SOX9 protein is stabilized by TGF-ß and regulates PAPSS2 mRNA expression in chondrocytes.

OBJECTIVE: We previously identified 3'-phosphoadenosine 5'-phosphosulfate synthase 2 (PAPSS2) as a transcriptional target of transforming growth factor ß (TGF-ß) in chondrocytes. PAPSS2 is required for proper sulfation of proteoglycans in cartilage. Defective sulfation in the matrix results in alterations in mechanical properties of the cartilage that would be expected to result in degeneration. The objective of this study was to identify factors that regulate PAPSS2 expression and compare to a known TGF-ß responsive gene, proteoglycan 4/lubricin (PRG4). In this study, TGF-ß-mediated regulation of SOX9 was characterized, and the involvement of SOX9 in regulation of PAPSS2 mRNA was investigated. DESIGN: Primary bovine articular chondrocytes grown in micromass culture and ATDC5 cells were used as the model system. Adenoviruses were used to express SOX9 and SMAD3. siRNA was used to knock-down Sox9 and Smad3. Western blot and real-time quantitative RT-PCR (qPCR) were used to measure changes in protein and mRNA levels in response to treatment. RESULTS: Over-expression of SOX9 was sufficient to up-regulate PAPSS2 mRNA. TGF-ß treatment of SOX9-expressing cells resulted in enhanced up-regulation of PAPSS2 mRNA, suggesting that SOX9 cooperates with TGF-ß signaling. Furthermore, Sox9 was required for full TGF-ß-mediated induction of Papss2. In contrast, PRG4 was regulated by SMAD3 but not SOX9. SOX9 protein levels were increased after treatment with TGF-ß, although SOX9 mRNA was not. SOX9 protein was post-translationally stabilized after treatment with TGF-ß. CONCLUSIONS: TGF-ß stabilizes SOX9 protein, and SOX9 is sufficient and necessary for TGF-ß-mediated regulation of PAPSS2 mRNA, providing a novel mechanism for TGF-ß-mediated gene regulation in chondrocytes.

Novel effects of sarcopenic osteoarthritis on metabolic syndrome, insulin resistance, osteoporosis, and bone fracture: the national survey.

UNLABELLED: This study compared the effects sarcopenic osteoarthritis on metabolic syndrome, insulin resistance, osteoporosis, and bone fracture. By using national survey data, we suggest that the relationship between sarcopenia and metabolic syndrome or insulin resistance is potentiated by the severity of osteoarthritis and is independent of body weight. INTRODUCTION: Sarcopenia and osteoarthritis are known risk factors for metabolic syndrome. However, their combined effects on metabolic syndrome, insulin resistance and osteoporosis remain uncertain. METHODS: We used data from the fifth Korean National Health and Nutrition Examination Survey using a total of 3158 adults (age >50 years). Sarcopenia was defined as a skeletal muscle index score (appendicular skeletal muscle mass/body weight) within the fifth percentile of sex-matched younger reference participants. Radiographic knee osteoarthritis was defined as a Kellgren-Lawrence (K-L) grade of 2 or greater. Metabolic syndrome was diagnosed using the National Cholesterol Education Program criteria. Insulin resistance was evaluated using the homeostasis model assessment-estimated insulin resistance index (HOMA-IR). Osteoporosis was defined using the World Health Organization T-score criteria. RESULTS: In multivariable logistic regression analysis, the sarcopenic osteoarthritis group had a higher odds ratio (OR) for metabolic syndrome (OR = 11.00, 95 % confidential interval (CI) = 2.12-56.99, p = 0.013) than the non-sarcopenic osteoarthritis (OR = 1.02, 95 % CI = 0.65-1.62, p = 0.972) and sarcopenic non-osteoarthritis groups (OR = 7.15, 95 % CI = 1.57-32.53, p = 0.027). Similarly, sarcopenic osteoarthritis had a greater OR of highest HOMA-IR quartiles (OR = 8.19, 95 % CI = 2.03-33.05, p = 0.003) than the other groups. Overall, the association between the K-L grade and body mass index was significant; however, this significance was lower in individuals with sarcopenia and was lost in those with sarcopenic osteoarthritis. Additionally, osteoporosis and bone fracture were not associated to sarcopenic osteoarthritis (p > 0.05). CONCLUSIONS: These results suggest that the relationship between sarcopenia and metabolic syndrome or insulin resistance is potentiated by the severity of osteoarthritis and is independent of body weight.

