RanBPM (Ran-binding protein in the microtubule-organizing centre) was originally reported as a centrosome-associated protein in human cells. However, RanBPM protein containing highly conserved SPRY, LisH, CTLH and CRA domains is currently considered as a scaffolding protein with multiple cellular functions. A plant homologue of RanBPM has not yet been characterized.

Vascular Smooth Muscle Cells (VSMCs), due to their plasticity and ability to shift from a physiological contractile-quiescent phenotype to a pathological proliferating-activated status, play a central role in the onset and progression of atherosclerosis and cardiovascular diseases. PDGF-BB, among a series of cytokines and growth factors, has been identified as the critical factor in this phenotypic switch. In order to obtain new insights on the molecular effects triggered by PDGF-BB, a hammerhead ribozyme targeting the membrane receptor PDGFR-β was applied to inhibit PDGF pathway in porcine VSMCs.

Retinoic acids regulate the reverse cholesterol transport by inducing the ATP binding cassette transporter A1 (ABCA1) dependent cholesterol efflux in macrophages, neuronal as well as intestine cells. In the present study, we aim to test the effect of all trans retinoic acid (ATRA) on ABCA1 expression in human CD4+ T cells and the involvement of cholesterol in ATRA mediated anti-HIV effect.

The use of insecticides to control malaria vectors is essential to reduce the prevalence of malaria and as a result, the development of insecticide resistance in vector populations is of major concern. Anopheles arabiensis is one of the main African malaria vectors and insecticide resistance in this species has been reported in a number of countries. The aim of this study was to investigate the detoxification enzymes that are involved in An. arabiensis resistance to DDT and pyrethroids.

Sustained HER2 signaling at the cell surface is an oncogenic mechanism in a significant proportion of breast cancers. While clinically effective therapies targeting HER2 such as mAbs and tyrosine kinase inhibitors exist, tumors overexpressing HER2 eventually progress despite treatment. Thus, abrogation of persistent HER2 expression at the plasma membrane to synergize with current approaches may represent a novel therapeutic strategy.

The ascomycete Ophiostoma piceae produces a sterol esterase (OPE) with high affinity towards p-nitrophenol, glycerol and sterol esters. Its hydrolytic activity on natural mixtures of triglycerides and sterol esters has been proposed for pitch biocontrol in paper industry since these compounds produce important economic losses during paper pulp manufacture.

Microcystis aeruginosa is a species of cyanobacteria commonly found in a number of countries and frequently related to animal poisoning episodes due to its capacity to produce the cyanotoxin known as microcystin. Despite vast literature on microcystin structures and their deleterious effects, little is known about its synthesis by cyanobacteria. Therefore, this study used proteomic tools to compare two M. aeruginosa strains, contrasting them for microcystin production.

Cytosine methylation is an important chromatin modification that maintains genome integrity and regulates gene expression through transcriptional gene silencing. Major players in de novo methylation guided by siRNAs (known as RNA-directed DNA methylation, or RdDM), maintenance methylation, and active demethylation have been identified in Arabidopsis. However, active demethylation only occurs at a subset of RdDM loci, raising the question of how the homeostasis of DNA methylation is achieved at most RdDM loci. To identify factors that regulate the levels of cytosine methylation, we aimed to establish a transgenic reporter system that allows for forward genetic screens in Arabidopsis.

Nogo-A is a member of the reticulon family of membrane-associated proteins and plays an important role in axonal remodeling. The present study aimed to investigate alterations in Nogo-A expression following traumatic brain injury (TBI)-induced inflammation and neuronal damage.

Early studies suggested that TR4 nuclear receptor might play important roles in the skeletal development, yet its detailed mechanism remains unclear.

Recently it was discovered that mutations in the UBQLN2 gene were a cause of an X-linked dominant type of familial amyotrophic lateral sclerosis (ALS). We investigated the frequency of mutations in this gene in a cohort of 92 families with ALS in the Netherlands. Eight families were excluded because of male-to-male transmission. In the remaining 84 familial ALS cases no mutations were discovered in UBQLN2. Hence, UBQLN2 was not found to be a cause of familial ALS in the Netherlands.

Caenopores belong to the saposin-like protein superfamily in Caenorhabditis elegans with 33 putative antimicrobial and pore-forming proteins. In this study, we analysed one selected member of this multifarious protein family, namely SPP-3, in detail, as its coding gene has been described to be inducible after bacterial challenge. The recombinant protein was antimicrobially active against a wide range of gram-negative and gram-positive bacteria and displayed membrane-permeabilizing and pH-dependent pore-forming activity. Promoter activity of the respective gene, spp-3, was localized to the intestine and the head neuron SDQR. While gene silencing had no apparent effect on the number of surviving Escherichia coli bacteria in the intestine, it increased the egg laying significantly. Accordingly, SPP-3 is a protein with antimicrobial activity that is presumably part of the redundant armamentarium of effector proteins in the worm's intestine, may help to protect neurons, and appears to be involved in regulating reproduction.

