNA expression in splenic T cells of high IgA-prone mice: a murine model of IgA nephropathy with glomerulosclerosis.

BACKGROUND/AIMS: Recently, we established a high serum IgA-prone inbred (HIGA) mouse strain as a murine model of spontaneous IgA nephropathy by selective mating of high serum IgA ddY mice, and found that they showed enhanced production of glomerular extracellular matrix components with increased expression of TGF-beta mRNA and protein in the kidneys. In this study, we examined the roles of lymphocytes in the development of high serum IgA in this strain. METHODS: We performed flow cytometric analyses of T and B cells in splenic mononuclear cells (SMNCs) from these mice using BALB/c mice as normal controls. We also compared serum TGF-beta1 concentrations and TGF-beta mRNA expression levels in the B-cell-depleted (T-cell-rich) fraction of SMNCs in these mice. RESULTS: HIGA mice showed significantly fewer CD3-positive cells compared with BALB/c mice when young, but not when aged. The CD4/CD8 ratio of HIGA mice was lower than that of BALB/c mice, but this difference was not significant. Although the number of B220-positive cells did not vary significantly, the ratio of surface IgA-positive B cells was significantly increased in both young and adult HIGA mice. The B-cell-depleted SMNCs from HIGA mice exhibited higher levels of expression of TGF-beta and TGF-beta1 mRNA than controls from a young age, which were maintained throughout life, but there were no differences in PDGF, MCP-1 or bFGF expression between these two strains. In contrast to local mRNA expression, serum TGF-beta1 concentration was decreased in HIGA mice compared with BALB/c controls. CONCLUSION: These findings suggest that the mating procedure performed to establish HIGA mice selected for a unique phenotype of local up-regulation of TGF-beta production in the kidneys, as well as T cells that may contribute to both the early and consistently high serum IgA expression and enhanced production of renal extracellular matrix components in HIGA mice. Additionally, TGF-beta1 may act locally, not systemically, in a paracrine or autocrine manner.

Animals↗

PubMed2001

Altered cultured mesangial cell phenotypes from RF/J mice: a spontaneous immune complex mediated glomerulonephritis with progressive glomerulosclerosis.

BACKGROUND/AIM: RF/J mice are a model of spontaneous immune complex mediated glomerulonephritis showing massive extracellular matrix accumulation and progressive glomerulosclerosis. The aim of this study was to investigate whether there is an altered cultured mesangial cell (MC) phenotype in RF/J mice associated with these glomerular changes. METHODS: The nature of cultured MCs from RF/J mice in the proliferative response to platelet-derived growth factor (PDGF) BB was compared with that of normal mice (BALB/c) by 3H-thymidine incorporation. The binding of PDGF-BB was examined with Scatchard analysis, and the messenger RNAs (mRNAs) of PDGF beta-receptor, collagen I, collagen IV, and fibronectin were detected using Northern blot analysis in the MCs of each mouse. RESULTS: The 3H-thymidine incorporation of MCs from RF/J mice showed significantly lower responses to PDGF-BB stimulations with concentrations ranging from 0.5 to 10.0 ng/ml in comparison with those of BALB/c mice which exhibited a proportional dose- dependent increase of the incorporation (p < 0.05 for 0.5 ng/ml PDGF-BB, p < 0.01 for 1.0-10.0 ng/ml). According to the Scatchard analysis, MCs from BALB/c mice showed aKD of 105 pM of PDGF-BB binding to its receptors, and the density of receptors was 5.82 fmol/10(5) cells. However, no binding PDGF-BB site on the surface of MCs from RF/J mice was noted. Northern blot analysis of MCs from RF/J mice indicated negative expression of detectable PDGF-beta receptor mRNA. As for matrix protein messages, MCs from RF/J mice did not express mRNA of type I collagen, but did express a higher amount of type IV collagen and fibronectin in comparison with MCs from normal BALB/c mice. CONCLUSIONS: An altered phenotype in MCs of RF/J mice was demonstrated, possibly contributing to the characteristic pathological glomerular changes. However, the precise association remains to be clarified.

Animals↗

PubMed2001

Initial symptoms of acute radiation syndrome in the JCO criticality accident in Tokai-mura.

A criticality accident occurred on September 30, 1999, at the uranium conversion plant in Tokai-mura (Tokai-village), Ibaraki Prefecture, Japan. When the criticality occurred, three workers saw a "blue-white glow," and a radiation monitor alarm was sounded. They were severely exposed to neutron and gamma-ray irradiation, and subsequently developed acute radiation syndrome (ARS). One worker reported vomiting within minutes and loss of consciousness for 10-20 seconds. This worker also had diarrhea an hour after the exposure. The other worker started to vomit almost an hour after the exposure. The three workers, including their supervisor, who had no symptoms at the time, were brought to the National Mito Hospital by ambulance. Because of the detection of gamma-rays from their body surface by preliminary surveys and decreased numbers of lymphocytes in peripheral blood, they were transferred to the National Institute of Radiological Sciences (NIRS), which has been designated as a hospital responsible for radiation emergencies. Dose estimations for the three workers were performed by prodromal symptoms, serial changes of lymphocyte numbers, chromosomal analysis, and 24Na activity. The results obtained from these methods were fairly consistent. Most of the data, such as the dose rate of radiation, its distribution, and the quality needed to evaluate the average dose, were not available when the decision for hematopoitic stem cell transplantation had to be made. Therefore, prodromal symptoms may be important in making decisions for therapeutic strategies, such as stem-cell transplantation in heavily exposed victims.

Amylases↗