Efficacy of HVT-IBD vector vaccine compared to attenuated live vaccine using in-ovo vaccination against a Korean very virulent IBDV in commercial broiler chickens.

The production performance, efficacy, and safety of two types of vaccines for infectious bursal disease virus (IBDV) were compared with in-ovo vaccination of Cobb 500 broiler chickens for gross and microscopic examination of the bursa of Fabricius, bursa/body weight (b/B) ratio, flow cytometry, and serologic response to Newcastle disease virus (NDV) vaccination. One vaccine was a recombinant HVT-IBD vector vaccine (HVT as for herpesvirus of turkeys) and the other was an intermediate plus live IBDV vaccine. A significant difference was detected at 21 d. Eight of 10 chickens that received the IBDV live vaccine had severe bursal lesions and a relatively low b/B ratio of 0.95, and an inhibited NDV vaccine response. On the other hand, the HVT-IBD vector vaccine resulted in mild bursal lesions and a b/B ratio of 1.89. Therefore, the live vaccine had lower safety than that of the HVT-IBD vector vaccine. To determine the protective efficacy, chickens were intraocularly challenged at 24 d. Eight of 10 chickens in the IBDV live vaccination group showed gross and histological lesions characterized by hemorrhage, cyst formation, lymphocytic depletion, and a decreased b/B ratio. In contrast, the HVT-IBD vector vaccinated chickens showed mild gross and histological lesions in three of 10 chickens with a b/B ratio of 1.36, which was similar to that of the unchallenged controls. Vaccinated chickens showed a significant increase in IBDV antibody titers, regardless of the type of vaccine used. In addition, significantly better broiler flock performance was observed with the HVT-IBD vector vaccine compared to that of the live vaccine. Our results revealed that the HVT-IBD vector vaccine could be used as an alternative vaccine to increase efficacy, and to have an improved safety profile compared with the IBDV live vaccine using in-ovo vaccination against the Korean very virulent IBDV in commercial broiler chickens.

Changes of olfactory abilities in relation to age: odor identification in more than 1400 people aged 4 to 80 years.

The currently presented large dataset (n = 1,422) consists of results that have been assembled over the last 8 years at science fairs using the 16-item odor identification part of the "Sniffin' Sticks". In this context, the focus was on olfactory function in children; in addition before testing, we asked participants to rate their olfactory abilities and the patency of the nasal airways. We reinvestigated some simple questions, e.g., differences in olfactory odor identification abilities in relation to age, sex, self-ratings of olfactory function and nasal patency. Three major results evolved: first, consistent with previously published reports, we found that identification scores of the youngest and the oldest participants were lower than the scores obtained by people aged 20-60. Second, we observed an age-related increase in the olfactory abilities of children. Moreover, the self-assessed olfactory abilities were related to actual performance in the smell test, but only in adults, and self-assessed nasal patency was not related to the "Sniffin' Sticks" identification score.

Enhanced antitumor immunotherapeutic effect of B-cell-based vaccine transduced with modified adenoviral vector containing type 35 fiber structures.

For successful clinical tumor immunotherapy outcomes, strong immune responses against tumor antigens must be generated. Cell-based vaccines compromise one strategy with which to induce appropriate strong immune responses. Previously, we established a natural killer T-cell (NKT) ligand-loaded, adenoviral vector-transduced B-cell-based anticancer cellular vaccine. To enhance tumor antigen delivery to B cells, we established a modified adenoviral vector (Ad-k35) that encoded a truncated form of the breast cancer antigen Her2/neu (Ad-k35HM) in which fiber structure was substituted with adenovirus serotype 35. We observed increased tumor antigen expression with Ad-k35HM in both human and murine B cells. In addition, an Ad-k35HM-transduced B-cell vaccine elicited strong antigen-specific cellular and humoral immune responses that were further enhanced with the additional loading of soluble NKT ligand KBC009. An Ad-k35HM-transduced, KBC009-loaded B-cell vaccine efficiently suppressed the in vivo growth of established tumors in a mouse model. Moreover, the vaccine elicited human leukocyte antigen (HLA)-A2 epitope-specific cytotoxic T-cell responses in B6.Cg (CB)-Tg (HLA-A/H2-D) 2Enge/Jat mice. These findings indicated that the Ad-k35 could be appropriate for the preclinical and clinical development of B-cell-based anticancer immunotherapies.

