VEGF inhibits tumor cell invasion and mesenchymal transition through a MET/VEGFR2 complex

Inhibition of VEGF signaling leads to a proinvasive phenotype in mouse models of glioblastoma multiforme (GBM) and in a subset of GBM patients treated with bevacizumab. Here, we demonstrate that vascular endothelial growth factor (VEGF) directly and negatively regulates tumor cell invasion through enhanced recruitment of the protein tyrosine phosphatase 1B (PTP1B) to a MET/VEGFR2 heterocomplex, thereby suppressing HGF-dependent MET phosphorylation and tumor cell migration. Consequently, VEGF blockade restores and increases MET activity in GBM cells in a hypoxia-independent manner, while inducing a program reminiscent of epithelial-to-mesenchymal transition highlighted by a T-cadherin to N-cadherin switch and enhanced mesenchymal features. Inhibition of MET in GBM mouse models blocks mesenchymal transition and invasion provoked by VEGF ablation, resulting in substantial survival benefit.     

Mitochondrial Antioxidants Alleviate Oxidative and Nitrosative Stress in a Cellular Model of Sepsis

Purpose  

Mitochondrial dysfunction plays a key role in sepsis.     

Heavy–light chain interrelations of MS-associated immunoglobulins probed by deep sequencing and rational variation

The mechanisms triggering most of autoimmune diseases are still obscure. Autoreactive B cells play a crucial role in the development of such pathologies and, in particular, production of autoantibodies of different specificities. The combination of deep-sequencing technology with functional studies of antibodies selected from highly representative immunoglobulin combinatorial libraries may provide unique information on specific features in the repertoires of autoreactive B cells. Here, we have analyzed cross-combinations of the variable regions of human immunoglobulins against the myelin basic protein (MBP) previously selected from a multiple sclerosis (MS)-related scFv phage-display library. On the other hand, we have performed deep sequencing of the sublibraries of scFvs against MBP, Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1), and myelin oligodendrocyte glycoprotein (MOG). Bioinformatics analysis of sequencing data and surface plasmon resonance (SPR) studies have shown that it is the variable fragments of antibody heavy chains that mainly determine both the affinity of antibodies to the parent autoantigen and their cross-reactivity. It is suggested that LMP1-cross-reactive anti-myelin autoantibodies contain heavy chains encoded by certain germline gene segments, which may be a hallmark of the EBV-specific B cell subpopulation involved in MS triggering.     

New insights into the karyotypic evolution in muroid rodents revealed by multicolor banding applying murine probes

Muroid rodents are composed of a wide range of species characterized by extensive karyotypic evolution. Even if this group includes such important laboratory animal models as domestic mouse (Mus musculus), Norway rat (Rattus norvegicus), Chinese hamster (Cricetulus griseus), and golden hamster (Mesocricetus auratus), comparative cytogenetic studies between rodents are difficult due to the characteristic rapid karyotypic evolution. Molecular cytogenetic methods can help resolve problems of comparing muroid chromosomes. Here, we used cross-species comparative multicolour banding with probes obtained from mouse chromosomes 3, 6, 18, and 19 to study the karyotypes of nine muroid species from the three subfamilies Murinae, Cricetinae, and Arvicolinae. Results from multicolour banding with these murine probes (mcb) allowed us to improve the comparative homology maps between these species and to obtain new insights into their karyotypic evolution. We identified evolutionary conserved chromosomal breakpoints and revealed four previously unrecognized homologous segments, four inversions, and 14 evolutionary new centromeres in the nine muroid species studied. We found Mus apomorphic rearrangements, not seen in other muroids, and defined several subfamily specific chromosome breaks, characteristic for Arvicolinae and Cricetinae. We show that mcb libraries are an effective tool both for the cytogenetic characterisation of important laboratory models such as the rat and hamster as well as elucidating the complex phylogenomics relationships of muroids.     

Fatal persistence of West Nile virus subtype Kunjin in the brains of flavivirus resistant mice

Flaviviruses cause febrile illnesses in humans that may progress to encephalitis and death. Both viral and host factors determine the level of virus replication and outcome of infection. In mice, genetically determined resistance conferred by the flavivirus resistance locus (Flv) is responsible for the restricted flavivirus replication and prevention of disease development. Majority of flaviviruses express significant virulence, replicate to high titers and cause high mortality in susceptible mice, while congenic resistant mice endure the infection, show significantly reduced levels of virus replication and remain healthy. In contrast, infection with West Nile virus subtype Kunjin (KUNV) causes morbidity and fatal outcomes even in mice that are naturally resistant to flaviviruses. There are two possible mechanisms that could account for such an unforeseen virulence of KUNV in resistant mice: (a) an abrogation of Flv-controlled natural resistance leading to high virus replication, or (b) massive virus-induced immunopathology in the brain. To identify the cause(s) of fatality of KUNV infection, disease progression, virus replication and brain histopathology were studied in parallel in resistant and congenic susceptible mice. While KUNV replicated to high titers causing early fatalities in susceptible mice, it showed only reduced replication associated with the delayed morbidity in resistant mice indicating no abrogation of the Flv resistance. No evidence of excessive immune cell infiltration and tissue damage following KUNV infection were found. However, incomplete KUNV clearance not previously described was perceived as an important source of pathogenesis in resistant mice.     

