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Background: The burden of stroke is becoming an important issue in Asian countries, where half of the world's population lives. In Western populations, standard-dose intravenous tissue plasminogen activator (tPA) at a dose of 0.9 mg/kg administered up to 4.5 h following acute ischaemic stroke has been shown to improve clinical outcomes. However, the dosage of tPA in Asian populations varies, partly influenced by treatment costs in developing countries and the perceived racial differences in treatment response. Furthermore, no randomized controlled trial of intravenous tPA thrombolysis has been carried out in Asia with the exceptions of observational and multicentre registries in Japan. Summary of the Review: This review aims to examine the current use of low-dose tPA in Asian populations with particular emphasis on its clinical benefits. Historically, the findings of 2-chain tPA (duteplase) trials in the early 1990s were the basis of low-dose tPA in Japan, and standard-dose tPA was chosen taking into account a pilot dose escalation study. Most of the trials incorporating low-dose tPA suggested that low-dose tPA has clinical efficacy comparable with the standard dose and that it is associated with a lower incidence of symptomatic haemorrhage. It follows that consensus regarding the dosage of tPA is wanting. Conclusions: Standard-dose tPA would be a reasonable choice in Asian populations until the results are available from randomized controlled studies comparing low-dose with standard-dose tPA. © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Neurotoxicity of amyloid β (Aβ) plays an important role in Alzheimer's disease (AD) pathogenesis. In this study, we researched the potential protective effects of resistin against Aβ neurotoxicity in mouse Neuro2a (N2a) cells transfected with the Swedish amyloid precursor protein (Sw-APP) mutant and Presenilin exon 9 deletion mutant (N2a/D9), which overproduced Aβ with abnormal intracellular Aβ accumulation. The results show increased levels of ROS, NO, protein carbonyls, and 4HNE in N2a/D9 cells, which were attenuated by resistin treatment in a dose dependent manner. We also found that resistin could improve mitochondrial function in N2a/D9 cells through increasing the level of ATP and mitochondrial membrane potential. MTT and LDH assay indicated that N2a/D9 cells show increased vulnerability to H2O2-induced insult, which could be ameliorated by resistin. Mechanically, we found that resistin prevented apoptosis signals through reducing the ratio of Bax/Bcl2, the level of cleaved caspase-3, and attenuating cytochrome C release. Finally, the results demonstrated that resistin did not change the production of Aβ1–40 and Aβ1–42 in N2a/D9 cells, which suggests that the protective effects of resistin are independent of APP metabolism. This raises the possibility of novel AD therapies using resistin. [Copyright &y& Elsevier]

Background:Cancer-associated inflammation, in the form of local and systemic inflammatory responses, appear to be linked to tumour necrosis and have prognostic value in patients with colorectal cancer. However, their relationship with circulating biochemical mediators is unclear. The aim of the present study was to examine the interrelationships between circulating mediators, in particular interleukin-6 (IL-6) and tumour necrosis, and local and systemic inflammatory responses in patients undergoing resection for colorectal cancer.Methods:Data were collected from preoperative blood tests for 118 patients who underwent resection for colorectal cancer. Analysis of circulating IL-6, IL-10, vascular endothelial growth factor (VEGF), differential white cell count, C-reactive protein, and albumin were carried out. Routine pathology specimens were examined for tumour characteristics including necrosis and the extent of the inflammatory cell infiltrate. Body composition was examined using body mass index (BMI), total body fat, subcutaneous body fat, visceral fat, and skeletal muscle mass.Results:Circulating IL-6 concentrations were significantly associated with increased T stage (P<0.05), tumour necrosis (P<0.001), IL-10 (P<0.001), VEGF (P<0.001), modified Glasgow Prognostic Score (mGPS; P<0.001), white cell (P<0.01) and platelet (P<0.01) counts, and low skeletal muscle index (P<0.01). When normalised for T stage, tumour necrosis was associated with IL-6 (P<0.001), IL-10 (P<0.01), VEGF (P<0.001), mGPS (P<0.001), neutrophil-lymphocyte ratio (NLR; P<0.05), white cell (P<0.001), neutrophil (P<0.05), and platelet counts (P<0.005), and skeletal muscle index (P<0.001).Conclusion:The present study provides, for the first time, supportive evidence for the hypothesis that tumour necrosis, independent of T stage, is associated with elevated circulating IL-6 concentrations, thereby modulating both local and systemic inflammatory responses including angiogenesis that, in turn, may promote tumour progression and metastases. [ABSTRACT FROM AUTHOR]

Highlights: [•] The detailed kinetics of ASC responses to an oral cholera vaccine was analyzed. [•] The ASC response kinetics differs considerably after primary and booster vaccination. [•] ASC responses determined 7 days after booster vaccination may be underestimated. [•] Detection of early ASC responses may be used as a tool to assess immunological memory. [ABSTRACT FROM AUTHOR]

Human copper transporter 1 (hCTR1) is a homotrimer of a 190-amino acid monomer having three transmembrane domains believed to form a pore for copper permeation through the plasma membrane. The hCTR1-mediated copper transport mechanism is not well understood, nor has any measurement been made of the rate at which copper ions are transported by hCTR1. In this study, we estimated the rate of copper transport by the hCTR1 trimer in cultured cells using 64Cu uptake assays and quantification of plasma membrane hCTR1. For endogenous hCTR1, we estimated a turnover number of about 10 ions/ trimer/s. When overexpressed in HEK293 cells, a second transmembrane domain mutant of hCTR1 (H139R) had a 3-fold higher Km value and a 4-fold higher turnover number than WT. Truncations of the intracellular C-terminal tail and an AAA substitution of the putative metal-binding HCH C-terminal tripeptide (thought to be required for transport) also exhibited elevated transport rates andKm values when compared withWT hCTR1. UnlikeWThCTR1, H139R and the C-terminal mutants did not undergo regulatory endocytosis in elevated copper. hCTR1 mutants combining methionine substitutions that block transport (M150L,M154L) on the extracellular side of the pore and the high transport H139R or AAA intracellular side mutations exhibited the blocked transport of M150L,M154L, confirming that Cu+ first interacts with the methionines during permeation. Our results show that hCTR1 elements on the intracellular side of the hCTR1 pore, including the carboxyl tail, are not essential for permeation, but serve to regulate the rate of copper entry. [ABSTRACT FROM AUTHOR]

Purpose: Dopamine D receptors (DRs) have two affinity states for endogenous dopamine, referred to as high-affinity state (D), which has a high affinity for endogenous dopamine, and low-affinity state (D). The density of D can be measured with ( R)-2-CHO- N-n-propylnorapomorphine ([C]MNPA), while total density of D and D (DRs) can be measured with [C]raclopride using positron emission tomography (PET). Thus, the ratio of the binding potential ( BP) of [C]MNPA to that of [C]raclopride ([C]MNPA/[C]raclopride) may reflect the proportion of the density of D to that of DRs. In the caudate and putamen, [C]MNPA/[C]raclopride reflects the proportion of the density of D to that of DRs. To evaluate the reliability of the PET paradigm with [C]MNPA and [C]raclopride, we investigated the test-retest reproducibility of non-displaceable BP ( BP) measured with [C]MNPA and of [C]MNPA/[C]raclopride in healthy humans. Methods: Eleven healthy male volunteers underwent two sets of PET studies on separate days that each included [C]MNPA and [C]raclopride scans. BP values in the caudate and putamen were calculated. Test-retest reproducibility of BP of [C]MNPA and [C]MNPA/[C]raclopride was assessed by intra-subject variability (absolute variability) and test-retest reliability (intraclass correlation coefficient: ICC). Results: The absolute variability of [C]MNPA BP was 5.30 ± 3.96 % and 12.3 ± 7.95 % and the ICC values of [C]MNPA BP were 0.72 and 0.82 in the caudate and putamen, respectively. The absolute variability of [C]MNPA/[C]raclopride was 6.11 ± 3.68 % and 11.60 ± 5.70 % and the ICC values of [C]MNPA/[C]raclopride were 0.79 and 0.80 in the caudate and putamen, respectively. Conclusion: In the present preliminary study, the test-retest reproducibility of BP of [C]MNPA and of [C]MNPA/[C]raclopride was reliable in the caudate and putamen. [ABSTRACT FROM AUTHOR]