Acute kidney injury after infrarenal abdominal aortic aneurysm surgery: a comparison of AKIN and RIFLE criteria for risk prediction.

BACKGROUND: Although both Acute Kidney Injury Network (AKIN) and risk, injury, failure, loss, and end-stage (RIFLE) kidney disease criteria are frequently used to diagnose acute kidney injury (AKI), they have rarely been compared in the diagnosis of AKI in patients undergoing surgery for infrarenal abdominal aortic aneurysm (AAA). This study investigated the incidence of, and risk factors for, AKI, defined by AKIN and RIFLE criteria, and compared their ability to predict mortality after infrarenal AAA surgery. METHODS: This study examined 444 patients who underwent infrarenal AAA surgery between January 1999 and December 2011. Risk factors for AKI were assessed by multivariable analyses, and the impact of AKI on overall mortality was assessed by a Cox's proportional hazard model with inverse probability of treatment weighting (IPTW). Net reclassification improvement (NRI) was used to assess the performance of AKIN and RIFLE criteria in predicting overall mortality. RESULTS: AKI based on AKIN and RIFLE criteria occurred in 82 (18.5%) and 55 (12.4%) patients, respectively. The independent risk factors for AKI were intraoperative red blood cell (RBC) transfusion and chronic kidney disease (CKD) by AKIN criteria, and age, intraoperative RBC transfusion, preoperative atrial fibrillation, and CKD by RIFLE criteria. After IPTW adjustment, AKI was related to 30 day mortality and overall mortality. NRI was 15.2% greater (P=0.04) for AKIN than for RIFLE criteria in assessing the risk of overall mortality. CONCLUSIONS: Although AKI defined by either AKIN or RIFLE criteria was associated with overall mortality, AKIN criteria showed better prediction of mortality in patients undergoing infrarenal AAA surgery.

The transmembrane domain of the rice small protein OsS1Fa1 is responsible for subcellular localization and drought tolerance.

OsS1Fa1, a homologue of spinach S1Fa, is a small protein in rice that contains four distinct conserved motifs and participates in drought tolerance. However, the biological functions of these conserved motifs have not been characterized to date. Therefore, we investigated the roles of these conserved domains in the localization and cellular function of OsS1Fa1. We analysed the subcellular localization of OsS1Fa1 using confocal laser scanning microscopy (CLSM), following particle bombardment and bacterial infiltration. An E. coli in vivo reconstituted sumoylation assay was conducted to investigate sumoylation of OsS1Fa1. We characterized the function of the transmembrane domain of OsS1Fa1 in drought tolerance using transgenic Arabidopsis plants. Fluorescence analysis showed that OsS1Fa1 localized to the nuclear and cytoplasmic membranes. Mutation and cell fractionation analyses revealed that the membrane localization domain determined the subcellular localization of OsS1Fa1. The rice homologue OsS1Fa2 and Arabidopsis orthologs AtS1Fa1, AtS1Fa2, and AtS1Fa3 also exhibited similar localization patterns as OsS1Fa1. Sumoylation analysis demonstrated that OsS1Fa1 was conjugated with the small ubiquitin-related modifier (SUMO). Transgenic analysis showed that overexpression of OsS1Fa1(TMm1), a mutant form of the transmembrane domain of OsS1Fa1, in Arabidopsis did not enhance drought stress tolerance, whereas OsS1Fa1 overexpression improved the drought tolerance of transgenic Arabidopsis. Our data indicate that rice and Arabidopsis S1Fa1 proteins localize in the nuclear and cytoplasmic membranes, and that transmembrane domain determines subcellular localization and plays an important role in drought stress tolerance.

Adverse events and development of tuberculosis after 4 months of rifampicin prophylaxis in a tuberculosis outbreak.