Diseases involving the medial temporal lobes (MTL) such as Alzheimer's disease and mesial temporal sclerosis pose an ongoing diagnostic challenge because of the difficulty in identifying conclusive imaging features, particularly in pre-clinical states. Abnormal neuronal connectivity may be present in the circuitry of the MTL, but current techniques cannot reliably detect those abnormalities. Diffusion tensor imaging (DTI) has shown promise in defining putative abnormalities in connectivity, but DTI studies of the MTL performed to date have shown neither dramatic nor consistent differences across patient populations. Conventional DTI methodology provides an inadequate depiction of the complex microanatomy present in the medial temporal lobe because of a typically employed low isotropic resolution of 2.0-2.5 mm, a low signal-to-noise ratio (SNR), and echo-planar imaging (EPI) geometric distortions that are exacerbated by the inhomogeneous magnetic environment at the skull base. In this study, we pushed the resolving power of DTI to near-mm isotropic voxel size to achieve a detailed depiction of mesial temporal microstructure at 3 T. High image fidelity and SNR at this resolution are achieved through several mechanisms: (1) acquiring multiple repetitions of the minimum field of view required for hippocampal coverage to boost SNR; (2) utilizing a single-refocused diffusion preparation to enhance SNR further; (3) performing a phase correction to reduce Rician noise; (4) minimizing distortion and maintaining left-right distortion symmetry with axial-plane parallel imaging; and (5) retaining anatomical and quantitative accuracy through the use of motion correction coupled with a higher-order eddy-current correction scheme. We combined this high-resolution methodology with a detailed segmentation of the MTL to identify tracks in all subjects that may represent the major pathways of the MTL, including the perforant pathway. Tractography performed on a subset of the data identified similar tracks, although they were lesser in number. This detailed analysis of MTL substructure may have applications to clinical populations.

Tyrosine phosphorylation is a dynamic reversible post-translational modification that regulates many aspects of cell biology. To understand how this modification controls biological function, it is necessary to not only identify the specific sites of phosphorylation, but also to quantify how phosphorylation levels on these sites may be altered under specific physiological conditions. Due to its sensitivity and accuracy, mass spectrometry (MS) has widely been applied to the identification and characterization of phosphotyrosine signaling across biological systems. In this review we highlight the advances in both MS and phosphotyrosine enrichment methods that have been developed to enable the identification of low level tyrosine phosphorylation events. Computational and manual approaches to ensure confident identification of phosphopeptide sequence and determination of phosphorylation site localization are discussed along with methods that have been applied to the relative quantification of large numbers of phosphorylation sites. Finally, we provide an overview of the challenges ahead as we extend these technologies to the characterization of tyrosine phosphorylation signaling in vivo. With these latest developments in analytical and computational techniques, it is now possible to derive biological insight from quantitative MS-based analysis of signaling networks in vitro and in vivo. Application of these approaches to a wide variety of biological systems will define how signal transduction regulates cellular physiology in health and disease.

Alcohol use disorders are associated with increased lung infections and exacerbations of chronic lung diseases. Whereas the effects of cigarette smoke are well recognized, the interplay of smoke and alcohol in modulating lung diseases is not clear. Because innate lung defense is mechanically maintained by airway cilia action and protein kinase C (PKC)-activating agents slow ciliary beat frequency (CBF), we hypothesized that the combination of smoke and alcohol would decrease CBF in a PKC-dependent manner. Primary ciliated bronchial epithelial cells were exposed to 5% cigarette smoke extract plus100 mmol/L ethanol for up to 24 hours and assayed for CBF and PKCε. Smoke and alcohol co-exposure activated PKCε by 1 hour and decreased both CBF and total number of beating cilia by 6 hours. A specific activator of PKCε, DCP-LA, slowed CBF after maximal PKCε activation. Interestingly, activation of PKCε by smoke and alcohol was only observed in ciliated cells, not basal bronchial epithelium. In precision-cut mouse lung slices treated with smoke and alcohol, PKCε activation preceded CBF slowing. Correspondingly, increased PKCε activity and cilia slowing were only observed in mice co-exposed to smoke and alcohol, regardless of the sequence of the combination exposure. No decreases in CBF were observed in PKCε knockout mice co-exposed to smoke and alcohol. These data identify PKCε as a key regulator of cilia slowing in response to combined smoke and alcohol-induced lung injury.