Protracted and partial implementation of treatment with intravenous tissue plasminogen activator (tPA) within 4.5 h after acute stroke onset results in potentially eligible patients not receiving optimal treatment. The goal of this study was to review the performance of various organisational models of acute stroke care delivery, and subsequent attempts to improve implementation of tPA treatment. Publications comprehensively reporting on organisational models to improve implementation of i.v. tPA treatment of acute ischemic stroke patients were selected. The efficacy of organisational models was assessed using process outcome measures: thrombolysis rates, time-dependent operational endpoints (time delays), functional outcomes: safety (rate of symptomatic intracranial hemorrhage, mortality rates) and clinical outcome at 90 days (modified Rankin Scale). Fifty-eight published studies assessing organisational models were identified. Four dominant models of acute stroke care delivery were discerned, i.e., primary and comprehensive stroke centres, telemedicine, and the mobile stroke unit. Performance reported for these models suggest a large variation in administration of thrombolytic therapy (0.7-30 %). Time delays and functional outcomes found varied considerably, just like safety and mortality (0.0-11.5 %, and 3.4-31.9 %, respectively). These findings suggest that improving organisational models for tPA treatment may improve acute stroke care. However, implementation may be hampered by regional variation in acute stroke care capacity, expertise, and a fragmented approach towards organising stroke care. [ABSTRACT FROM AUTHOR]

The article discusses a study which compared the effectiveness of endovascular therapy used after the administration of intravenous tissue plasminogen activator (t-PA) for patients with moderate-to-severe acute ischemic stroke, than intravenous t-PA alone. The study concluded similar safety outcomes and no significant difference in functional independence with endovascular therapy after intravenous t-PA, as compared with intravenous t-PA alone.

The article discusses a study which investigated the effectiveness of endovascular treatment, including the options of a mechanical device and intraarterial tissue plasminogen activator (t-PA) in patients with acute ischemic stroke, than the currently available treatment with intravenous t-PA. The results of the study indicate that endovascular therapy is not superior to standard treatment with intravenous t-PA.

Abstract: Introduction: Prevention of arterial thrombotic diseases has high priority in developed countries. Taurine (2-aminomethylsulfonic acid), which is rich in sea foods, showed antithrombotic effect in animal models of thrombosis. The present study aimed to investigate such effect in healthy human volunteers. Methods and Results: In 101 healthy Japanese people the overall thrombotic status was accessed from non-anticoagulated blood sample by the Global Thrombosis Test (GTT). There was no significant correlation between taurine concentration in urine samples and GTT-Occlusion Times (OT; mainly reactivity of platelets). In contrast, a significant inverse correlation was demonstrated between urine taurine concentrations and GTT-Lysis Times (LT; showing spontaneous thrombolytic activity). Conclusions: Our findings suggest that taurine enhances endogenous thrombolytic activity which could be a mechanism of the earlier observed cardioprotective and antithrombotic effect. [Copyright &y& Elsevier]

The events leading to the degradation of the endogenous PrPC (normal cellular prion protein) have been the subject of numerous studies. Two cleavage processes, α-cleavage and β-cleavage, are responsible for the main C- and N-terminal fragments produced from PrPC. Both cleavage processes occur within the Nterminus of PrPC, a region that is significant in terms of function. α-Cleavage, an enzymatic event that occurs at amino acid residues 110 and 111 on PrPC, interferes with the conversion of PrPC into the prion diseaseassociated isoform, PrPSc (abnormal disease-specific conformation of prion protein). This processing is seen as a positive event in terms of disease development. The study of β-cleavage has taken some surprising turns. β-Cleavage is brought about by ROS (reactive oxygen species). The C-terminal fragment produced, C2, may provide the seed for the abnormal conversion process, as it resembles in size the fragments isolated from prion-infected brains. There is, however, strong evidence that β-cleavage provides an essential process to reduce oxidative stress. β-Cleavage may act as a double-edged sword. By β-cleavage, PrPC may try to balance the ROS levels produced during prion infection, but the C2 produced may provide a PrPSc seed that maintains the prion conversion process. [ABSTRACT FROM AUTHOR]

Previously, it was shown that the type 1 deiodinase (D1) is subject to substrate-dependent inactivation that is blocked by pretreatment with the inhibitor of D1 catalysis, propylthiouracil (PTU). Using HepG2 cells with endogenous D1 activity, we found that while considerable D1-mediated catalysis of reverse tri-iodothyronine (rT3) is observed in intact cells, there was a significant loss of D1 activity in sonicates assayed from the same cells in parallel. This rT3-mediated loss of D1 activity occurs despite no change in D1 mRNA levels and is blocked by PTU treatment, suggesting a requirement for catalysis. Endogenous D1 activity in sonicates was inactivated in a dose-dependent manner in HepG2 cells, with a w50% decrease after 10 nM rT3 treatment. Inactivation of D1 was rapid, occurring after only half an hour of rT3 treatment. D1 expressed in HEK293 cells was inactivated by rT3 in a similar manner. 75Se labeling of the D1 selenoprotein indicated that after 4 h rT3-mediated inactivation of D1 occurs without a corresponding decrease in D1 protein levels, though rT3 treatment causes a loss of D1 protein after 8-24 h. Bioluminescence resonance energy transfer studies indicate that rT3 exposure increases energy transfer between the D1 homodimer subunits, and this was lost when the active site of D1 was mutated to alanine, suggesting that a post-catalytic structural change in the D1 homodimer could cause enzyme inactivation. Thus, both D1 and type 2 deiodinase are subject to catalysis-induced loss of activity although their inactivation occurs via very different mechanisms. [ABSTRACT FROM AUTHOR]

Abnormalities of bone metabolism and increased vascular calcification are common in chronic kidney disease (CKD) and important causes of morbidity and mortality. The Wnt signaling pathway may play a role in the bone and vascular disturbances seen in CKD, termed collectively 'CKD-MBD.' The aim of the study was to investigate the possible association of circulating concentrations of the secreted Wnt signaling inhibitors DKK1 and sclerostin with BMD and arterial stiffness in predialysis CKD. Seventy-seven patients (48 M, 29 F), mean age 57 (SD = 14) years with CKD stages 3B ( n = 32) and 4 ( n = 45) were studied. Sclerostin, DKK1, PTH, and 1,25(OH)D were analyzed. BMD was measured at the lumbar spine (LS), femoral neck (FN), total hip (TH), and forearm (FARM). Arterial stiffness index was determined by contour analysis of digital volume pulse (SI). There was a positive correlation between sclerostin and age ( r = 0.47, p < 0.000). Sclerostin was higher in men than women ( p = 0.013). Following correction for age and gender, there was a negative association between GFR and sclerostin ( p = 0.002). We observed a positive association between sclerostin and BMD at the LS ( p = 0.0001), FN ( p = 0.004), and TH ( p = 0.002). In contrast, DKK1 was negatively associated with BMD at the FN ( p = 0.038). A negative association was seen between DKK1 and SI ( p = 0.027). Our data suggest that the Wnt pathway may play a role in CKD-MBD. Prospective studies are required to establish the clinical relevance of sclerostin and DKK1 as serological markers in CKD. [ABSTRACT FROM AUTHOR]

Despite the large number of the outstanding researches, pathogenesis of osteonecrosis remains unknown. During the last decades the hypothesis that increased intravascular coagulation may be the pathogenetic mechanism which leads to osteonecrosis is gaining constantly support. Both primary factors of hypercoagulability, such as resistance to activated protein C, protein C and protein S deficiency, low levels of tissue plasminogen activator, high levels of plasminogen activator inhibitor, von Willebrand factor, lipoprotein (a), and secondary factors of hypercoagulability with factors potentially activating intravascular coagulation, such as pregnancy, antiphospholipid antibodies, systemic lupus erythematosus, hemoglobinopathies and sickle cell disease, and hemato-oncologic diseases are discussed in this article. Although coagulation abnormalities in patients with hip osteonecrosis might represent increased risk factors for the development of bone necrosis by predisposing the patient to thromboembolic phenomena, further investigation is needed to indicate the definite correlation between factors leading to increased intravascular coagulation and pathogenesis of osteonecrosis. [ABSTRACT FROM AUTHOR]