We screened tuberculosis (TB) contacts as an outbreak investigation with tuberculin skin test (TST) and interferon-gamma release assay (IGRA). We evaluated adverse events and TB incidence in all persons screened after rifampicin (RFP) prophylaxis, and specifically assessed the new TB cases in relation to initial TST and IGRA results. The 180 contacts were divided into four groups: TST+/IGRA+ (n = 101), TST+/IGRA- (n = 22), TST-/IGRA+ (n = 16), and TST-/IGRA- (n = 41). RFP treatment (4 months) was prescribed only to the TST+/IGRA+ group. Of 87 contacts who initiated prophylaxis, adverse events occurred in 21 contacts (24.1%) including hepatotoxicity (11.5%), flu-like syndrome (5.7%), and thrombocytopenia (3.4%). TB developed in two TST+/IGRA+ subjects after completion of prophylaxis, including one multidrug-resistant (MDR)-TB case during 21.8 months of follow-up. Adverse events were frequent, and development of TB including MDR-TB occurred after RFP prophylaxis.

The diagnostic value of the sagittal multiplanar reconstruction CT images for nasal bone fractures.

AIM: To compare the diagnostic performance of sagittal multiplanar reconstruction (MPR) images and axial images for the detection of a nasal bone fracture. MATERIALS AND METHODS: This prospective study included 533 consecutive patients who underwent three-dimensional images with 64-section multidetector-row CT for the evaluation of a facial bone fracture between June 2007 and May 2008 (366 males; 167 females; mean age +/- standard deviation 31.1+/-21.2 years; age range 1-92 years). Two observers independently scored the possibility of a nasal bone fracture on axial and sagittal images. Receiver operating characteristic (ROC) curve analysis was performed. RESULTS: The Az values of the sagittal images were higher than those of the axial images for both observers (p=0.002 and 0.010, respectively) with higher accuracy (p<0.001 and 0.016, respectively). The sensitivities of sagittal images were superior to those of axial images, especially for type 1simple nasal bone fractures with no or minimal displacement (observer 1, 98.6 versus 72.8%; observer 2, 84.9 versus 71%). CONCLUSION: Sagittal MPR facial bone CT images provided superior diagnostic performance, and their addition to axial images is useful for the evaluation of nasal bone fractures.

Changes of Neurotransmitters in Youth with Internet and Smartphone Addiction: A Comparison with Healthy Controls and Changes after Cognitive Behavioral Therapy.

BACKGROUND AND PURPOSE: Neurotransmitter changes in youth addicted to the Internet and smartphone were compared with normal controls and in subjects after cognitive behavioral therapy. In addition, the correlations between neurotransmitters and affective factors were investigated. MATERIALS AND METHODS: Nineteen young people with Internet and smartphone addiction and 19 sex- and age-matched healthy controls (male/female ratio, 9:10; mean age, 15.47 ± 3.06 years) were included. Twelve teenagers with Internet and smartphone addiction (male/female ratio, 8:4; mean age, 14.99 ± 1.95 years) participated in 9 weeks of cognitive behavioral therapy. Meshcher-Garwood point-resolved spectroscopy was used to measure γ-aminobutyric acid and Glx levels in the anterior cingulate cortex. The γ-aminobutyric acid and Glx levels in the addicted group were compared with those in controls and after cognitive behavioral therapy. The γ-aminobutyric acid and Glx levels correlated with clinical scales of Internet and smartphone addiction, impulsiveness, depression, anxiety, insomnia, and sleep quality. RESULTS: Brain parenchymal and gray matter volume-adjusted γ-aminobutyric acid-to-creatine ratios were higher in subjects with Internet and smartphone addiction (P = .028 and .016). After therapy, brain parenchymal- and gray matter volume-adjusted γ-aminobutyric acid-to-creatine ratios were decreased (P = .034 and .026). The Glx level was not statistically significant in subjects with Internet and smartphone addiction compared with controls and posttherapy status. Brain parenchymal- and gray matter volume-adjusted γ-aminobutyric acid-to-creatine ratios correlated with clinical scales of Internet and smartphone addictions, depression, and anxiety. Glx/Cr was negatively correlated with insomnia and sleep quality scales. CONCLUSIONS: The high γ-aminobutyric acid levels and disrupted balance of γ-aminobutyric acid-to-Glx including glutamate in the anterior cingulate cortex may contribute to understanding the pathophysiology and treatment of Internet and smartphone addiction and associated comorbidities.

Activation of the spinal sigma-1 receptor enhances NMDA-induced pain via PKC- and PKA-dependent phosphorylation of the NR1 subunit in mice.

