Platelets promote epileptic seizures by modulating brain serotonin level, enhancing neuronal electric activity, and contributing to neuroinflammation and oxidative stress.

The drugs currently available for treating epilepsy are only partially effective in managing this condition. Therefore, it is crucial to investigate new pathways that induce and promote epilepsy development. Previously, we found that platelets interact with neuronal glycolipids and actively secrete pro-inflammatory mediators during central nervous system (CNS) pathological conditions such as neuroinflammation and traumatic brain injury (TBI). These factors increase the permeability of the blood-brain barrier (BBB), which may create a predisposition to epileptic seizures. In this study, we demonstrated that platelets substantially enhanced epileptic seizures in a mouse model of pentylenetetrazole (PTZ) -induced seizures. We found that platelets actively secreted serotonin, contributed to increased BBB permeability, and were present in the CNS parenchyma during epileptic seizures. Furthermore, platelets directly stimulated neuronal electric activity and induced the expression of specific genes related to early neuronal response, neuroinflammation, and oxidative phosphorylation, leading to oxidative stress in neurons. The intracranial injection of physiological numbers of platelets that mimicked TBI-associated bleeding was sufficient to induce severe seizures, which resembled conventional PTZ-induced epileptic activity. These findings highlight a conceptually new role of platelets in the development of epileptic seizures, and indicate a potential new therapeutic approach targeting platelets to prevent and treat epilepsy.

Fresh evidence for major brain gangliosides as a target for the treatment of Alzheimer's disease.

Although it was suggested that gangliosides play an important role in the binding of amyloid fragments to neuronal cells, the exact role of gangliosides in Alzheimer's disease (AD) pathology remains unclear. To understand the role of gangliosides in AD pathology in vivo, we crossed st3gal5-deficient (ST3-/-) mice that lack major brain gangliosides GM1, GD1a, GD3, GT1b, and GQ1b with 5XFAD transgenic mice that overexpress 3 mutant human amyloid proteins AP695 and 2 presenilin PS1 genes. We found that ST3-/- 5XFAD mice have a significantly reduced burden of amyloid depositions, low level of neuroinflammation, and did not exhibit neuronal loss or synaptic dysfunction. ST3-/- 5XFAD mice performed significantly better in a cognitive test than wild-type (WT) 5XFAD mice, which was comparable with WT nontransgenic mice. Treatment of WT 5XFAD mice with the sialic acid-specific Limax flavus agglutinin resulted in substantial improvement of AD pathology to a level of ST3-/- 5XFAD mice. Thus, our findings highlight an important role for gangliosides as a target for the treatment of AD.

In Vitro-In Vivo Extrapolation of Key Transporter Activity at the Blood-Brain Barrier.

Understanding the quantitative implications of P-glycoprotein and breast cancer resistance protein efflux is a key hurdle in the design of effective, centrally acting or centrally restricted therapeutics. Previously, a comprehensive physiologically based pharmacokinetic model was developed to describe the in vivo unbound brain-to-plasma concentration ratio as a function of efflux activity measured in vitro. In the present work, the predictive utility of this framework was examined through application to in vitro and in vivo data generated on 133 unique compounds across three preclinical species. Two approaches were examined for the scaling of efflux activity to in vivo, namely relative expression as determined by independent proteomics measurements and relative activity as determined via fitting the in vivo neuropharmacokinetic data. The results with both approaches indicate that in vitro efflux data can be used to accurately predict the degree of brain penetration across species within the context of the proposed physiologically based pharmacokinetic framework.

Absolute Quantification of Toxicological Biomarkers via Mass Spectrometry.

With the advent of "-omics" technologies there has been an explosion of data generation in the field of toxicology, as well as many others. As new candidate biomarkers of toxicity are being regularly discovered, the next challenge is to validate these observations in a targeted manner. Traditionally, these validation experiments have been conducted using antibody-based technologies such as Western blotting, ELISA, and immunohistochemistry. However, this often produces a significant bottleneck as the time, cost, and development of successful antibodies are often far outpaced by the generation of targets of interest. In response to this, there recently have been several developments in the use of triple quadrupole (QQQ) mass spectrometry (MS) as a platform to provide quantification of proteins. This technology does not require antibodies; it is typically less expensive and quicker to develop assays and has the opportunity for more accessible multiplexing. The speed of these experiments combined with their flexibility and ability to multiplex assays makes the technique a valuable strategy to validate biomarker discovery.