Abstract: Blood coagulation is a complex and highly coordinated process that is constantly altered and impacted by procoagulant and anticoagulant “players.” It is vital that these components work in concert to maintain a balance to keep coagulation in check. Several important endogenous anticoagulants will be discussed in this review including tissue factor pathway inhibitor, antithrombin, protein C, and protein S in origin, structure, mechanism of action, effects of deficiency, and current knowledge in veterinary medicine. [Copyright &y& Elsevier]

Abstract: A 2-acrylamido-2-methyl-1-propanesulfonic acid-co-ethylene glycol dimethacrylate (Fe3O4/SiO2/P(AMPS-co-EGDMA)) copolymer was prepared and used as a magnetic solid phase extraction (MSPE) medium for recovery of endogenous cytokinins (CKs) from plant extracts. This magnetic porous polymer was characterized by electron microscopy, nitrogen sorption experiments, elemental analysis and Fourier-transformed infrared spectroscopy. It was demonstrated to have high extraction capacity toward CKs in plants due to its specificity, surface area and porous structure. Coupled with hydrophilic interaction chromatography–tandem mass spectrometry (HILIC–MS/MS), a rapid, simple, and effective MSPE–HILIC–MS/MS analytical method for the quantitative analysis of endogenous CKs in Oryza sativa (O. sativa) roots was successfully established. Good linearities were obtained for all CKs investigated with correlation coefficients (R 2)>0.9975. The results showed that LODs (S/N=3) were ranged from 0.18 to 3.65pgmL−1. Reproducibility of the method was obtained with intra-day and inter-day relative standard deviations (RSDs) less than 16.1% and the recoveries in plant samples ranged from 72.8% to 115.5%. Finally, the MSPE–HILIC–MS/MS method was applied to several plant samples, and the amounts of endogenous CKs in O. sativa roots, leaves and Arabidopsis thaliana (A. thaliana) were successfully determined. [Copyright &y& Elsevier]

Background: Cardiac allograft vasculopathy (CAV) is the principal cause of long-term graft failure following heart transplantation. Early identification of patients at risk of CAV is essential to target invasive follow-up procedures more effectively and to establish appropriate therapies. We evaluated the prognostic value of the first heart biopsy (median: 9 days post-transplant) versus all biopsies obtained within the first three months for the prediction of CAV and graft failure due to CAV. Methods and Findings: In a prospective cohort study, we developed multivariate regression models evaluating markers of atherothrombosis (fibrin, antithrombin and tissue plasminogen activator [tPA]) and endothelial activation (intercellular adhesion molecule-1) in serial biopsies obtained during the first three months post-transplantation from 172 patients (median follow-up = 6.3 years; min = 0.37 years, max = 16.3 years). Presence of fibrin was the dominant predictor in firstbiopsy models (Odds Ratio [OR] for one- and 10-year graft failure due to CAV = 38.70, p = 0.002, 95% CI = 4.00-374.77; and 3.99, p = 0.005, 95% CI = 1.53-10.40) and loss of tPA was predominant in three-month models (OR for one- and 10-year graft failure due to CAV = 1.81, p = 0.025, 95% CI = 1.08-3.03; and 1.31, p = 0.001, 95% CI = 1.12-1.55). First-biopsy and three-month models had similar predictive and discriminative accuracy and were comparable in their capacities to correctly classify patient outcomes, with the exception of 10-year graft failure due to CAV in which the three-month model was more predictive. Both models had particularly high negative predictive values (e.g., First-biopsy vs. three-month models: 99% vs. 100% at 1-year and 96% vs. 95% at 10-years). Conclusions: Patients with absence of fibrin in the first biopsy and persistence of normal tPA in subsequent biopsies rarely develop CAV or graft failure during the next 10 years and potentially could be monitored less invasively. Presence of early risk markers in the transplanted heart may be secondary to ischemia/reperfusion injury, a potentially modifiable factor. [ABSTRACT FROM AUTHOR]

Objective: Myosin binding protein C (MYBPC3) plays a role in ventricular relaxation. The aim of the study was to investigate the association between cardiac myosin binding protein C (MYBPC3) gene polymorphisms and diastolic heart failure (DHF) in a human case-control study. Methods: A total of 352 participants of 1752 consecutive patients from the National Taiwan University Hospital and its affiliated hospital were enrolled. 176 patients diagnosed with DHF confirmed by echocardiography were recruited. Controls were matched 1-to-1 by age, sex, hypertension, diabetes, renal function and medication use. We genotyped 12 single nucleotide polymorphisms (SNPs) according to HapMap Han Chinese Beijing databank across a 40 kb genetic region containing the MYBPC3 gene and the neighboring DNA sequences to capture 100% of haplotype variance in all SNPs with minor allele frequencies ≥5%. We also analyzed associations of these tagging SNPs and haplotypes with DHF and linkage disequilibrium (LD) structure of the MYBPC3 gene. Results: In a single locus analysis, SNP rs2290149 was associated with DHF (allele-specific p = 0.004; permuted p = 0.031). The SNP with a minor allele frequency of 9.4%, had an odds ratio 2.14 (95% CI 1.25-3.66; p = 0.004) for the additive model and 2.06 for the autosomal dominant model (GG+GA : AA, 95% CI 1.17-3.63; p = 0.013), corresponding to a population attributable risk fraction of 12.02%. The haplotypes in a LD block of rs2290149 (C-C-G-C) was also significantly associated with DHF (odds ratio 2.10 (1.53-2.89); permuted p = 0.029). Conclusions: We identified a SNP (rs2290149) among the tagging SNP set that was significantly associated with early DHF in a Chinese population. [ABSTRACT FROM AUTHOR]

The molecular mechanisms underlying cerebral angiogenesis have not been fully investigated. Using primary mouse brain endothelial cells (MBECs) and a capillary-like tube formation assay, we investigated whether the sonic hedgehog (Shh) signaling pathway is coupled with the plasminogen/plasmin system in mediating cerebral angiogenesis. We found that incubation of MBECs with recombinant human Shh (rhShh) substantially increased the tube formation in naïve MBECs. This was associated with increases in tissue plasminogen activator (tPA) activation and reduction of plasminogen activator inhibitor 1 (PAI-1). Blockage of the Shh pathway with cyclopamine abolished the induction of tube formation and the effect of rhShh on tPA and PAI-1. Addition of PAI-1 reduced rhShh-augmented tube formation. Genetic ablation of tPA in MBECs impaired tube formation and downregulated of vascular endothelial growth factor (VEGF) and angiopoietin 1 (Ang1). Addition of rhShh to tPA-/- MBECs only partially restored the tube formation and upregulated Ang1, but not VEGF, although rhShh increased VEGF and Ang1 expression on wild-type MBECs. Complete restoration of tube formation in tPA-/- MBECs was observed only when both exogenous Shh and tPA were added. The present study provides evidence that tPA and PAI-1 contribute to Shh-induced in vitro cerebral angiogenesis. [ABSTRACT FROM AUTHOR]