ATP7B mutations result in Cu storage in the liver and brain in Wilson disease (WD). Atox1 and COMMD1 were found to interact with ATP7B and involved in copper transport in the hepatocyte. To understand the molecular etiology of WD, we analyzed ATP7B, Atox1 and COMMD1 genes. Direct sequencing of (i) ATP7B gene was performed in 112 WD patients to identify the spectrum of disease-causing mutations in the French population, (ii) Atox1 gene was performed to study the known polymorphism 5'UTR-99T>C in 78 WD patients with two ATP7B mutations and (iii) COMMD1 gene was performed to detect the nucleotide change c.492GAT>GAC. MLPA (Multiplex Ligation-dependent Probe Amplification) analysis was performed in WD patients presenting only one ATP7B mutation. Among our 112 WD unrelated patients, 83 different ATP7B gene mutations were identified, 27 of which were novel. Two ATP7B mutations were identified in 98 WD cases, and one mutation was identified in 14 cases. In two of these 14 WD patients, we identified the deletion of exon 4 of the ATP7B gene by MLPA technique. In 78 selected patients of the cohort with two mutations in ATP7B, we have examined genotype-phenotype correlation between the detected changes in Atox1 and COMMD1 genes, and the presentation of the WD patients. Based on the data of this study, no major role can be attributed to Atox1 and COMMD in the pathophysiology or clinical variation of WD.

Toxic death is defined as study treatment-related mortality and as such is considered as an iatrogenic death. This belongs to unnatural death where an autopsy is advised. Until now, conventional autopsy is the gold standard to discriminate between pre- and post-mortem discrepancies.

E2F1 deletion leads to increased mitochondrial number and function, increased body temperature in response to cold and increased resistance to fatigue with exercise. Since E2f1-/- mice show increased muscle performance, we examined the effect of E2f1 genetic inactivation in the mdx background, a mouse model of Duchenne muscular dystrophy (DMD). E2f1-/-;mdx mice demonstrated a strong reduction of physiopathological signs of DMD, including preservation of muscle structure, decreased inflammatory profile, increased utrophin expression, resulting in better endurance and muscle contractile parameters, comparable to normal mdx mice. E2f1 deficiency in the mdx genetic background increased the oxidative metabolic gene program, mitochondrial activity and improved muscle functions. Interestingly, we observed increased E2F1 protein levels in DMD patients, suggesting that E2F1 might represent a promising target for the treatment of DMD.

The specialized tightly controlled microcirculation of craniofacial neurosensory organs is an essential evolutionary adaptation and yet a dilemma where angiogenic remodeling occurs. Despite extreme plasticity of neurosensory structures, the capacity to reconcile barrier phenotype of the microcirculation with an angiogenic cascade is not known. Here we provide primary evidence for such a response in an elemental neurosensory structure, human dental pulp, following chronic carious insult. In response to hypoxic challenge neurosensory odontoblasts express hypoxia-inducible factor-1α and notch-1. Associated radial rearrangement of astrocyte-like telacytes that communicate through a cell-poor zone with the microvasculature is observed. Activated pericytes characterized by expression of α-smooth muscle actin are located adjacent to the telacyte attachment to the vasculature. In this location, endothelial expression of sonic hedgehog parallels expression of notch-1 by pericytes. The angiogenic response is initiated by pericyte contraction and altered endothelial polarity and proliferation leading to intussusception of endothelial cells and extensive remodeling of basement membrane with upregulation of laminin-8 and laminin-5. These responses guide intravascular loop formation that maintains both intact basement membrane and tight junctions. This initial phase is followed by formation of anastomoses that enhance the hemodynamic capacity of the intravascular loops. The formation of anastomoses is mediated by extension of cytonemes from pericytes guided by MHC-II(+)/CD-163(+) microglia aligned with the telacytes. The cytonemes seek out pericytes on adjacent intravascular loops to initiate migration of endothelial cells. These findings support a new paradigm for understanding angiogenic capacity of neurosensory structures and aberrations of this response manifest as neurovasculopathies.

A novel recombinant human growth hormone (rhGH) fusion protein (VRS-317) was designed to minimize receptor-mediated clearance through a reduction in receptor binding without mutations to rhGH by genetically fusing with XTEN amino acid sequences to the N-terminus and the C-terminus of the native hGH sequence. Although in vitro potency of VRS-317 was reduced approximately 12-fold compared with rhGH, in vivo potency was increased because of the greatly prolonged exposure to the target tissues and organs. VRS-317 was threefold more potent than daily rhGH in hypophysectomized rats and fivefold more potent than daily rhGH in juvenile monkeys. In juvenile monkeys, a monthly dose of 1.4 mg/kg VRS-317 (equivalent to 0.26 mg/kg rhGH) caused a sustained pharmacodynamic response for 1 month equivalent to 0.05 mg/kg/day rhGH (1.4 mg/kg rhGH total over 28 days). In monkeys, VRS-317, having a terminal elimination half-life of approximately 110 h, was rapidly and near-completely absorbed, and was well tolerated with no observed adverse effects after every alternate week subcutaneous dosing for 14 weeks. VRS-317 also did not cause lipoatrophy in pig and monkey studies. VRS-317 is currently being studied in GH-deficient patients to confirm the observations in these animal studies.