BACKGROUND AND PURPOSE: Previously we demonstrated that the spinal sigma-1 receptor (Sig-1 R) plays an important role in pain transmission, although the exact mechanism is still unclear. It has been suggested that Sig-1 R agonists increase glutamate-induced calcium influx through N-methyl-D-aspartate (NMDA) receptors. Despite data suggesting a link between Sig-1 Rs and NMDA receptors, there are no studies addressing whether Sig-1 R activation directly affects NMDA receptor sensitivity. EXPERIMENTAL APPROACH: We studied the effect of intrathecal (i.t.) administration of Sig-1 R agonists on protein kinase C (PKC) and protein kinase A (PKA) dependent phosphorylation of the NMDA receptor subunit NR1 (pNR1) as a marker of NMDA receptor sensitization. In addition, we examined whether this Sig-1 R mediated phosphorylation of NR1 plays an important role in sensory function using a model of NMDA-induced pain. KEY RESULTS: Both Western blot assays and image analysis of pNR1 immunohistochemical staining in the spinal cord indicated that i.t. injection of the Sig-1 R agonists, PRE-084 or carbetapentane dose dependently enhanced pNR1 expression in the murine dorsal horn. This increased pNR1 expression was significantly reduced by pretreatment with the specific Sig-1 R antagonist, BD-1047. In another set of experiments Sig-1 R agonists further potentiated NMDA-induced pain behaviour and pNR1 immunoreactivity and this was also reversed with BD-1047. CONCLUSIONS AND IMPLICATIONS: The results of this study suggest that the activation of spinal Sig-1 R enhances NMDA-induced pain via PKC- and PKA-dependent phosphorylation of the NMDA receptor NR 1 subunit.

Relationship between blood pressure parameters and pulse wave velocity in normotensive and hypertensive subjects: invasive study.

Blood pressure (BP) is one of the most important contributing factors to pulse wave velocity (PWV), a classic measure of arterial stiffness. Although there have been many non-invasive studies to show the relation between arterial stiffness and BP, the results are controversial. The aim of this study is to evaluate the role of BP as an influencing factor on PWV using invasive method. We observed 174 normotensive and untreated hypertensive subjects using coronary angiography. Arterial stiffness was assessed through aorto-femoral PWV by foot-to-foot velocity method using fluid-filled system. And BP was measured by pressure wave at the right common femoral artery. From univariate analysis, age, diabetes mellitus (DM), hypertension, waist, waist-to-hip ratio, total cholesterol-to-high-density lipoprotein cholesterol ratio, systolic BP (SBP), pulse pressure (PP) and mean arterial pressure (MAP) showed significant association with PWV. To avoid multiple colinearity among SBP, PP and MAP, we performed multiple regression analysis predicting PWV thrice. Age, DM and each BP were significantly and consistently correlated to PWV. In the first and third modules, compared to age, SBP and MAP were less strong predictors, respectively. However, PP was the stronger predictor than age and DM in the second module. Lastly, we simultaneously forced MAP and PP with other variables in the fourth multivariate analysis. Age, DM and PP remained significantly correlated with PWV, but the significance of MAP was lost. This is the first invasive study to suggest that PP has the strongest correlation with PWV among a variety of BP parameters.

P569Diastolic dyssynchrony is associated with exercise intolerance in hypertensive patients with left ventricular hypertrophy.