Background and purpose: Therapy for stroke with intravenous tissue plasminogen activator (IV-tPA) is hampered by tight licensing restrictions; some of them have been discussed in recent literature. We assessed the safety and effectiveness of off-label IV-tPA in the clinical settings. Methods: Retrospective analysis of all the patients treated with IV-tPA at our Stroke Unit. Patients were divided into two groups by licence criteria [on-label group (OnLG), off-label group (OffLG)]. Primary outcome measures were symptomatic intracranial haemorrhages (sICH), major systemic haemorrhages, modified Rankin scale (mRS) and mortality rate at 3 months. Results: Five hundred and five patients were registered, 269 (53.2%) were assigned to OnLG and 236 (46.9%) to OffLG. Inclusion criteria for the OffLG were aged >80 years (129 patients), time from onset of symptoms to treatment over 3 h (111), prior oral anticoagulant treatment with International Normalised Ratio ≤ 1.7 (41), combination of previous stroke and diabetes mellitus (14), surgery or severe trauma within 3 months of stroke (13), National Institutes of Health Stroke Scale score over 25 (11), intracranial tumours (5), systemic diseases with risk of bleeding (7) and seizure at the onset of stroke (2). No significant differences were identified between both groups regarding the proportion of sICH (OnLG 2.2% vs. OffLG 1.6%, P = 0.78) or the 3-month mortality rate (11.1% vs. 19%: odds ratio (OR), 1.49; 95% CI, 0.86-2.55; P = 0.14). Multivariate analysis showed no significant differences in functional independence at 3 months between both groups (mRS <3 64.3% vs. 50.4%: OR mRS >2 1.7; 95% CI, 0.96-2.5; P = 0.07). Conclusion: Intravenous thrombolysis may be safe and efficacious beyond its current label restrictions. [ABSTRACT FROM AUTHOR]

Abstract: Sulfated low molecular weight lignins (LMWLs), designed as oligomeric mimetics of low molecular weight heparins (LMWHs), have been found to bind in exosite II of thrombin . To assess whether sulfated LMWLs recognize other heparin-binding proteins, we studied their effect on serine proteases of the coagulation, inflammatory and digestive systems. Using chromogenic substrate hydrolysis assay, sulfated LMWLs were found to potently inhibit coagulation factor XIa and human leukocyte elastase, moderately inhibit cathepsin G and not inhibit coagulation factors VIIa, IXa, and XIIa, plasma kallikrein, activated protein C, trypsin, and chymotrypsin. Competition studies show that UFH competes with sulfated LMWLs for binding to factors Xa and XIa. These results further advance the concept of sulfated LMWLs as heparin mimics and will aid the design of anticoagulants based on their novel scaffold. [Copyright &y& Elsevier]

Cofactors are intimately involved in steroid-regulated gene expression. Two critical questions are (1) the steps at which cofactors exert their biological activities and (2) the nature of that activity. Here we show that a new mathematical theory of steroid hormone action can be used to deduce the kinetic properties and reaction sequence position for the functioning of any two cofactors relative to a concentration limiting step (CLS) and to each other. The predictions of the theory, which can be applied using graphical methods similar to those of enzyme kinetics, are validated by obtaining internally consistent data for pair-wise analyses of three cofactors (TIF2, sSMRT, and NCoR) in U2OS cells. The analysis of TIF2 and sSMRT actions on GR-induction of an endogenous gene gave results identical to those with an exogenous reporter. Thus new tools to determine previously unobtainable information about the nature and position of cofactor action in any process displaying first-order Hill plot kinetics are now available. [ABSTRACT FROM AUTHOR]

The commentary emphasizes the immediate necessity to find alternative sources for the production of pharmaceutical heparin to meet the ton-amount world's demand of the glycosaminoglycan. The recent development of a synthetic approach brings considerable new technical and scientifically relevant knowledge to the field, with a strong potential for application in the future. However, the artificial approach does not offer a rapid alternative for the current world crisis affecting the production of heparin, which has to respond to an increasing worldwide demand. It is important to call attention for the availability of marine organisms that are rich sources of heparin analogs with significant anticoagulant activity, low bleeding effect that have been cultivated in very large amounts for years in different parts of the world. Additionally, alternative sources of mammalian heparin, such as bovine intestine and lung have been continually used in countries from South America, Africa and Asia, since the outbreak of the BSE without any report of prion contamination in humans. Recently, it has been shown that bovine and porcine intestinal heparins are composed by different proportions of a mixture of the same fractions that can be simply separated by anion exchange chromatography. In other words, high quality porcine heparin can be obtained from bovine tissue. We believe that alternative animal sources of heparin are currently a more realistic solution than artificial heparin to respond to the increasing demand for heparin. [ABSTRACT FROM AUTHOR]

The Protein C anticoagulant pathway regulates blood coagulation by preventing the inadequate formation of thrombi. It has two main plasma components: protein C and protein S. Individuals with protein C or protein S deficiency present a dramatically increased incidence of thromboembolic disorders. Here, we present the results of a genome-wide association study (GWAS) for protein C and protein S plasma levels in a set of extended pedigrees from the Genetic Analysis of Idiopathic Thrombophilia (GAIT) Project. A total number of 397 individuals from 21 families were typed for 307,984 SNPs using the Infinium® 317 k Beadchip (Illumina). Protein C and protein S (free, functional and total) plasma levels were determined with biochemical assays for all participants. Association with phenotypes was investigated through variance component analysis. After correcting for multiple testing, two SNPs for protein C plasma levels (rs867186 and rs8119351) and another two for free protein S plasma levels (rs1413885 and rs1570868) remained significant on a genome-wide level, located in and around the PROCR and the DNAJC6 genomic regions respectively. No SNPs were significantly associated with functional or total protein S plasma levels, although rs1413885 from DNAJC6 showed suggestive association with the functional protein S phenotype, possibly indicating that this locus plays an important role in protein S metabolism. Our results provide evidence that PROCR and DNAJC6 might play a role in protein C and free protein S plasma levels in the population studied, warranting further investigation on the role of these loci in the etiology of venous thromboembolism and other thrombotic diseases. [ABSTRACT FROM AUTHOR]

Objective: An increased tendency towards thromboembolic events is observed in patients with Cushing's syndrome. There are much fewer publications available about thromboembolic complications in patients with subclinical Cushing's syndrome (SCS). Therefore, a question arises whether hemostatic disturbances appear in this particular disease phase. Aim of study: Estimation of protein C (PC), free protein S (FPS), antithrombin (AT) activity, thrombomodulin (TM) concentration and activated PC resistance (APCR) in patients with SCS. Materials and methods: We studied 35 patients with SCS. The control group consisted of 33 healthy volunteers. The activity of PC, AT, FPS, APCR and the concentration of TM was estimated in all representatives Results: The comparison of the examined coagulation parameters between the patients with SCS and the healthy individuals revealed significantly higher mean PC activity and mean FPS activity in the SCS group. Mean TM concentration was significantly lower in patients with SCS compared with the control group. The differences in APCR and AT activity were not significant. We did not prove any statistically significant correlations between the examined coagulation parameters and hormonal parameters. We did not find any correlation between the concentration of cortisol and basic coagulation parameters such as international normalized ratio, activated partial thromboplastin time or fibrinogen in the group with SCS either. Conclusions: The patients with SCS present disturbances in endogenous anticoagulation system defined as PC, FPS activity and TM concentration. This finding suggests an impact of mild autonomic cortisol overproduction on coagulation system. [ABSTRACT FROM AUTHOR]

Eur J Clin Invest 2011; 41 (11): 1210-1219 Abstract Background In models of acute ischaemia, erythropoietin (EPO) administration has been found to attenuate vascular injury largely through reduced apoptosis, suppressed inflammation and increased nitric oxide availability. We studied the association between circulating endogenous EPO and no-reflow in patients with first ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI). Methods Blood sampling was performed before PPCI. Consecutive patients with ( n = 24) or without ( n = 24) evidence of angiographic no-reflow after PPCI were enrolled. Angiographic no-reflow was defined as Thrombolysis in Myocardial Infarction (TIMI) flow ≤ 2 or as TIMI flow = 3 but with myocardial blush grade < 2. We also assessed electrocardiographic (ECG) no-reflow as ≤ 50% resolution of maximal ST elevation 60 min after PPCI. Results Baseline characteristics did not correlate significantly with EPO concentrations. In contrast, both angiographic and ECG no-reflow correlated with lower EPO levels at univariate analysis [median (interquartile): 4·2 (0·6-9·5) vs. 12·2 (5·2-20·3) mIU mL−1, P = 0·001, and 4·0 (0·6-7·1) vs. 9·3 (1·0-12·6) mIU mL−1, P = 0·01, respectively]. At multivariable analysis, decreasing EPO tertiles and left anterior descending as the infarct-related artery were the only factors that predicted both angiographic and ECG no-reflow ( P = 0·017 and P = 0·02 for EPO; P < 0·005 and P > 0·05 for left anterior descending artery, respectively). Conclusions We found an independent, graded, inverse relation between endogenous EPO levels and angiographic and ECG no-reflow following PPCI. In animal models of ischaemia, EPO has been found to be protective. In humans, endogenous EPO may contribute to offset the mechanisms responsible for no-reflow. [ABSTRACT FROM AUTHOR]

