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Biomedical subjects

K Satoh

Publications and source records attributed to K Satoh.

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

Total sleep deprivation induces an acute and transient increase in NK cell activity in healthy young volunteers.

STUDY OBJECTIVES: To investigate the effects of one night's total sleep deprivation (TSD) on NK cell activity, with rigorous control of circadian phase of sampling points as well as physical exercise level in association with sleep deprivation. DESIGN: The mean sleep onset time of each subject before starting the study was defined as his 0000 h. This study was composed of a Sleep-Sleep session (sleep times, 00:00 h - 08:00 h and 24:00 h - 32:00 h) and a Sleep-Wake session (sleep time, 00:00 h - 08:00 h) with TSD (24:00 h - 32:00 h) placed in a cross-over design with 2-week interval between each session. In each session, the subjects were rested in the supine position under dim light from - 06:00 h to 36:00 h (for 42 hours). SETTING: University-based sleep and chronobiology laboratory PARTICIPANTS: 10 healthy adult men (mean age, 20.9 y; age range, 19-23 y) INTERVENTIONS: NA. MEASUREMENTS AND RESULTS: NK cell activity was measured every 4 hours from 12:00 h. NK cell activity during TSD (at 28:00 h) has been revealed to significantly increase (p=0.01) compared with the corresponding value in the Sleep-Sleep session. This effect was weaker at their usual waking time 32:00 h (p=0.07), and disappeared until 36:00 h (4 hours after awakening). The circadian rhythm phases (dim light melatonin onset time) were coincident between the 2 sessions. CONCLUSIONS: The present findings suggest that one night TSD induces an acute and transient increase in NK cell activity that is not influenced by the effects of circadian rhythm or the amount of physical exercise undertaken during TSD.

Adult↗
PubMed2001

Wheat germ agglutinin-induced platelet activation via platelet endothelial cell adhesion molecule-1: involvement of rapid phospholipase C gamma 2 activation by Src family kinases.

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a 130K transmembrane glycoprotein that belongs to the immunoglobulin gene superfamily and is expressed on the surface of hematological or vascular cells, including platelets and endothelial cells. Although the importance of this adhesion molecule in various cell-cell interactions is established, its function in platelets remains ill-defined. In the process of clarifying the mechanism by which the lectin wheat germ agglutinin (WGA) activates platelets, we unexpectedly discovered that PECAM-1 is involved in signal transduction pathways elicited by this N-acetyl-D-glucosamine (NAGlu)-reactive lectin. WGA, which is a very potent platelet stimulator, elicited a rapid surge in Syk and phospholipase C (PLC)-gamma 2 tyrosine phosphorylation and the resultant intracellular Ca(2+) mobilization; collagen, as reported, induced these responses, but in a much slower and weaker manner. WGA strongly induced tyrosine phosphorylation of a 130-140K protein, which was confirmed to be PECAM-1 by immunoprecipitation and immunodepletion studies. WGA-induced PECAM-1 tyrosine phosphorylation occurred rapidly, strongly and in a manner independent of platelet aggregation or cell-cell contact; these characteristics of PECAM-1 phosphorylation were not mimicked at all by receptor-mediated platelet agonists. In addition, WGA was found to associate with PECAM-1 itself, and anti-PECAM-1 antibody, as well as NAGlu, specifically inhibited WGA-induced platelet aggregation. In PECAM-1 immunoprecipitates, Src family tyrosine kinases existed, and a kinase activity was detected, which increased upon WGA stimulation. Furthermore, the Src family kinase inhibitor PP2 inhibited WGA-induced platelet aggregation, Ca(2+) mobilization, and PLC-gamma 2 tyrosine phosphorylation. Finally, WGA induced PECAM-1 tyrosine phosphorylation and cytoskeletal reorganization in vascular endothelial cells. Our results suggest that (i) PECAM-1 is involved in WGA-induced platelet activation, (ii) PECAM-1 clustering by WGA activates unique and strong platelet signaling pathways, leading to a rapid PLC activation via Src family kinases, and (iii) WGA is a useful tool for elucidating PECAM-1-mediated signaling with wide implications not confined to platelets.