BACKGROUND: Left ventricular hypertrophy (LVH) is associated with intra-ventricular dyssynchrony at systolic phase during exercise in hypertensive patients. However, dypsnea on exertion is much more correlated with diastolic phase. We investigated whether LVH is associated with diastolic dyssynchrony during exercise in patients with hypertension. METHODS: Ninety hypertensive patients with exertional dyspnea and 30 control individuals were enrolled. Exercise stress echocardiography was performed using a symptom limited, multistage supine bicycle test. To evaluate the diastolic dyssynchrony of LV, we calculated the standard deviation (SD) of the averaged time from Q wave to myocardial early diastolic velocity in 12 segments. (TPe-SD, ms). Therefore, diastolic dyssynchrony index was SD of TPe. And also, we applied modified SD (SD/heart rate). RESULTS: There was no significant difference in systolic blood pressure (BP) and heart rate between the two groups. TPe-SD was significantly higher in patients with LVH at rest (27 ± 11.0 vs. 18.7 ± 7.4 ms, p<0.005) with exaggeration of the degree at peak exercise (42.0 ± 10.6 vs. 30.6 ± 12.4 ms, p<0.001). When applying modified SD, the difference is much more increased (80.0 ± 17.6 vs. 49.0 ± 21.3 ms, p <0.001). Multiple regression analysis showed LV mass index (ß=0.515, P=0.001) and E/E' at peak exercise (ß= -0.253, P=0.025) were independently associated with LV dyssynchrony during diastolic phase when controlled for age, sex, and systolic BP at peak exercise. CONCLUSION: Intra-ventricular diastolic dyssynchrony during exercise is significantly associated with exercise duration in hypertensive patients with LVH. And this result could explain that the patients with exertional dyspnea are more common in LVH group.Univariate and multivariate analysis forUnivariate AnalysisMultivariate Analysisßp valueßp valueAge-0.288*0.037-0.355*0.001Sex0.161*0.0200.250*0.034LVMI (g/m2)-0.787*0.008-0.515*0.001LAVI(mL)-0.4400.065-0.1750.075E' at peak ex.0.2160.589Diastolic dyssynchrony-0.725*0.030-0.253*0.025S' at peak ex.0.7100.073 LVMI, left ventricular mass index; LAVI, left atrium volume index; E, early diastolic mitral inflow velocity; E', early diastolic longitudinal tissue velocity; S', early systolic longitudinal tissue velocity.Univariate and multivariate analysis for.LVMI, left ventricular mass index; LAVI, left atrium volume index; E, early diastolic mitral inflow velocity; E', early diastolic longitudinal tissue velocity; S', early systolic longitudinal tissue velocity.

Inflammation, insulin resistance, and glucose intolerance in acute myocardial infarction patients without a previous diagnosis of diabetes mellitus.

We examined the prevalence of impaired glucose metabolism and its association with inflammation and insulin resistance (IR) in acute myocardial infarction (AMI) patients without a previous diagnosis of diabetes. This prospective study enrolled 52 AMI patients, and 75-g oral glucose tolerance testing was performed on 30 patients at discharge and again 3 months later. We also measured serum adiponectin, high sensitive C-reactive protein, and IL-6 on both occasions. Data were compared with those of 30 type 2 diabetic patients without a history of AMI. Forty percent and 36.7% of AMI patients had impaired glucose tolerance (IGT) at discharge and at 3 months, respectively. The corresponding proportions for newly diagnosed diabetes are 33.0% and 30.0%. At discharge, AMI patients with IGT or diabetes showed higher high sensitive C-reactive protein and IL-6 levels compared with AMI patients with normal glucose tolerance or control type 2 diabetic patients. Furthermore, AMI patients with IGT or diabetes exhibited higher IR and lower serum adiponectin levels than AMI patients with normal glucose tolerance at 3 months after discharge. Previously undiagnosed diabetes and IGT are common in Korean patients with AMI. These glycometabolic abnormalities are associated with inflammation, IR, and serum adiponectin levels.

The antithrombotic efficacy of AT-1459, a novel, direct thrombin inhibitor, in rat models of venous and arterial thrombosis.

The antithrombotic efficacy of AT-1459, a novel, direct thrombin inhibitor (Ki = 4.9 nM) was evaluated in rat models of venous thrombosis combined with a bleeding time test and arterial thrombosis. After drugs were given by i. v. bolus injection plus a continuous infusion, the ID50, (a dose that exhibits 50% inhibition of thrombus formation over each vehicle group) values of AT-1459, argatroban, and dalteparin were 0.04 mg/kg plus 0.04 mg/kg/h, 0.1 mg/kg plus 0.4 mg/ kg/h, and 13.0 IU/kg plus 26.0 IU/kg/h, respectively, in the venous thrombosis study. The BT2 (a dose that causes 2-fold prolongation of bleeding time over each vehicle group) values of AT-1459, argatroban, and dalteparin were 0.9 mg/kg plus 0.9 mg/kg/h, 1.0 mg/kg plus 0.6 mg/kg/h, and 345.5 IU/kg plus 691.0 IU/kg/h in the rat tail transection model. The ratios of BT2/ID50 of AT-1459, argatroban, and dalteparin were 22.5, 10.0, and 26.6, respectively. In a rat model of arterial thrombosis induced by topical FeCl2 application, intravenous administration of AT-1459, argatroban, and dalteparin improved the vessel patency significantly (P < 0.01) at 0.6 mg/kg plus 0.6 mg/kg/h, 0.6 mg/kg plus 2.4 mg/kg/h, and 300 IU/kg plus 600 IU/kg/h, respectively. The oral antithrombotic effect of AT-1459 lasted for 6 after administering 30 mg/kg and improved the vessel patency significantly 1 h after administering the same dose in venous and arterial thrombosis models, respectively, with a rapid onset of action. Warfarin also inhibited thrombus weight and improved the vessel patency significantly after oral administration of 0.3 mg/kg for three consecutive days in the same study. The antithrombotic and hemorrhagic effects of all drugs studied were correlated with plasma concentration or clotting times. These results suggest that AT-1459 may be clinically useful as an orally available antithrombotic agent for the prevention of venous and arterial thrombosis.