In most other studies the release of amino acid neurotransmitters and modulators in vitro has been studied mostly using labeled preloaded compounds. For several reasons the estimated release may not reliably reflect the release of endogenous compounds. The magnitudes of the release cannot thus be quite correctly estimated using radioactive labels. The basal and K-evoked release of the neuroactive endogenous amino acids γ-aminobutyrate (GABA), glycine, taurine, glutamate and aspartate was now studied in slices from the striatum from 7-day-old to 3-month-old mice under control (normoxic) and ischemic conditions. The release of alanine, threonine and serine was assessed as control. GABA and glutamate release was much greater in 3-month-old than in 7-day-old mice, whereas with taurine the situation was the opposite. Ischemia markedly enhanced the release of all these three amino acids. The release of aspartate and glycine was markedly enhanced as well whereas no effects were discernible in the release of glutamine, alanine, serine and threonine. K stimulation (50 mM) enhanced the release of GABA, glutamate, taurine, aspartate and glycine in most cases, except with taurine in 3-month-old mice under the ischemic conditions and with aspartate in 7-day-old mice under the control conditions. K stimulation did not affect the release of glutamine, alanine, serine or threonine. The results on endogenous amino acids are qualitatively similar to those obtained in our earlier experiments with labeled preloaded amino acids. In conclusion, in developing mice only inhibitory taurine is released in such amounts that may counteract the harmful effects of excitatory amino acids in ischemia. [ABSTRACT FROM AUTHOR]

Angiotensin (Ang)-(1-7) Ang-(1-7) is formed from angiotensin II by angiotensin-converting enzyme 2 (ACE2) and modulates the renin-angiotensin system. We evaluated whether the Ang-(1-7)-Mas axis in the rostral ventrolateral medulla (RVLM) contributes to neural mechanisms of blood pressure (BP) regulation. We microinjected Ang-(1-7), Ang-(1-7)-Mas receptor antagonist A-779, and ACE2 inhibitor DX600 into the RVLM of anesthetized Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHRs). Unilateral Ang-(1-7) microinjection induced a significantly greater increase in AP (arterial blood pressure) in SHR than in WKY. Bilateral A-779 microinjection induced a significantly greater decrease in AP and renal sympathetic nerve activity in SHR than in WKY. Bilateral DX600 microinjection induced a significantly greater decrease in AP in SHR than in WKY. Our results suggest that endogenous Ang-(1-7) in the RVLM contributes to maintain AP and renal sympathetic nerve activity both in SHR and WKY and that its activity might be enhanced in SHR. [ABSTRACT FROM AUTHOR]

Owing to its ability to generate the clot-dissolving protease plasmin, tissue plasminogen activator (tPA) is the only approved drug for the acute treatment of ischemic stroke. However, tPA also promotes hemorrhagic transformation and excitotoxic events. High mobility group box-1 protein (HMGB-1) is a non-histone transcription factor and a pro-inflammatory cytokine, which has also been shown to bind to both tPA and plasminogen. We thus investigated the cellular and molecular effects through which HMGB-1 could influence the vascular and parenchymal effects of tPA during ischemia. We demonstrate that HMGB-1 not only increases clot lysis by tPA, but also reduces the passage of vascular tPA across the blood-brain barrier, as well as tPA-driven leakage of the blood-brain barrier. In addition, HMGB-1 prevents the pro-neurotoxic effect of tPA, by blocking its interaction with N-methyl-D-aspartate (NMDA) receptors and the attendant potentiation of NMDA-induced neuronal Ca2+ influx. In conclusion, we show in vitro that HMGB-1 can promote the beneficial effects of tPA while counteracting its deleterious properties. We suggest that derivatives of HMGB-1, devoid of pro-inflammatory properties, could be used as adjunctive therapies to improve the overall benefit of tPA-mediated thrombolysis following stroke. [ABSTRACT FROM AUTHOR]

Cellular FLICE (FADD-like IL-1beta-converting enzyme)-inhibitory protein (c-FLIP) is a major resistance factor and critical anti-apoptotic regulator that inhibits tumor necrosis factor-alpha (TNF-alpha), Fas-L, and TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis as well as chemotherapy-triggered apoptosis in malignant cells. c-FLIP is expressed as long (c-FLIPL), short (c-FLIPS), and c-FLIPR splice variants in human cells. c-FLIP binds to FADD and/or caspase-8 or -10 in a ligand-dependent and-independent fashion, which in turn prevents death-inducing signaling complex (DISC) formation and subsequent activation of the caspase cascade. Moreover, c-FLIPL and c-FLIPS are known to have multifunctional roles in various signaling pathways, as well as activating and/or upregulating several cytoprotective signaling molecules. Upregulation of c-FLIP has been found in various tumor types, and its downregulation has been shown to restore apoptosis triggered by cytokines and various chemotherapeutic agents. Hence, c-FLIP is an important target for cancer therapy. For example, small interfering RNAs (siRNAs) that specifically knockdown the expression of c-FLIPL in diverse human cancer cell lines augmented TRAIL-induced DISC recruitment and increased the efficacy of chemotherapeutic agents, thereby enhancing effector caspase stimulation and apoptosis. Moreover, small molecules causing degradation of c-FLIP as well as decreasing mRNA and protein levels of c-FLIPL and c-FLIPS splice variants have been found, and efforts are underway to develop other c-FLIP-targeted cancer therapies. This review focuses on (1) the functional role of c-FLIP splice variants in preventing apoptosis and inducing cytokine and drug resistance; (2) the molecular mechanisms that regulate c-FLIP expression; and (3) strategies to inhibit c-FLIP expression and function. [ABSTRACT FROM AUTHOR]

Abstract: In order to unravel the functions of receptor-like proteins in Arabidopsis (AtRLPs), including the CLAVATA2 (CLV2) receptor, we employed a reverse genetics approach. In previous studies, we assembled a genome-wide collection of AtRLP gene T-DNA insertion mutants and reported on the biological roles of a few AtRLPs closely related to CLV2. In this study, we showed that over-expression of CLV2 in wild-type plants unexpectedly resulted in a multi-carpel phenotype, mimicking the clv2 mutant, which suggests a co-suppression effect on the endogenous CLV2. We also found evidence that the phenotypes of various clv2 alleles are significantly affected by the genetic background of Arabidopsis ecotypes, which, however, did not depend on the ER locus. [ABSTRACT FROM AUTHOR]

Sequences governing RNA splicing are difficult to study in situ due to the great difficulty of traditional targeted mutagenesis. Zinc-finger nuclease (ZFN) technology allows for the rapid and efficient introduction of site-specific mutations into mammalian chromosomes. Using a ZFN pair along with a donor plasmid to manipulate the outcomes of DNA repair, we introduced several discrete, targeted mutations into the fourth intron of the endogenous BAX gene in Chinese hamster ovary cells. Putative lariat branch points, the polypyrimidine tract, and the splice acceptor site were targeted. We recovered numerous otherwise isogenic clones carrying the intended mutations and analyzed the effect of each on BAX pre-mRNA splicing. Mutation of one of three possible branch points, the polypyrimidine tract, and the splice acceptor site all caused exclusion of exon five from BAX mRNA. Interestingly, these exon-skipping mutations allowed usage of cryptic splice acceptor sites within intron four. These data demonstrate that ZFN-mediated gene editing is a highly effective tool for dissection of pre-mRNA splicing regulatory sequences in their endogenous context. [ABSTRACT FROM AUTHOR]