Acetylglucosamine↗
PubMed2001

Effects of lidocaine on blood flow and endoneurial fluid pressure in a rat model of herniated nucleus pulposus.

STUDY DESIGN: An experimental study was conducted to evaluate the effects of lidocaine on nucleus pulposus-induced pathophysiologic changes. OBJECTIVES: To investigate the effects of lidocaine on blood flow in the hind paws and endoneurial fluid pressure in the dorsal root ganglia in a rat model of herniated nucleus pulposus, and to clarify the therapeutic mechanisms of nerve root infiltration. SUMMARY OF BACKGROUND DATA: It has been shown experimentally that application of nucleus pulposus to the nerve roots increases endoneurial fluid pressure and decreases blood flow in the dorsal root ganglia and the corresponding hind paw. These changes are thought to be an important pathogenic mechanism associated with sciatica caused by disc herniation. Nerve root infiltration is one of the nonoperative effective therapies for radiculopathy caused by disc herniation. However, the therapeutic mechanisms still are unknown. METHODS: For this study, 21 Sprague-Dawley rats were used. Autologous nucleus pulposus was applied to the nerve root with a piece of Spongel containing lidocaine (lido group) or physiologic saline solution (control group). In Series 1 of this study (Blood Flow in the Hind Paw), blood flow in the corresponding hind paws was monitored continuously using a laser Doppler flowmeter before application of the test solutions, and every 5 minutes thereafter for an additional 3 hours in both the control (n = 5) and lido (n = 5) groups. In Series 2 of this study (Endoneurial Fluid Pressure in the Dorsal Root Ganglion), endoneurial fluid pressure was recorded with a servo-null micropipette system using glass micropipettes before and 3 hours after application of the test solutions in both the control (n = 6) and lido (n = 5) groups. After measurements, dorsal root ganglia were assessed for histology. RESULTS: In Series 1, blood flow in the corresponding hind paw in the control group showed significant reduction as compared with that of the Lido group, starting about 90 minutes after application (P < 0.01-0.05). Hind paw blood flow in the lido group did not show any reduction during measurements. In Series 2, the value of endoneurial fluid pressure in the lido group 3 hours after application was significantly lower than in the control group (P < 0.01). Interstitial (endoneurial) edema in the dorsal root ganglion in the lido group appeared to be qualitatively less than in the control group. CONCLUSIONS: The data indicate that lidocaine reduces the pathophysiologic changes in the dorsal root ganglion and hind paws induced by nucleus pulposus. These effects of lidocaine may relate to the mechanisms underlying the therapeutic effects of nerve root infiltration.

Anesthetics, Local↗
PubMed2001

Expression of beta-synuclein in normal human astrocytes.

Although alpha- and beta-synucleins are expressed predominantly in presynaptic nerve terminals, recent studies have demonstrated that alpha-synuclein is also expressed in cultured astrocytes and oligodendrocytes. We determined whether beta-synuclein might be expressed in astrocytes. Beta-synuclein mRNA and protein were detected in normal human astrocytes in culture, and immunofluorescent staining showed that beta-synuclein protein was expressed within the cytoplasm and nucleus. Furthermore, beta-synuclein immunoreactivity was present in astrocytes, but not in oligodendrocytes, in normal human brain tissues. Ultrastructurally, beta-synuclein immunoreactivity was found in the cytoplasm of astrocytes, in association with the plasma membrane, ribosomes, rough endoplasmic reticulum and the nuclear outer membrane. The novel expression of beta-synuclein in astrocytes may provide an important insight about the role of this protein.

Astrocytes↗
PubMed2001

Soluble interleukin-6 receptor alpha inhibits the cytokine-Induced fractalkine/CX3CL1 expression in human vascular endothelial cells in culture.