Proteomic mapping in live Drosophila tissues using an engineered ascorbate peroxidase.

Characterization of the proteome of organelles and subcellular domains is essential for understanding cellular organization and identifying protein complexes as well as networks of protein interactions. We established a proteomic mapping platform in live Drosophila tissues using an engineered ascorbate peroxidase (APEX). Upon activation, the APEX enzyme catalyzes the biotinylation of neighboring endogenous proteins that can then be isolated and identified by mass spectrometry. We demonstrate that APEX labeling functions effectively in multiple fly tissues for different subcellular compartments and maps the mitochondrial matrix proteome of Drosophila muscle to demonstrate the power of APEX for characterizing subcellular proteomes in live cells. Further, we generate "MitoMax," a database that provides an inventory of Drosophila mitochondrial proteins with subcompartmental annotation. Altogether, APEX labeling in live Drosophila tissues provides an opportunity to characterize the organelle proteome of specific cell types in different physiological conditions.

Hepatic response to chronic hypoxia in experimental rat model through HIF-1 alpha, activator protein-1 and NF-kappa B.

Chronic liver diseases are commonly associated with tissue hypoxia that may cause inflammation, oxidative stress, liver cell injury and increased nuclear transcriptional regulation. The hepatic response to chronic hypoxia at the molecular level has not yet been clearly understood until now. The aim of this study is to investigate whether nuclear transcription factors [hypoxia-inducible factor-1 (HIF-1α), activator protein-1 (AP-1), nuclear factor-kappa B (NF-κB)] exhibit activity changes during hepatic response to chronic hypoxia. Blood and liver samples were collected from adult Sprague-Dawley rats living in atmospheric air or 10% oxygen for four weeks. Levels of serum alanine aminotransferase (ALT), 8-isoprostane and nitrotyrosine were measured. The activities of nuclear transcription factors and the expression of downstream genes (iNOS, eNOS, ET-1 and VEGF) were measured using RT-PCR, Western blotting and Gel shift analysis. Results showed that serum ALT level, 8-isoprostane level and formation of nitrotyrosine were within normal range at all time-points. In the hypoxic liver, DNA-binding activities of HIF-1α, NF-κB and AP-1 increased significantly. Expression levels of iNOS, VEGF and ET-1 progressively increased from day 7 to day 28. eNOS was also elevated in the hypoxic liver. In conclusion, our study suggests that increased activity of HIF-1α, AP-1 and NF-κB may partly play a significant role in the hepatic response to oxidative stress and liver injury under chronic hypoxia. The increased expression of VEGF, ET-1, iNOS and eNOS may be partly due to the compensatory mechanism in the vascular beds of the liver in response to chronic hypoxia.

Contributions of a disulfide bond and a reduced cysteine side chain to the intrinsic activity of the high-density lipoprotein receptor SR-BI.

The high-density lipoprotein (HDL) receptor scavenger receptor class B, type I (SR-BI), binds HDL and mediates selective cholesteryl ester uptake. SR-BI's structure and mechanism are poorly understood. We used mass spectrometry to assign the two disulfide bonds in SR-BI that connect cysteines within the conserved Cys(321)-Pro(322)-Cys(323) (CPC) motif and connect Cys(280) to Cys(334). We used site-specific mutagenesis to evaluate the contributions of the CPC motif and the side chain of extracellular Cys(384) to HDL binding and lipid uptake. The effects of CPC mutations on activity were context-dependent. Full wild-type (WT) activity required Pro(322) and Cys(323) only when Cys(321) was present. Reduced intrinsic activities were observed for CXC and CPX, but not XXC, XPX, or XXX mutants (X ≠ WT residue). Apparently, a free thiol side chain at position 321 that cannot form an intra-CPC disulfide bond with Cys(323) is deleterious, perhaps because of aberrant disulfide bond formation. Pro(322) may stabilize an otherwise strained CPC disulfide bond, thus supporting WT activity, but this disulfide bond is not absolutely required for normal activity. C(384)X (X = S, T, L, Y, G, or A) mutants exhibited altered activities that varied with the side chain's size: larger side chains phenocopied WT SR-BI treated with its thiosemicarbazone inhibitor BLT-1 (enhanced binding, weakened uptake); smaller side chains produced almost inverse effects (increased uptake:binding ratio). C(384)X mutants were BLT-1-resistant, supporting the proposal that Cys(384)'s thiol interacts with BLT-1. We discuss the implications of our findings on the functions of the extracellular loop cysteines in SR-BI and compare our results to those presented by other laboratories.