Background: Recombinant tissue plasminogen activator (rt-PA) is one of the most important thrombolytic agents used in patients with vascular occlusions such as acute ischemic stroke or myocardial infarction. A variety of recombinant protein expression systems have been developed for heterologous gene expression in prokaryotic and eukaryotic hosts. In recent years, Leishmania tarentolae (L. tarentolae), a nonpathogenic trypanosomatid protozoa, has come under consideration because of its safety and immunogenicity as a vaccine vector and special attributes in the expression of complex proteins. This study was done to improve rt-PA expression in this protozoon and create the opportunity for the replacement of rt-PA gene with other genes for the production of other complex proteins. Methods: Two expression cassettes were used for the integration of two copies of t-PA cDNA, one copy in each cassette, into the parasite genome by electroporation. The transformed clones were selected by antibiotic resistancy. The expression of active secreted rt-PA was confirmed by Western blot analysis and Chromolize assay. Results: Appearance of a 64 kD band in nitrocellulose membrane in the Western blot analysis confirmed the presence of full-length rt-PA in the culture media. Chromolize assay showed the expression levels of active recombinant t-PA in single and double transfected L. tarentolae clones- 375 IU/ml and 480 IU/ml of the culture media, respectively. Conclusion: The produced rt-PA in the culture media containing the transfected cells was at least seven times higher than what has been reported in previous studies on L. tarentolae or on some other eukaryotic systems. [ABSTRACT FROM AUTHOR]

The proteins, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1), act in concert to balance thrombus formation and degradation, thereby modulating the development of arterial thrombosis and excessive bleeding. PAI-1 is upregulated by the renin-angiotensin system (RAS), specifically by angiotensin II, the product of angiotensin converting enzyme (ACE) cleavage of angiotensin I, which is produced by the cleavage of angiotensinogen (AGT) by renin (REN). ACE indirectly stimulates the release of t-PA which, in turn, activates the corresponding fibrinolytic system. Single polymorphisms in these pathways have been shown to significantly impact plasma levels of t-PA and PAI-1 differently in Ghanaian males and females. Here we explore the involvement of epistatic interactions between the same polymorphisms in central genes of the RAS and fibrinolytic systems on plasma t-PA and PAI-1 levels within the same population (n = 992). Statistical modeling of pairwise interactions was done using two-way ANOVA between polymorphisms in the ETNK2, RENIN, ACE, PAI-1, t-PA, and AGT genes. The most significant interactions that associated with t-PA levels were between the ETNK2 A6135G and the REN T9435C polymorphisms in females (p = 0.006) and the REN T9435C and the TPA I/D polymorphisms (p = 0.005) in males. The most significant interactions for PAI-1 levels were with REN T9435C and the TPA I/D polymorphisms (p = 0.001) in females, and the association of REN G6567T with the TPA I/D polymorphisms (p = 0.032) in males. Our results provide evidence for multiple genetic effects that may not be detected using single SNP analysis. Because t-PA and PAI-1 have been implicated in cardiovascular disease these results support the idea that the genetic architecture of cardiovascular disease is complex. Therefore, it is necessary to consider the relationship between interacting polymorphisms of pathway specific genes that predict t-PA and PAI-1 levels. [ABSTRACT FROM AUTHOR]

Background: In Maine Coon (MC) cats the c.91G > C mutation in the gene MYBPC3, coding for cardiac myosin binding protein C (cMyBP-C), is associated with feline hypertrophic cardiomyopathy (fHCM). The mutation causes a substitution of an alanine for a proline at residue 31 (p.A31P) of cMyBP-C. The pattern of inheritance has been considered autosomal dominant based on a single pedigree. However, larger studies are needed to establish the significance of cats being heterozygous or homozygous for the mutation with respect to echocardiographic indices and the probability of developing fHCM. The objective of the present study was to establish the clinical significance of being homozygous or heterozygous for the p.A31P cMyBP-C mutation in young to middle-aged cats. Methods: The cohort consisted of 332 MC cats, 282 cats < 4 years (85%). All cats were examined by 2-D and Mmode echocardiography. DNA was extracted from blood samples or buccal swabs and screened for the p.A31P cMyBP-C mutation in exon 3 of the gene, using polymerase chain reaction followed by DNA sequencing. Results: The fHCM prevalence was 6.3% in the cohort. Eighteen cats were homozygous and 89 cats were heterozygous for the mutation. The odds ratio for having fHCM for homozygous cats was 21.6 (95% confidence interval 7.01-66.2) - when the group of equivocal cats was categorized as non-affected. Overall, 50% of the cats that were homozygous for the mutation had fHCM. p.A31P heterozygosity was not associated with a significant odds ratio for fHCM. In cats in the 4 to 6 years of age range a similar, non significant, odds ratio was seen in heterozygous cats. Only two cats over four years were homozygous and both were diagnosed with fHCM. Conclusion: As there is no significant odds ratio associated with being heterozygous for the pA31P cMyBP-C mutation at this age, the mutation must have a very low penetrance in this group. From our data it would appear that most MC cats that develop fHCM due to the p.A31P mutation prior to the age of approximately 6 years do so because they are homozygous for this mutation. [ABSTRACT FROM AUTHOR]

In the same way that peptide antigens are presented by major histocompatibility complex (MHC) molecules, glycolipid antigens can also activate the immune response via binding to CD1 proteins on antigen-presenting cells (APCs) and stimulate CD1- restricted T cells. In humans, there are fivemembers of the CD1 family, termed CD1a-e, of which CD1a-d are involved in glycolipid presentation at the cell surface, while CD1e is involved in the intracellular trafficking of glycolipid antigens. Both endogenous (self-derived) and exogenous (non-self-derived) glycolipids have been shown to bind to members of the CD1 family with varying degrees of specificity. In this paper we focus on the key glycolipids that bind to the different members of the CD1 family. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide is a colorless gas and has a strong odor of rotten eggs. It is absorbed by the upper respiratory tract mucosa, and it causes histotoxic hypoxemia and respiratory depression by exerting an inhibitory effect on cytochrome oxidase. To evaluate the role of toxicological data in distinguishing between the H2S blood concentration secondary to lethal poisoning and the endogenous H2S produced during putrefaction, we compared the postmortem H2S concentrations of six fatal H2S poisoning cases (8.7-28.6 mg/L) with the postmortem concentrations of endogenous H2S of 12 subjects who died from other causes (traffic-related deaths) (2.2-32.7 mg/L). These results will be of interest to the forensic community as it underlines the importance of considering circumstantial evidence along with the toxicological and pathological findings in the identification of H2S lethal poisoning. [ABSTRACT FROM AUTHOR]

To date, very few βy-crystallins have been identified and structurally characterized. Several of them have been shown to bind Ca2+ and thereby enhance their stability without any significant change in structure. Although Ca2+-induced conformational changes have been reported in two putative βy-crystallins from Caulobacter crescentus and Yersinia pestis, they are shown to be partially unstructured, and whether they acquire a βy-crystallin fold is not known. We describe here a βy-crystallin domain, hahellin, its Ca2+ binding properties and NMR structure. Unlike any other βy-crystallin, hahellin is characterized as a pre-molten globule (PMG) type of natively unfolded protein domain, It undergoes drastic conformational change and acquires a typical βy-crystallin fold upon Ca2+ binding and hence acts as a Ca2+-regulated conformational switch. However, it does not bind Mg2+. The intrinsically disordered Ca2+-free state and the close structural similarity of Ca2+-bound hahellin to a microbial βy-crystallin homologue. Protein S, which shows Ca2+-dependent stress response, make it a potential candidate for the cellular functions. This study indicates the presence of a new class of natively unfolded βy-crystallins and therefore the commencement of the possible functional roles of such proteins in this superfamily. [ABSTRACT FROM AUTHOR]