Soluble form of IL-6 receptor alpha (sIL-6R) is known to serve as an agonist, without exogenous IL-6, on endothelial cells which do not express IL-6R but have only IL-6 receptor beta chain, gp130. We investigated the effect of sIL-6R on fractalkine expression in human umbilical vein endothelial cells (HUVECs) in culture. sIL-6R markedly inhibited HUVEC fractalkine/CX3CL1 expression induced by interleukin (IL)-1alpha, tumor necrosis factor (TNF)-alpha, or interferon (IFN)-gamma. IL-1alpha-induced fractalkine expression was inhibited by sIL-6R in time- and concentration-dependent manners. The experiment using actinomycin D indicated that sIL-6R lowered the stability of fractalkine mRNA. The inhibitory effect of sIL-6R was reversed by anti-gp130 neutralizing antibody. sIL-6R inhibited adhesion of mononuclear cells (MNCs) to HUVEC monolayers stimulated with IFN-gamma, but it did not inhibit the adhesion to monolayers stimulated with IL-1alpha. MNC chemotactic activity of conditioned medium of HUVEC stimulated with IL-1alpha or IFN-gamma was inhibited by co-treatment with sIL-6R. sIL-6R may play a regulatory role in immune responses by modulating the interaction between leukocytes and the vascular endothelium.

Cell Adhesion↗
PubMed2001

DNA demethylase is expressed in ovarian cancers and the expression correlates with demethylation of CpG sites in the promoter region of c-erbB-2 and survivin genes.

The objectives of this study were to examine DNA demethylase (dMTase) expression in ovarian cancers and evaluate methylation of CpG sites in the promoter of the c-erbB-2 gene and survivin gene exon 1. Forty-three epithelial ovarian cancers and 43 non-cancerous ovarian tissues were studied for dMTase expression by RT-PCR. Genomic DNA was extracted and digested with HindIII and then HpaII. CpG site-sensitive primers were constructed to amplify the promoter of the c-erbB-2 gene and survivin gene exon 1. Immunohistochemical evaluation of ErbB-2 protein and RT-PCR for survivin were also performed. dMTase was positive in 88.4% of ovarian cancers but only in 9.3% of non-cancerous ovaries (P<0.001, Fisher's exact test). The expression was similarly observed in both early stage (stage I+II: 17/19) and advanced stage (stage III+IV: 21/24) groups of ovarian malignancy. It was found that 78.9% of dMTase-positive cancers had both c-erbB-2 promoter and survivin gene exon 1 unmethylated, whereas 40% of dMTase-negative cancers had both sites methylated. In non-cancerous ovaries, these sites were mostly methylated (90.6%) and the difference from cancer cases was highly significant (P<0.001). Immunohistochemical evaluation of ErbB-2 showed significant correlation of unmethylated c-erbB-2 promoter and ErbB-2 expression. The RT-PCR for survivin expression showed that 86% of cancers were positive and six cases were negative. Exon 1 was methylated in 83% of the survivin-negative cases. This is the first report of dMTase expression in ovarian cancers. The correlation of dMTase expression with unmethylation of c-erbB-2 promoter and survivin gene exon 1 suggests that these sites may be targets for demethylation by the enzyme. The up-regulation of oncogenes may be the consequence of epigenetic control of gene expression by the dMTase.

Adolescent↗
PubMed2001

Decreased bone mass and increased bone turnover with valproate therapy in adults with epilepsy.

BACKGROUND: Bone loss and hypovitaminosis D are reported in patients taking antiepileptic drugs, but little is known about changes in bone and calcium metabolism from valproic acid (VPA). OBJECTIVE: To assess the relationship of VPA to bone mass and calcium metabolism in 40 adults with epilepsy on long-term VPA monotherapy, 40 age- and sex-matched epileptic patients taking phenytoin (PHT), and 40 healthy control subjects. Bone mineral density (BMD) of the second metacarpal was determined as T- and Z-scores. RESULTS: BMD reduction from control values was 14% (12% in men, 16% in women) with VPA and 13% (12% in men, 15% in women) with PHT. Among patients on VPA, nine (23%) had T-scores below -2.5 SD, suggesting osteoporosis; 15 (37%) had T-scores between -1 and -2.5 SD, suggesting osteopenia. Serum concentrations of calcium were significantly higher with VPA than in PHT or control groups. Serum concentrations of bone Gla protein (a bone formation marker) and pyridinoline cross-linked carboxy-terminal telopeptide of type I collagen (ICTP; a bone resorption marker) associated with either drug significantly exceeded control values. Z-scores for BMD in the VPA group correlated negatively with calcium and ICTP. High ICTP correlated positively with ionized calcium, implying that increased bone resorption caused the latter. CONCLUSION: Long-term VPA monotherapy can increase bone resorption, leading to decreased BMD.