Infection and Alzheimer's disease: the APOE epsilon4 connection and lipid metabolism

Microorganisms, bacteria and viruses may infect and cause a range of acute and chronic diseases in humans dependent on the genetic background, age, sex, immune and health status of the host, as well as on the nature, virulence and dose of infectious agent. Late onset Alzheimer's disease (AD) is a progressive neurodegenerative illness of broad aetiology with a strong genetic component and a significant contribution of age, sex and life style factors. Both infectious diseases and AD are characterised by an increased production of an array of immune mediators, cytokines, chemokines and complement proteins by the host cells as well as by changes in the host lipid metabolism. In this review, we re-examine a dangerous liaison between several viral and bacterial infections and the most significant genetic factor for AD, APOE epsilon4, and the possible impact of this alliance on AD development. This connection was discussed in the broader context of lipid metabolism and in the light of different capacity of various infectious agents, their toxic lipophilic products and host lipoprotein particles for binding to cell receptor(s).     

Multiple mechanisms underlying the anticancer action of nanocrystalline fullerene

Using the rat glioma cell line C6 and the human glioma cell line U251, we demonstrate the multiple mechanisms underlying the in vitro anticancer effects of the C(60) fullerene water suspension (nano-C(60) or nC(60)) produced by solvent exchange method. Nano-C(60) in a dose-dependent manner reduced the tumor cell numbers after 24 h of incubation. The observed antiglioma action of nC(60) at high concentration (1 microg/ml) was due to a reactive oxygen species-mediated necrotic cell damage that was partly dependent on oxidative stress-induced activation of extracellular signal-regulated kinase (ERK). On the other hand, low-dose nC(60) (0.25 microg/ml) did not induce either necrotic or apoptotic cell death, but caused oxidative stress/ERK-independent cell cycle block in G(2)/M phase and subsequent inhibition of tumor cell proliferation. Treatment with either high-dose or low-dose nC(60) caused the appearance of acidified intracytoplasmic vesicles indicative of autophagy, but only the antiglioma effect of low-dose nC(60) was significantly attenuated by inhibiting autophagy with bafilomycin A1. Importantly, primary rat astrocytes were less sensitive than their transformed counterparts to a cytostatic action of low-dose nC(60). These data provide grounds for further development of nC(60) as an anticancer agent.     

Mutation analysis of the <Emphasis Type="Italic">MECP2</Emphasis> gene in patients of Slavic origin with Rett syndrome: novel mutations and polymorphisms

Rett syndrome (RTT), an X-linked dominant neurodevelopmental disorder in females, is caused mainly by de novo mutations in the methyl-CpG-binding protein 2 gene (MECP2). Here we report mutation analysis of the MECP2 gene in 87 patients with RTT from the Czech and Slovak Republics, and Ukraine. The patients, all girls, with classical RTT were investigated for mutations using bi-directional DNA sequencing and conformation sensitive gel electrophoresis analysis of the coding sequence and exon/intron boundaries of the MECP2 gene. Restriction fragment length polymorphism analysis was performed to confirm the mutations that cause the creation or abolition of the restriction site. Mutation-negative cases were subsequently examined by multiple ligation-dependent probe amplification (MLPA) to identify large deletions. Mutation screening revealed 31 different mutations in 68 patients and 12 non-pathogenic polymorphisms. Six mutations have not been previously published: two point mutations (323T>A, 904C>T), three deletions (189_190delGA, 816_832del17, 1069delAGC) and one deletion/inversion (1063_1236del174;1189_1231inv43). MLPA analysis revealed large deletions in two patients. The detection rate was 78.16%. Our results confirm the high frequency of MECP2 mutations in females with RTT and provide data concerning the mutation heterogeneity in the Slavic population.     

Metabolomics of the effect of AMPK activation by AICAR on human umbilical vein endothelial cells

AMP-activated protein kinase (AMPK) is a metabolic master switch expressed in a great number of cells and tissues. AMPK is thought to modulate the cellular response to different stresses that increase cellular AMP concentration. The adenosine analog, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) is an AMPK activator used in many studies to assess the effects of AMPK activation on cellular metabolism and function. However, the effect of AICAR on cell metabolism reaches many different pathways and metabolites, some of which do not seem to be fully related to AMPK activation. We have now for the first time used NMR metabolomics on human umbilical vein endothelial cells (HUVEC) for the study of the global metabolic impact of AMPK activation by AICAR. In our study, incubation with AICAR activates AMPK and is associated with, among others, broad metabolic alterations in energy metabolism and phospholipid biosynthesis. Using NMR spectroscopy and metabolic network tools, we analyzed the connections between the different metabolic switches activated by AICAR. Our approach reveals a strong interconnection between different phospholipid precursors and oxidation by-products. Metabolomics profiling is a useful tool for detecting major metabolic alterations, generating new hypotheses and provides some insight about the different molecular correlations in a complex system. The present study shows that AICAR induces metabolic effects in cell metabolism well beyond energy production pathways.