Background: Alzheimers disease (AD) has been strongly linked to an anomalous self-assembly of the amyloid-β peptide (Aβ). The correlation between clinical symptoms of AD and Aβ depositions is, however, weak. Instead small and soluble Aβ oligomers are suggested to exert the major pathological effects. In strong support of this notion, immunological targeting of Aβ oligomers in AD mice-models shows that memory impairments can be restored without affecting the total burden of Aβ deposits. Consequently a specific immunological targeting of Aβ oligomers is of high therapeutic interest. Methodology/Principal Findings: Previously the generation of conformational-dependent oligomer specific anti-Aβ antibodies has been described. However, to avoid the difficult task of identifying a molecular architecture only present on oligomers, we have focused on a more general approach based on the hypothesis that all oligomers expose multiple identical epitopes and therefore would have an increased binding to a multivalent receptor. Using the polyvalent IgM immunoglobulin we have developed a monoclonal anti-Aβ antibody (OMAB). OMAB only demonstrates a weak interaction with Aβ monomers and dimers having fast on and off-rate kinetics. However, as an effect of avidity, its interaction with Aβ-oligomers results in a strong complex with an exceptionally slow off-rate. Through this mechanism a selectivity towards Aβ oligomers is acquired and OMAB fully inhibits the cytotoxic effect exerted by Aβ(1-42) at highly substoichiometric ratios. Anti-Aβ auto-antibodies of IgM isotype are frequently present in the sera of humans. Through a screen of endogenous anti- Aβ IgM auto-antibodies from a group of healthy individuals we show that all displays a preference for oligomeric Aβ. Conclusions/Significance: Taken together we provide a simple and general mechanism for targeting of oligomers without the requirement of conformational-dependent epitopes. In addition, our results suggest that IgM anti-Aβ auto-antibodies may exert a more specific protective mechanism in vivo than previously anticipated. [ABSTRACT FROM AUTHOR]

Thrombospondin 1 (TSP1) has been shown to play a critical role in inhibiting angiogenesis, resulting in inhibition of tumor growth and metastases. To figure out TSP1's regulators will lead to reveal its biological function mechanistically. In this study, we show that E2F-1 could activate the transcription of TSP1 by both promoter assays and Northern blot. Analysis of various TSP1 promoter mutant constructs showed that a sequence located -144/-137 up-stream of the transcriptional initiation site, related to the consensus E2F-responsive sequence, is necessary for the activation. In consistence with upregulation of TSP-1 activity by over-expression of E2F-1, the knockdown of endogenous E2F-1 inhibited TSP-1 promoter activity significantly, implying that E2F-1 mediated regulation of TSP-1 is relevant in vivo. In addition, E2F-1 could also directly bind to the TSP1 promoter region covering -144/-137 region as revealed by ChIP assays. Furthermore, the E2F-1- induced activation of TSP1 gene transcription is suppressed by pRB1 in a dose-dependent manner. Taken together, the results demonstrate that TSP1 is a novel target for E2F1, which might imply that E2F-1 can affect angiogenesis by modulating TSP1 expression. [ABSTRACT FROM AUTHOR]

Background: A striatocapsular infarct (SCI) is a subcortical infarct in the territory of the lenticulostriate arteries, most likely due to transient occlusion of the main stem of the middle cerebral artery (MCA). Presence of the hyperdense middle cerebral artery sign (HMCAS) is a reliable marker of occlusion of the MCA. We hypothesized that SCIs are related to HMCAS at baseline, which subsequently disappears (HMCAS-D) on follow-up CT in stroke patients treated with intravenous rtPA. Methods: Baseline and 24-hour follow-up CTs were evaluated for HMCAS in acute ischemic stroke patients treated with intravenous rtPA and follow-up scans were also reviewed for the presence of isolated cortical (CIn), SCI, cortical and striatocapsular (CI-SCI) or lacunar infarct. We determined the incidence of SCI and the association between SCI and HMCAS on baseline and follow-up CT. Results: Of the 247 patients, 43 had an SCI (17.4%; 95% CI: 13.1–22.5). The presence of HMCAS at baseline was related to the occurrence of infarction with involvement of the striatocapsular region (SCI or CI-SCI) on follow-up CT (OR: 11.6; 95% CI: 5.9–22.8). HMCAS-D on follow-up scans was significantly related to SCI on follow-up CT compared to CI-SCI (OR: 4.9; 95% CI: 3.7–6.1). Conclusions: Occurrence of SCI and CI-SCI is associated with the presence of HMCAS on CT before thrombolysis, whereas HMCAS-D on follow-up CT is strongly related to the occurrence of SCI. Our findings support the causative role of transient occlusion of the MCA main stem in the pathogenesis of SCI. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Chalcones and dihydrochalcones exhibit chemopreventive and antitumor activity. TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a natural endogenous anticancer agent. We examined the cytotoxic and apoptotic effect of chalcones and dihydrochalcones on TRAIL-mediated apoptosis in LNCaP prostate cancer cells. The cytotoxicity was evaluated by the MTT and LDH assays. The apoptosis was detected using annexin V-FITC by flow cytometry and fluorescence microscopy. The ΔΨm was evaluated using DePsipher staining by fluorescence microscopy. Our study showed that two tested chalcones (chalcone and 2′,6′dihydroxy-4′-methoxychalcone) and three dihydrochalcones (2′,6′-dihydroxy-4′4-dimethoxydihydrochalcone, 2′,6′-dihydroxy-4′-methoxydihydrochalcone, and 2′,4′,6′-trihydroxydihydrochalcone, called phloretin) markedly augmented TRAIL-induced apoptosis and cytotoxicity in LNCaP cells and confirmed the significant role of chalcones in chemoprevention of prostate cancer. [ABSTRACT FROM AUTHOR]

Nutritional rickets (NR) remains the most common form of developmental bone disease, in spite of the efforts of clinicians and health care providers to reduce the incidence of the disease in Turkey. Today, it is well known that the etiology of NR exists along a spectrum ranging from isolated vitamin D deficiency to isolated calcium deficiency. In Turkey, almost all NR results from vitamin D deficiency that may have temporary but profound effects on short- and long-term skeletal development. Recent evidence suggests that vitamin D deficiency during infancy may predispose a patient to diseases such as diabetes mellitus, cancer, multiple sclerosis, etc. The factors responsible for the high prevalence of vitamin D deficiency in developing countries and its resurgence in developed countries include the following: limited sunshine exposure as individuals spend more time indoors watching television and working on computers or intentional sunshine avoidance for skin cancer prevention. Traditional clothing (covered dress) further limits the exposure to sunshine and thus decreases the endogenous synthesis of vitamin D. In Turkey, maternal vitamin D deficiency and breast feeding without supplementation are the most prominent reasons. The diagnosis of NR is established with a thorough history and physical examination and confirmed by laboratory evaluation. In conclusion, recent literature has drawn attention to the supplemental doses of vitamin D required to achieve a serum 25-hydroxyvitamin D level of at least 20 ng/ml (50 nmol/l), the serum concentration that is needed to optimize absorption of dietary calcium, suppress excess secretion of parathyroid hormone, and reduce fracture risk as well as prevent long-term negative effects. [ABSTRACT FROM AUTHOR]

Peroxisome proliferator-activated receptors (PPARs) are nuclear transcription factors that play central roles in metabolism and inflammation. Although a variety of compounds have been shown to activate PPARs, identification of physiologically relevant ligands has proven difficult. In silico studies of lipid derivatives reported here identify specific 5-lipoxygenase products as candidate physiologically relevant PPAR-α activators. Subsequent studies show both in vitro and in a murine model of inflammation that 5-lipoxygenase stimulation induces PPAR-α signaling and that this results specifically from production of the inflammatory mediator and chemoattractant leukotriene B4 (LTB4). Activation of PPAR- α is a direct effect of intracellularly generated LTB4 binding to the nuclear receptor and not of secreted LTB4 acting via its cell-surface receptors. Activation of PPAR-α reduces secretion of LTB4 by stimulating degradation of this fatty acid derivative. We also show that the LTB4 precursors leukotriene A4 (LTA4) and 5-hydroperoxyeicosatetrenoic acid (5- HPETE) activate PPAR-α but have no significant endogenous effect independent of conversion to LTB4. We conclude that LTB4 is a physiologically relevant PPAR-α activator in cells of the immune system. This, together with previous findings, demonstrates that different types of lipids serve as endogenous PPAR-α ligands, with the relevant ligand varying between functionally different cell types. Our results also support the suggestion that regulation of inflammation may involve balancing proinflammatory effects of LTB4, exerted through cell-surface receptors, and anti-inflammatory effects exerted through PPAR-α activation. [ABSTRACT FROM AUTHOR]