Adult↗
PubMed2001

Expression of survivin is correlated with cancer cell apoptosis and is involved in the development of human pancreatic duct cell tumors.

BACKGROUND: Survivin is a new member of the inhibitor of apoptosis family of antiapoptotic proteins. This protein was expressed selectively in all the most common human carcinomas but not in normal adult tissues. To our knowledge, the relation between survivin expression and apoptosis or tumorigenesis has not yet been studied in pancreatic neoplasms. METHODS: The authors investigated the expression of survivin in 4 pancreatic carcinoma cell lines and 56 human pancreatic tissues (5 cases of normal, 12 cases of chronic pancreatitis [CP], 26 cases of pancreatic duct cell adenocarcinoma [PDC], 16 lesions of 13 intraductal papillary-mucinous tumor [IPMT] by immunohistochemistry, immunoblotting, and reverse transcription-polymerase chain reaction (RT-PCR) to examine the association of its expression with tumor apoptosis and/or tumorigenesis. RESULTS: Survivin expression was found in the tumor cells but not in the nonneoplastic pancreatic tissues (normal and CP tissues). Survivin expression was observed in 20 of 26 cases of PDC (76.9%) and in 9 of 16 IPMT lesions that ranged from adenoma to invasive (56.3%) by immunohistochemistry. Survivin was more frequently expressed in malignant tumors than in benign tumors (P = 0.0089). In PDC, high levels of survivin expression were associated significantly with a reduction in the apoptotic index of the tumor cells (0.445% +/- 0.150% vs. 0.961% +/- 0.378%; P < 0.0001). CONCLUSIONS: These data suggest that the expression of survivin may be upregulated during an early step of tumorigenesis and during the development of cancer by reducing the cancer cell apoptosis.

Adenocarcinoma↗
PubMed2001

Mutations of either or both Cys876 and Cys888 residues of sarcoplasmic reticulum Ca2+-ATPase result in a complete loss of Ca2+ transport activity without a loss of Ca2+-dependent ATPase activity. Role of the CYS876-CYS888 disulfide bond.

Disulfide-containing peptides in pepsin digest of sarcoplasmic reticulum vesicles were identified by using a fluorogenic thiol-specific reagent 4-fluoro-7-sulfamoylbenzofurazan and a reductant tributylphosphine. Sequencing of the purified peptides revealed the presence of a Cys(876)-Cys(888) disulfide bond on the luminal loop connecting the 7th and 8th transmembrane helices (loop 7-8) of the Ca(2+)-ATPase (SERCA1a). We substituted either or both of these cysteine residues with alanine and made three mutants (C876A, C888A, C876A/C888A), in which the disulfide bond is disrupted. The mutants and the wild type were expressed in COS-1 cells, and functional analysis was performed with the microsomes isolated from the cells. Electrophoresis performed under reducing and non-reducing conditions confirmed the presence of Cys(876)-Cys(888) disulfide bond in the expressed wild type. All the three mutants possessed high Ca(2+)-ATPase activity. In contrast, no Ca(2+) transport activity was detected with these mutants. These mutants formed almost the same amount of phosphoenzyme intermediate as the wild type from ATP and from P(i). Detailed kinetic analysis showed that the three mutants hydrolyze ATP in the mechanism well accepted for the Ca(2+)-ATPase; activation of the catalytic site upon high affinity Ca(2+) binding, formation of ADP-sensitive phosphoenzyme, subsequent rate-limiting transition to ADP-insensitive phosphoenzyme, and hydrolysis of the latter phosphoenzyme. It is likely that the pathway for delivery of Ca(2+) from the binding sites into the lumen of vesicles is disrupted by disruption of the Cys(876)-Cys(888) disulfide bond, and therefore that the loop 7-8 having the disulfide bond is important for formation of the proper structure of the Ca(2+) pathway.