Evaluation of liver fibrosis by investigation of hepatic parenchymal perfusion using contrast-enhanced ultrasound: an animal study.

PURPOSE: To investigate the value of assessing the hepatic parenchymal perfusion in contrast-enhanced ultrasound (CEUS) for evaluating liver fibrosis, using an animal model. METHODS: Seventy Sprague-Dawley rats were divided into experimental (n = 35) and control (n = 35) groups. In the experimental group, liver fibrosis was induced by intraperitoneal injection of carbon tetrachloride. CEUS of the liver was performed at a 2-week interval for 14 weeks. Signal intensity of liver parenchyma was analyzed with time-intensity curves. Histologic examination of liver specimens of the animals was performed to assess the fibrosis stage. RESULTS: The peak signal intensity of hepatic parenchymal perfusion in stage 2-3 fibrosis was significantly lower than that in stage 0-1. The time to peak intensity of hepatic parenchymal perfusion was significantly longer in the experimental group than the control group, and in the stage 3 fibrosis than in stages 0-2 fibrosis. Using time to peak intensity of hepatic parenchymal perfusion to distinguish stage 3 fibrosis and stages 0-2 fibrosis, the optimum cutoff was 75,000 milliseconds with the sensitivity and specificity of 67% and 78%, respectively. CONCLUSIONS: This animal study showed that CEUS has the potential to be a complementary imaging tool in the evaluation of liver fibrosis.

Cyclooxygenase inhibitors protect D-galactosamine/lipopolysaccharide induced acute hepatic injury in experimental mice model.

We investigated the protective effects of two non-steroid anti-inflammatory drugs, indomethacin (COX-1 and COX-2 inhibitors) and nimesulide (specific COX-2 inhibitor) on the hepatic injury induced by lipopolysaccharide in d-galactosamine sensitized (Gal/LPS) mice. ICR male mice were injected with a single dose of Gal/LPS with or without pre-treatment of 3mg/kg indomethacin or 30 mg/kg nimesulide (single i.p. injection). Sixteen hours later, blood and liver tissues of mice were collected for histological, molecular, and biochemical analyses. Our results showed marked reduction of hepatic necrosis, serum ALT, and tissue TBARS levels in both indomethacin- and nimesulide-pre-treated mice when compared with Gal/LPS-treated mice. Western blot and RT-PCR analysis showed decreased levels of iNOS mRNA, iNOS protein, and nitrotyrosine formation in both COX inhibitor pre-treated groups when compared with Gal/LPS-treated group. There was an inverse relationship between COX-1 and COX-2 expressions, as well as between COX-2 and C/EBP-α expressions in COX inhibitors groups, Gal/LPS and control groups. COX inhibitors reduced the expression of TNF-α mRNA and the activity of NF-κB which were elevated by Gal/LPS treatment. We conclude that COX inhibitors protected the liver from Gal/LPS-induced hepatotoxicity. COX inhibitors could be considered as potential agents in the prevention of acute liver failure and sepsis.

Absolute quantification of toxicological biomarkers by multiple reaction monitoring.

With the advent of "-omics" technologies, there has been an explosion of data generation in the field of toxicology, as well as in many others. As new candidate biomarkers of toxicity are being regularly discovered, the next challenge is to validate these observations in a targeted manner. Traditionally, these validation experiments have been conducted using antibody-based technologies such as Western blotting, ELISA, and immunohistochemistry. However, this often produces a significant bottleneck as the time, cost, and development of successful antibodies are often far outpaced by the generation of targets of interest. In response to this, recently there have been several developments in the use of triple quadrupole (QQQ) mass spectrometry (MS) as a platform to provide quantification of proteins by multiple reaction monitoring (MRM). This technology does not require antibodies; it is typically less expensive and quicker to develop, and has the opportunity for more accessible multiplexing. The speed of these experiments combined with their flexibility and ability to multiplex assays makes the technique a valuable strategy to validate biomarker discovery.

Role of nitric oxide in the regulation of fibrogenic factors in experimental liver fibrosis in mice.