Chemical investigation of Trogopterus Feces has led to the isolation of two new isopimarane diterpenes, wulingzhic acid A (1) and wulingzhic acid B (2). Their structures were elucidated by chemical and extensive spectral analysis. Compounds 1 and 2 exhibited weak activity of antithrombin and moderate activity of antiplatelet aggregation in vitro. [Copyright &y& Elsevier]

BACKGROUND Microbubbles (MB) and ultrasound have been shown to enhance thrombolysis. We sought to evaluate safety and efficacy on middle cerebral artery (MCA) recanalization of local MB administration during intra-arterial (IA) thrombolysis and continuous transcranial Doppler (TCD) monitoring. METHODS Patients with acute M1-MCA occlusion were treated with intravenous tissue plasminogen activator (iv-tPA) and continuously monitored with TCD. If recanalization was not achieved during first-hour bridging IA-rescue was adopted: MB + tPA direct intraclot microcatheter infusion. TCD flow monitoring allowed continuous insonation at clot location. Recanalization was angiographically assessed (thrombolysis in cerebral infarction [TICI] score) and compared with simultaneous TCD data. IA procedures were stopped at 6 hours. Recanalization was reassessed at 12 hours (TCD). Neurological status was repeatedly assessed (National Institutes of Health Stroke Scale [NIHSS]). At three months, patients were considered independent if mRS ≤ 2. RESULTS Of the 18 included patients (mean age 72), 16 received standard iv-tPA (.9 mg/kg). Nine patients were recanalized during tPA infusion and 9 patients underwent IA-rescue procedures. Median pre-IA NIHSS score: 20. Median time to IA initiation was 175 ± 63 minutes. Mean IA doses were tPA = 10 ± 3 mg and MB = 3 ± 1 mL. TCD monitoring allowed direct visualization of massive MB arrival during every administration. In-procedure recanalization was observed in 78% ( n= 7): complete-TICI3 in 22% ( n= 2), partial-TICI2 in 56% ( n= 5). Perfect correlation was observed between TICI and TCD scores. At 12 hours complete recanalization increased to 56%, partial to 22%. One patient (11%) experienced symptomatic intracranial hemorrhage accounting for the only death. Median NIHSS evolution was 12 at 24 hours and 10 at discharge. At 3 months 4 patients (44%) were independent. CONCLUSION The combination of ultrasound and IA MB and tPA may be a strategy to enhance the thrombolytic effect and increase recanalization rates. [ABSTRACT FROM AUTHOR]

Background and Purpose: Whether leukoaraiosis on baseline CT is associated with an increased risk of symptomatic intracerebral haemorrhage (sICH) or poor outcome following tissue plasminogen activator (tPA) treatment for acute ischaemic stroke is still a matter of debate. Objective: To investigate the relationship between the presence and severity of leukoaraiosis on baseline CT and the risk of sICH and functional outcome after tPA treatment for acute ischaemic stroke. Methods: A single-center observational cohort study with a retrospective analysis on consecutive patients with ischaemic stroke treated with tPA in the period 2002–2008. Outcome measures were the occurrence of sICH and functional outcome at 3 months. Results: Of the 400 patients, 24% had leukoaraiosis on their baseline CT. Eleven patients (11%) with leukoaraiosis versus thirteen (4%) patients without leukoaraiosis had a sICH [odds ratio (OR) 2.85 95%-CI 1.23–6.60, P = 0.02]. Multivariate analysis showed a non-significant trend towards an association of leukoaraiosis and sICH (OR 1.9, 95%-CI 0.78–4.68, P = 0.16). Leukoaraiosis was independently associated with poor functional outcome (OR 2.39, 95%-CI 1.21–4.72, P = 0.01). No difference was observed in the outcome measures amongst patients with moderate or severe leukoaraiosis. Conclusion: Our study demonstrates that patients treated with tPA and leukoaraiosis on their baseline CT are at greater risk of sICH and have a worse functional outcome compared to patients without leukoaraiosis. It is important to note that these results should not lead to exclusion of patients with leukoaraiosis for tPA treatment. [ABSTRACT FROM AUTHOR]

This study investigated the influence of oral contraceptives (OC) on diffuse noxious inhibitory control (DNIC) in healthy women. Fifteen women taking OC and 17 normally menstruating women (No-OC) were tested during high and low endogenous estrogens sessions. Saliva was sampled for analysis of endogenous estradiol level. Mechanical pressure (test stimuli) was applied to the masseter muscle and finger. The pain induced by this pressure was assessed on a 0–10 numerical rating scale (NRS) before, during, and after immersion of the contralateral hand in ice-cold water (cold pressor test, CPT) to induce DNIC. For all subjects, pain induced by the test stimuli decreased significantly during the CPT ( P < 0.001). The decrease in general was larger in the No-OC group, with a significant difference between groups in the masseter muscle in the low session ( P < 0.027). There were no significant differences between groups or sessions in estradiol levels. These results indicate that endogenous pain modulation may be less effective in OC users. [ABSTRACT FROM AUTHOR]

Male moths are endowed with odorant receptors (ORs) to detect species-specific sex pheromones with remarkable sensitivity and selectivity. We serendipitously discovered that an endogenous OR in the fruit fly, Drosophila melanogaster, is highly sensitive to the sex pheromone of the silkworm moth, bombykol. Intriguingly, the fruit fly detectors are more sensitive than the receptors of the silkworm moth, although its ecological significance is unknown. By expression in the "empty neuron" system, we identified the fruit fly bombykol-sensitive OR as DmelOR7a (= DmOR7a). The profiles of this receptor in response to bombykol in the native sensilla (ab4) or expressed in the empty neuron system (ab3 sensilla) are indistinguishable. Both WT and transgenic flies responded with high sensitivity, in a dose-dependent manner, and with rapid signal termination. In contrast, the same empty neuron expressing the moth bombykol receptor, BmorOR1, demonstrated low sensitivity and slow signal inactivation. When expressed in the trichoid sensilla T1 of the fruit fly, the neuron housing BmorOR1 responded with sensitivity comparable to that of the native trichoid sensilla in the silkworm moth. By challenging the native bombykol receptor in the fruit fly with high doses of another odorant to which the receptor responds with the highest sensitivity, we demonstrate that slow signal termination is induced by overdose of a stimulus. As opposed to the empty neuron system in the basiconic sensilla, the structural, biochemical, and/or biophysical features of the sensilla make the T1 trichoid system of the fly a better surrogate for the moth receptor. [ABSTRACT FROM AUTHOR]

Secretion of glucocorticoid hormones during stress produces an array of physiological changes that are adaptive and beneficial in the short term. In the face of repeated stress exposure, however, habituation of the glucocorticoid response is essential as prolonged glucocorticoid secretion can produce deleterious effects on metabolic, immune, cardiovascular, and neurobiological function. Endocannabinoid signaling responds to and regulates the activity of the hypothalamic-pituitary-adrenal (HPA) axis that governs the secretion of glucocorticoids; however, the role this system plays in adaptation of the neuroendocrine response to repeated stress is not well characterized. Herein, we demonstrate a divergent regulation of the two endocannabinoid ligands, N-arachidonylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG), following repeated stress such that AEA content is persistently decreased throughout the corticolimbic stress circuit, whereas 2-AG is exclusively elevated within the amygdala in a stress-dependent manner. Pharmacological studies demonstrate that this divergent regulation of AEA and 2-AG contribute to distinct forms of HPA axis habituation. Inhibition of AEA hydrolysis prevented the development of basal hypersecretion of corticosterone following repeated stress. In contrast, systemic or intra-amygdalar administration of a CB1 receptor antagonist before the final stress exposure prevented the repeated stress-induced decline in corticosterone responses. The present findings demonstrate an important role for endocannabinoid signaling in the process of stress HPA habituation, and suggest that AEA and 2-AG modulate different components of the adrenocortical response to repeated stressor exposure. [ABSTRACT FROM AUTHOR]