Adenosine Triphosphate↗
PubMed2001

Expression of alpha-synuclein in a human glioma cell line and its up-regulation by interleukin-1beta.

Although alpha-synuclein is expressed primarily in neurons, it is a major component of oligodendroglial and astrocytic inclusions in several neurodegenerative diseases. Recent study has further demonstrated that alpha-synuclein is expressed in cultured rat oligodendrocytes. We determined whether alpha-synuclein might be expressed in astrocytic cells. alpha-Synuclein mRNA and protein were detected in U251 human astrocytic glioma cells and normal human astrocytes, and the levels were increased in the former, but not in the latter, by stimulation with interleukin-1beta in a time- and concentration-dependent manner. Serum deprivation also led to an increase of alpha-synuclein mRNA and protein in U251 cells. Immunofluorescent staining confirmed the cell-associated alpha-synuclein. These findings suggest that human astrocytes can produce alpha-synuclein in culture and that certain inflammatory cytokines and cell stress increase alpha-synuclein expression.

Astrocytes↗
PubMed2001

Role of Fc receptor gamma-chain in platelet glycoprotein Ib-mediated signaling.

Interaction between von Willebrand factor (vWF) and glycoprotein Ib (GPIb) stimulates tyrosine kinases and subsequent tyrosine phosphorylation events in human platelets. This study found that the combination of vWF and botrocetin, by interacting with GPIb, induced tyrosine phosphorylation of Fc receptor gamma-chain (FcR gamma-chain), Syk, linker for activation of T cells (LAT), and phospholipase C gamma2 (PLCgamma2). Pretreatment of platelets with 10 microM PP1 completely inhibited these tyrosine phosphorylation events. On GPIb stimulation, Src and Lyn formed a complex with FcR gamma-chain and Syk, suggesting that Src and Lyn are involved in FcR gamma-chain tyrosine phosphorylation and downstream signals. In spite of the PLCgamma2 tyrosine phosphorylation, however, there was no intracellular calcium release and inositol 1,4,5-trisphosphate production. In Brij 35 lysates, FcR gamma-chain was found to constitutively associate with GPIb. The number of GPIb expressed on FcR gamma-chain-deficient platelets was comparable to that of the wild-type, as assessed by flow cytometry. However, tyrosine phosphorylation of Syk, LAT, and PLCgamma2 in response to vWF plus botrocetin was significantly suppressed, suggesting that FcR gamma-chain mediates activation signals related to GPIb. Compared with the aggregation response of wild-type platelets, that of FcR gamma-chain-deficient platelets in response to vWF plus botrocetin was impaired, implying that FcR gamma-chain is required for the full activation of platelets mediated by GPIb. (Blood. 2001;97:3836-3845)

Adaptor Proteins, Signal Transducing↗
PubMed2001

Amino acid residues that are critical for in vivo catalytic activity of CtpA, the carboxyl-terminal processing protease for the D1 protein of photosystem II.

CtpA, a carboxyl-terminal processing protease, is a member of a novel family of endoproteases that includes a tail-specific protease from Escherichia coli. In oxygenic photosynthetic organisms, CtpA catalyzes C-terminal processing of the D1 protein of photosystem II, an essential event for the assembly of a manganese cluster and consequent light-mediated water oxidation. We introduced site-specific mutations at 14 conserved residues of CtpA in the cyanobacterium Synechocystis sp. PCC 6803 to examine their functional roles. Analysis of the photoautotrophic growth capabilities of these mutants, their ability to process precursor D1 protein and hence evolve oxygen, along with an estimation of the protease content in the mutants revealed that five of these residues are critical for in vivo activity of CtpA. Recent x-ray crystal structure analysis of CtpA from the eukaryotic alga Scenedesmus obliquus (Liao, D.-I., Qian, J., Chisholm, D. A., Jordan, D. B. and Diner, B. A. (2000) Nat. Struct. Biol. 7, 749-753) has shown that the residues equivalent to Ser-313 and Lys-338, two of the five residues mentioned above, form the catalytic center of this enzyme. Our in vivo analysis demonstrates that the three other residues, Asp-253, Arg-255, and Glu-316, are also important determinants of the catalytic activity of CtpA.