Previously, we have shown that an increased expression level of iNOS but a reduction in the expression of eNOS is associated with increased oxidative stress markers in CCl4-induced experimental liver fibrosis. The present study aimed to investigate the effect of L-arginine and 5-methylisothiourea hemisulfate (SMT) in the expression of profibrogenic factors in chronic liver injury. ICR mice were treated with CCl4 with or without treatment of L-arginine, an NO donor, or SMT, an iNOS inhibitor. The expression of matrix metalloptroteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), α-smooth muscle actin (α-SMA), tumor necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2) were investigated by RT-PCR. The activity of the MMP-2 and MMP-9 were measured by zymography. Our results showed that CCl4-treated mice showed significant up-regulation of expression of pro-fibrogenic factors, TNF-α and COX-2. Treatment with L-arginine or SMT showed a significant reduction in CCl4-induced expression of these pro-fibrogenic factors, TNF-α and COX-2. In conclusion, both SMT and L-arginine effectively attenuated the progression of CCl4-induced liver fibrosis. SMT suppresses iNOS mediated NO production. However, L-arginine augments NO production. The similar effect of the two drugs on liver fibrosis indicates that there may be two distinct pathways of NOS mediated fibrogenesis in chronic liver injury by iNOS and eNOS. Our results suggest that eNOS-mediated liver fibrogenesis may play a more important role than that of iNOS in chronic liver injury. Taken together, these results support the contention that NO plays an active role in the progression of liver fibrosis and hepatocellular damage.

Differential proteomics incorporating iTRAQ labeling and multi-dimensional separations.

Considerable effort is currently being expended to integrate newly developed "omics"-based approaches (proteomics, transcriptomics, and metabonomics) into preclinical safety evaluation workflows in the hope that more sensitive prediction of toxicology can be achieved as reported by Waters and Fostel (Nat. Rev. Genet. 5(12):936-948, 2004) and Craig et al. (J. Proteome Res. 5(7):1586-1601, 2006). Proteomic approaches are well placed to contribute to this effort as (a) proteins are the metabolically active products of genes and, as such, may provide more sensitive and direct predictive information on drug-induced liabilities and (b) they have the potential to determine tissue leakage markers in peripheral fluids. Here, we describe a workflow for proteomic semi-quantitative expression profiling of liver from rats treated with a known hepatotoxicant using a multiplexed isobaric labeling strategy and multi-dimensional liquid chromatography.

Prioritization of candidate protein biomarkers from an in vitro model system of breast tumor progression toward clinical verification.

The use of in vitro cell culture model systems has revealed many potential mediators and candidate biomarkers of various disease phenotypes. To be of clinical utility, the expression of these candidates must be assessed in patient samples such as tissue, urine or blood. However, typical "omic" experiments may produce candidates in such large numbers that it is usually impossible to test all of these in clinical samples. Here, we present a proteomic approach to discover and prioritize candidate biomarkers that are more likely to be found in serum. Using a combination of experimental and in silico approaches, we have demonstrated this approach using an isogenic cell culture model of breast cancer invasion. Differential proteomics (2D-DIGE) was used to discover a number of candidate biomarkers and a subset of these were identified as "extracellular". We tested the validity of this approach by screening serum from breast cancer patients for these candidates and then verified the presence of several of these "extracellular" proteins. This approach provides a pragmatic approach to prioritizing candidates that may be most suitable for downstream assays such as multiple reaction monitoring.

Identification and functional validation of therapeutic targets for malignant melanoma.

Despite remarkable effort, malignant melanoma still remains a potent killer. Millions of dollars have been spent on clinical trials that have not succeeded in achieving significant patient benefit. The thorough validation of drug targets at an earlier stage is, therefore, an essential step in the development of new therapies. Since the development of microarray experiments, putative drug targets are being identified in a high-throughput manner. Though high-throughput functional validation methods are currently being established, a more specific, pre-clinical analysis of promising target genes remains inevitable. For this, a broad range of increasingly sophisticated functional models is available. In vitro, the microenvironment of skin can be simulated through various two or three-dimensional models. In vivo assays range from xenograft studies to the establishment of transgenic organisms. Here, we provide a summary of functional interrogation approaches in melanoma research, focusing on the application of these strategies to the development of new effective therapies.

Endothelial nitric oxide synthase is a critical factor in experimental liver fibrosis.