Regulation of estrogen receptor levels by ligand-induced release of compound(s) in MCF-7 cells.

In MCF-7 cells, estradiol (E2) and pure antiestrogens (AEs) decrease estrogen receptor alpha (ER) levels, while AEs with partial estrogenic activity lead to ER alpha accumulation. Using immunocytochemistry, we found that cells pre-exposed to one of such ligands, when plated with untreated cells, led to similar ER changes in the latter. Conditioned media (CMs) prepared from stimulated cells displayed identical regulatory effects even after strong dilution; they also modulated ERE-dependent transcriptional activity. Evaluation of residual ligand concentrations in CMs rejected the possibility of a major interference of the former. Cycloheximide, which inhibits E2-induced down-regulation, failed to block the influence of CM(E2) in agreement with this view. DCC-treatment of CMs abrogated their effects, suggesting the release of hydrophobic compound(s) which regulate ER and/or amplify the effect of extremely low amounts of residual ligands. Such a release appears independent of ER since CMs from MDA-MB-231 cells (ER-negative) were effective as their autologous media on MCF-7 cells.

Estrogenic and antiestrogenic regulation of the half-life of covalently labeled estrogen receptor in MCF-7 breast cancer cells.

Effect of estrogens and antiestrogens (AEs) on estrogen receptor (ER) half-life was analyzed in MCF-7 cells by assessing its progressive disappearance after covalent labeling in situ with [3H]tamoxifen aziridine ([3H]TAZ). Cells were incubated for 1 h with 20 nM [3H]TAZ either in the absence or presence of a 500-fold excess of unlabeled estradiol (E2) (non-specific binding). The entire ER population was labeled by this method as established by subsequent incubation of the cells with [125I]E2. [3H]TAZ labeled cells were maintained in culture for additional 5 h in the absence (control) or presence of increasing amounts (0.1 nM - 1 microM) of either a given estrogen (E2, estrone, diethylstilbestrol, bisphenol), a pure AE (RU 58 668, ICI 164 384) or an AE with residual estrogenic activity (RU 39 411, 4-hydroxytamoxifen, keoxifene). The progressive disappearance of nuclear and cytosolic [3H]TAZ-ER complex during 5 h incubation were assessed by their immunoprecipitation with anti-ER monoclonal antibody (H 222) followed by scintillation counting or SDS-PAGE and fluorography. Fading of labeled receptors was extremely slow (approximately 10% loss after 6 h) in absence of any hormone/antihormone indicating a long half-life of the [3H]TAZ-ER complex. Addition of estrogens as well as pure AEs led to a dramatic reduction of the half-life while AEs with residual estrogenic activity were extremely less efficient in this regard providing an explanation for the ability of latter compounds to up-regulate the receptor since they do not affect ER mRNA synthesis and stability. Receptor disappearance induced by estrogens was closely related to their binding affinity for ER. Newly synthesized ER emerged during the treatment with hormones or antihormones seems to be implicated in the phenomenon since [3H]TAZ was covalently bound and could, therefore, not be displaced by these compounds. Induction of synthesis of a short half-life peptide(s) with degradative activity was demonstrated by addition of cycloheximide or puromycine (both at 50 microM) which completely blocked ER disappearance. The fact that no cleavage products of ER were detected by SDS-PAGE suggested a lysosomial hydrolysis. Hence, hormonal modulation of only a part of ERs may down-regulate their total population until it reaches the steady-state level.

Molecular characterization of a rab-related small GTP binding protein cDNA from rice (Oryza sativa L. IR-36).