Algal Proteins↗
PubMed2001

Follicle-stimulating hormone promotes the growth of human epithelial ovarian cancer cells through the protein kinase C-mediated system.

We have previously described that follicle-stimulating hormone (FSH) stimulated the growth of human epithelial ovarian cancer tissues and cells. In order to determine the signaling pathway on FSH action in ovarian cancer, we used an epithelial ovarian cancer cell line (HRA line) which constitutively FSH receptors (FSHRs). FSH significantly increased cell proliferation (230.1 +/- 20.5%, P < 0.05) and (3)H-thymidine uptake (443.5 +/- 35.1%, P < 0.01). 1-(5-Isoquinolinesulfonyl)-2-methyipiperazine (H7, 1 5 nM), staurosponine (STR, 5 nM) and calphostin C (5 nM), specific protein kinase C (PKC) inhibitors, significantly suppressed the FSH-stimulated cell growth (120.2-140.2%, P < 0.05) and (3)H-thymidine uptake (140.5-173.9%, P < 0.05), whereas N-(2-guanidinoethyl)-5-isoquinoline-sulfon-amide (HA1004, l5 nM), which is a derivant of H7 and inhibits most of protein kinases except PKC, showed no effect on the FSH-stimulated cell growth and (3)H-thymidine uptake. A pretreatment with 12-0-tetradecanoylphorbol-13 acetate (TPA, 100 ng/ml) or STR (20 nM) significantly suppressed the subsequent FSH-stimulated cell growth (TPA; 152.3 +/-10.3%, STR; 160.4 +/- 15.9%, P < 0.05) and (3)H-thymidine uptake (TPA; 250.4 +/-18.3%, STR; 208.7 +/- 15.9%, P < 0.05). STR abolished the suppression of TPA preincubation on the subsequent FSH-stimulated cell growth and (3)H-thymidine uptake. HRA cells constitutively expressed PKCalpha but not PKCbeta nor PKCgamma. The levels of either expression of PKCalpha protein and mRNA were significantly amplified by FSH. These data suggest that stimulation of PKCalpha transcription is involved in the FSH-stimulated cell growth and DNA synthesis in epithelial ovarian cancer cells.

Cell Division↗
PubMed2001

Synergistic stimulation, by tumor necrosis factor-alpha and interferon-gamma, of fractalkine expression in human astrocytes.

Fractalkine is a CX3C chemonkine that appears to be a neuron-to-microglia signal molecule in the central nervous system. We studied the expression of fractalkine in normal human astrocytes in culture, by using semi-quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. We found that tumor-necrosis factor alpha (TNF-alpha) and interferon-gamma (IFN-gamma) synergistically enhance the expression of fractalkine. The expression of both fractalkine mRNA and protein was increased in time- and concentration-dependent manners in the cells co-stimulated with TNF-alpha and IFN-gamma. Cycloheximide, an inhibitor of protein synthesis, and dexamethasone had no effect on the synergy of the stimulation of fractalkine expression. We conclude that normal human astrocytes produce fractalkine by co-stimulation with pro-inflammatory cytokines and it may serve as a potential signal for immune and inflammatory responses in the central nervous system.

Astrocytes↗
PubMed2001

Potentiation of ibudilast inhibition of platelet aggregation in the presence of endothelial cells.

Although communications between platelets and endothelial cells or other blood cells are important in in vivo thrombus formation, laboratory platelet function tests are usually performed in isolation from these surrounding cells. In this study, we evaluated the effect of an antiplatelet drug, ibudilast (3-isobutyryl-2-isopropylpyrazolo[1,5-a]pyridine), on platelet aggregation in the presence and absence of human umbilical vein endothelial cells (HUVECs) and with the use of platelet-rich plasma (PRP) or whole blood as platelet samples. Stimulation-dependent platelet aggregation was weakened in the presence of HUVECs, which was especially prominent when the thrombin receptor-activating peptide SFLL (compared with ADP and epinephrine) was used as an aggregating agent. Ibudilast hardly affected SFLL-induced platelet aggregation (in PRP), while this antiplatelet agent was found to clearly inhibit this SFLL-induced response in a concentration-dependent manner, in the presence of HUVECs. Ibudilast tended to inhibit ADP- or epinephrine-induced platelet aggregation in the presence of HUVECs, but the effects were not statistically significant. Enhanced inhibition by ibudilast of SFLL-induced platelet aggregation (in the presence of HUVECs) was reproduced with the use of whole blood samples when a screen filtration pressure method was employed. It is suggested that the platelet aggregation studies in the presence of endothelial cells and/or other blood cells provide us with valuable information on platelet reactivity in vivo and improvement of antiplatelet therapy.