Reduced expression of endothelial nitric oxide synthase (eNOS) in chronic liver disease can reduce hepatic perfusion and accelerate fibrosis. The relationship between eNOS expression and liver fibrogenesis remains unclear. We investigated whether L-arginine attenuated chronic liver fibrosis through eNOS expression. Chronic liver injury was induced by administration of carbon tetrachloride (CCl(4)) to mice for 8 weeks. 5-Methylisothiourea hemisulphate (SMT), an iNOS inhibitor, or L-arginine, a NOS substrate were injected subcutaneously. CCl(4)-induced hepatotoxicity, oxidative stress and accumulation of collagen were detected in the liver. The expression levels of inducible NOS (iNOS) and nuclear factor kappa-B (NF-kappaB) activity in the liver after CCl(4) treatment were increased but eNOS expression and activator protein-1 (AP-1) activity were decreased. Both SMT and L-arginine effectively reduced CCl(4) induced oxidative stress and collagen formation, but L-arginine showed a significantly greater suppression of collagen formation, iNOS expression and NF-kappaB activity. L-arginine also restored the level of eNOS and AP-1 activity. L-arginine was more effective than SMT in suppressing liver fibrosis. L-arginine might improve NO production which facilitates hepatic blood flow and thus retards liver fibrogenesis. Our results showed that the reduced eNOS expression in CCl(4)-treated mice was reversed by L-arginine. Furthermore, L-arginine also reversed the reduced AP-1 activity, an eNOS promoter.

Construction of Multi-dimensional Arterial Health Status Map based on Molecular and Clinical Measurements, Fuzzy System and Data Cubes.

Atherosclerosis results from inflammatory processes involving biomarkers, such as lipid profile, haemoglobin A1C, oxidative stress, coronary artery calcium score and flow-mediated endothelial response through nitric oxide. This paper proposes a health status coefficient, which comprehends molecular and clinical measurements concerning atherosclerosis to provide a measure of arterial health. An arterial health status map is produced to map the multi-dimensional measurements to the health status coefficient. The mapping is modeled by a fuzzy system embedded with the health domain expert knowledge. The measurements obtained from the pilot study are used to tune the fuzzy system. The inferred arterial health coefficients are stored into the data cubes of a multi-dimensional database. Due to this adaptability and transparency of fuzzy system, the health status map can be easily updated when the refinement of fuzzy rule base is needed or new measurements are obtained.

Breast cancer proteomics: clinical perspectives.

Breast cancer is the one of leading causes of cancer-related deaths in women within economically developed regions of the world. A major focus of present research into this malignancy is the identification of new biomarkers and drug targets to improve detection and treatment. Proteomics represents one of the latest technological developments in this context. It aims to analyse the complex circuitry of the breast cancer proteome. Here, the authors review how breast cancer proteomics has progressed so far, with emphasis on its potential application to clinically relevant scenarios.

Expression and functions of vasoactive substances regulated by hypoxia-inducible factor-1 in chronic hypoxemia.

The aims of the present review are to summarize and to discuss the role of hypoxia-inducible factor-1 (HIF-1) and the expression and functions of vasoactive substances in chronic hypoxemia with specific focus in the liver and the carotid body. Vascular remodelling and vasoactive substances play important functional roles in the adaptive response to chronic hypoxemia for the maintenance of oxygen homeostasis in all systems in man. HIF-1 regulates the gene expression of vasoactive substances such as vascular endothelial growth factor (VEGF), endothelin-1 (ET-1) and enzymes for producing nitric oxide (NO). Recent studies have shown the effect of chronic hypoxia on the expression of HIF-1alpha and HIF-1-target genes in multiple organ systems including the liver and the carotid body. Results are consistent with increases in the hematocrit levels, pulmonary arterial pressure and right heart mass developed during chronic hypoxia. In addition, the carotid body is also hyperplastic and increases in organ mass with increased levels of HIF-1alpha and the vasoactive substances. These molecules increase the mitotic activity and modulate the excitability of the chemoreceptor. Intriguingly, the liver morphology, serum alanine aminotransferase and 8-isoprostane levels are within normal range in chronic hypoxia, suggesting the absence of significant oxidative stress. Yet, the HIF-1alpha is upregulated and the mRNA and protein levels of VEGF, ET-1, inducible and constitutive NO synthases are elevated in the liver during chronic hypoxia. In conclusion, the adaptive response to long-term hypoxemia involves compensatory mechanisms mediated by expressing significant levels of HIF-1alpha and vasoactive substances regulated by HIF-1.