To study physiological roles of plant small GTP binding proteins, we isolated a cDNA clone (ORrab2) encoding the rab-related small GTP binding protein from rice (Oryza sativa L. IR-36) by using human cDNA rab2 as a probe. The deduced amino acid sequence of the ORrab2 gene shared all the conserved regions, important for GTPase/GTP binding activities, with those of other small GTP binding proteins. ORrab2 is a 1028 bp long cDNA, encodes a 23.2 kDa protein which shows 85.2% similarity on the amino acid sequence level to the Hrab2 protein, and was used as a probe. Through Southern and Northern blot analyses, we found that ORrab2 is a single copy gene and actively expressed at the stages of cell division and elongation. We investigated GTP binding abilities by a filter assay procedure. Deletion of a binding motif, GDTGVGKS, within an ORrab2 protein showed a significant decrease of GTP binding affinity, suggesting its important role in nucleotide binding.

Assessment of coronary flow reserve with transthoracic Doppler echocardiography: comparison among adenosine, standard-dose dipyridamole, and high-dose dipyridamole.

Coronary flow reserve (CFR), defined as a ratio of hyperemic-to-basal coronary flow velocity, provides important information about the functional aspect of coronary circulation. However, it usually is determined by invasive methods during catheterization. Recent studies have shown that transthoracic Doppler echocardiography (TTDE) may be useful in the measurement of coronary flow velocity in the distal portion of the left anterior descending coronary artery (LAD). The vasodilators used for hyperemia are adenosine and dipyridamole. However, the coronary vasodilative response and systemic hemodynamic effects of the two agents have not been directly compared with TTDE. We assessed blood flow velocity and vascular resistance in the distal LAD by TTDE during an intravenous 2-minute adenosine infusion (140 microg/kg/min) and low- (0.56 mg/kg) and high-dose dipyridamole (0. 84 mg/kg) infusion in 25 patients with patent LAD. Coronary flow velocity was successfully recorded in 20 patients (80%) during baseline and the consecutive vasodilator-infusion period. Compared with low-dose dipyridamole, adenosine infusion induced a higher CFR (3.7 +/- 0.87 vs 2.73 +/- 0.65; P <.05) and a lower coronary resistance index (0.31 +/- 0.04 vs 0.35 +/- 0.08; P <.05). But by increasing the dipyridamole dose to 0.84 mg/kg, the values of the CFR and coronary resistance index became comparable to those of adenosine infusion (2.85 +/- 0.78 vs 3.03 +/- 0.7, P = not significant [NS]; 0.33 +/- 0.04 vs 0.32 +/- 0.09, P = NS; respectively). We conclude that adenosine seems to be a favorable vasodilator for the measurement of CFR with TTDE.

Identification and quantification of molecular species of diacyl glyceryl ether by reversed-phase high-performance liquid chromatography with refractive index detection and mass spectrometry.

We have developed a method to identify and quantify the molecular species of diacyl glyceryl ether (DAGE) using high-performance liquid chromatography (HPLC) equipped with a refractive index detector and an electrospray ionization and time of flight mass spectrometer (LC-RI-MS). An octadecyl silica column with a mixture of acetonitrile and dichloromethane (65:35, v/v) as an eluant was used for the HPLC. When the LC-RI-MS method was applied to a mixture of synthetic DAGEs; 1-O-hexadecyl-2,3-dioleoylglycerol (O-16:0-18:1-18:18:1), 1-O-octadecyl-2,3-dioleoylglycerol (O-18:0-18:1-18:1), 1-O-octadecenyl-2,3-dioleoylglycerol (O-18:1-18:1-18:1), 1-O-octadecyl-2,3-didocodahexaenoylglycerol (O-18:0-22:6-22:6), and 1-O-octadecenyl-2,3-didocosahexaenoylglycerol (O-18:1-22:6-22:6), good separation and quantification were obtained on the refractive index chromatogram. A pseudo-molecular ion [M+NH4]+ and a monoacyl glyceryl ether ion [M-RCO2] + were observed for all synthetic DAGEs on the mass spectrum. It was found that the fatty acids and glyceryl ether in DAGE could be easily identified by these mass spectra. When this LC-RI-MS method was applied to the DAGEs extracted from muscle of Stromateus stellatus, approximately 18 peaks were observed on LC-RI-MS chromatograms and the major molecular species of DAGEs were identified as O-16:0-18:1-18:1.