Blood Platelets↗
PubMed2001

Involvement of Hic-5 in platelet activation: integrin alphaIIbbeta3-dependent tyrosine phosphorylation and association with proline-rich tyrosine kinase 2.

Hic-5 and paxillin, members of the LIM protein family, have been shown to be localized in focal adhesion and to have a role in integrin-mediated signalling. In the present study we examined the involvement of Hic-5 in human platelet activation: platelets express Hic-5 but not paxillin, whereas human umbilical-vein vascular endothelial cells and MEG-01 cells express mainly paxillin. When platelets were stimulated with thrombin, collagen or the stable thromboxane A(2) analogue U46619, Hic-5 was markedly tyrosine-phosphorylated, in a manner dependent on integrin alphaIIbbeta3-mediated aggregation. In addition, direct activation of protein kinase C with PMA resulted in tyrosine phosphorylation of Hic-5 only when platelets were fully aggregated with the exogenous addition of fibrinogen. Furthermore, PMA-induced Hic-5 tyrosine phosphorylation was also observed when platelets adhered to immobilized fibrinogen. In studies on immunoprecipitation and immunodepletion, Hic-5 seemed to associate with proline-rich tyrosine kinase 2 (Pyk2) but only marginally with focal adhesion kinase. When platelets were stimulated with thrombin, both Hic-5 and Pyk2 translocated to the cytoskeleton from the cytosol and membrane fractions in a manner dependent on alphaIIbbeta3-mediated aggregation. Finally, on stimulation with PMA, Hic-5, as well as Pyk2, translocated to the cell periphery, where a meshwork of actin filaments assembled after adhesion to immobilized fibrinogen. Our results suggest that Hic-5 might be important in platelet aggregation and adhesion, in a manner dependent on alphaIIbbeta3-mediated outside-in signalling, through association with Pyk2.

Blood Platelets↗
PubMed2001

An oxidized derivative of cholesterol increases the release of soluble vascular cell adhesion molecule-1 from human umbilical vein endothelial cells in culture.

Treatment of human umbilical vein endothelial cells (HUVECs) with 7-ketocholesterol resulted in an increased release of soluble vascular cell adhesion molecule-1 (VCAM-1) into culture medium. 7-Ketocholesterol did not enhance the expression of mRNA for VCAM-1. 7 beta-Hydroxy- or 25-hydroxycholesterol had no effect on soluble VCAM-1 levels. Western blot analysis revealed that soluble VCAM-1, in the conditioned medium of both 7-ketocholesterol-stimulated and control cells, had a molecular size of 100 kDa. Stimulation of the TNF-alpha-treated HUVECs with 7-ketocholesterol further increased the levels of soluble VCAM-1 in the culture medium. Again, 7-ketocholesterol did not affect the VCAM-1 mRNA level, which was enhanced by TNF-alpha. Pretreatment of the cells with tissue inhibitor of membrane metalloproteinase-2 (TIMP-2) completely inhibited the release of VCAM-1 in response to 7-ketocholesterol but TIMP-1 had no effect. Adherence of mononuclear cells to TNF-stimulated HUVEC monolayers was slightly inhibited by 7-ketocholesterol, but this oxysterol did not affect the basal adherence to non-stimulated HUVECs. Immunofluorescent staining of the cells confirmed diffuse perinuclear distribution of VCAM-1 in HUVECs treated with TNF-alpha, but 7-ketocholesterol did not affect the intensity or distribution of immunofluorescence. We conclude that 7-ketocholesterol releases VCAM-1 from the endothelium probably by a proteolytic process.

Blotting, Western↗