GBF1, a Guanine Nucleotide Exchange Factor for Arf,Is Crucial for Coxsackievirus B3 RNA Replication

The replication of enteroviruses is sensitive to brefeldin A (BFA), an inhibitor of endoplasmic reticulum-to-Golgi network transport that blocks activation of guanine exchange factors (GEFs) of the Arf GTPases. Mammalian cells contain three BFA-sensitive Arf GEFs: GBF1, BIG1, and BIG2. Here, we show that coxsackievirus B3 (CVB3) RNA replication is insensitive to BFA in MDCK cells, which contain a BFA-resistant GBF1 due to mutation M832L. Further evidence for a critical role of GBF1 stems from the observations that viral RNA replication is inhibited upon knockdown of GBF1 by RNA interference and that replication in the presence of BFA is rescued upon overexpression of active, but not inactive, GBF1. Overexpression of Arf proteins or Rab1B, a GTPase that induces GBF1 recruitment to membranes, failed to rescue RNA replication in the presence of BFA. Additionally, the importance of the interaction between enterovirus protein 3A and GBF1 for viral RNA replication was investigated. For this, the rescue from BFA inhibition of wild-type (wt) replicons and that of mutant replicons of both CVB3 and poliovirus (PV) carrying a 3A protein that is impaired in binding GBF1 were compared. The BFA-resistant GBF1-M832L protein efficiently rescued RNA replication of both wt and mutant CVB3 and PV replicons in the presence of BFA. However, another BFA-resistant GBF1 protein, GBF1-A795E, also efficiently rescued RNA replication of the wt replicons, but not that of mutant replicons, in the presence of BFA. In conclusion, this study identifies a critical role for GBF1 in CVB3 RNA replication, but the importance of the 3A-GBF1 interaction requires further study.Enteroviruses are small, nonenveloped, positive-stranded RNA viruses that include many important pathogens, such as poliovirus (PV), coxsackievirus, echovirus, and human rhinovirus. Following virus entry and uncoating, the 7.5-kb enteroviral RNA genome is directly translated into a large polyprotein. This polyprotein is proteolytically processed by the virus-encoded proteases 2A^pro, 3C^pro, and 3CD^pro into the structural P1 region proteins and the nonstructural P2 and P3 region proteins that are involved in viral RNA replication.All RNA viruses with a positive-stranded genome induce the remodeling of cellular membranes to create a scaffold for genomic RNA replication. The organelle origin and morphology of these membranous replication sites, however, appear to vary for different viruses. Enteroviruses replicate their RNA genomes in nucleoprotein complexes that are associated with small vesicular membrane structures (6). The enteroviral proteins 2B, 2C, and 3A have been implicated in vesicle formation (4, 6, 27), but the mechanism and pathway of membrane reorganization are poorly understood. There are strong indications that these vesicular membranous structures, which are referred to here as “vesicles,” are derived from the early secretory pathway. Vesicles produced in PV-infected cells may form at the endoplasmic reticulum (ER) by the cellular COP-II budding machinery and may therefore share components with the membranous vesicles mediating ER-to-Golgi network transport (26). Further support for the involvement of the secretory pathway stems from the observation that brefeldin A (BFA), a well-known inhibitor of ER-to-Golgi network transport, completely inhibits enteroviral RNA replication (17, 20). In addition, the autophagocytic pathway appears to contribute to the formation of the membrane vesicles, many of which exhibit a double-membrane morphology characteristic of autophagosomes (18, 27). The utilization of individual components or reactions from different membrane metabolic pathways, rather than subversion of an entire pathway in toto, may represent a common strategy for building viral replication machinery.BFA inhibits activation of the small monomeric GTPase ADP ribosylation factor 1 (Arf1), a major regulator of intracellular protein transport (2). Arf1 cycles between an inactive, GDP-bound, cytosolic state and an active, GTP-bound, membrane-associated state, and this cycling is catalyzed by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (13). BFA blocks the activities of the large GEFs GBF1, BIG1, and BIG2 by stabilizing an intermediate, abortive complex with inactive Arf1 (23), thus efficiently preventing activation of Arf1 and eventually formation of transport intermediates.Not only the fact that BFA blocks enteroviral replication suggests a role for Arf1 and/or its large GEFs in this process; recently, it was shown that Arf1 accumulates on membranes during PV infection (3). Arf1 translocation to membranes can be induced independently by enterovirus protein 3A or 3CD in vitro (5), but the underlying mechanisms seem to differ; the 3A protein specifically triggers the recruitment of GBF1 to membranes, most likely through a direct interaction with this GEF (32, 33), whereas 3CD recruits BIG1 and BIG2 to membranes (3). Here, we report the involvement of Arf1 and its large BFA-sensitive GEFs in coxsackievirus B3 (CVB3) replication.     

Synthesis, structure and electron-mediator properties of the mono- and difunctionalized macrobicyclic iron(II) tris-dioximates with thiol terminated ribbed spacer substituents

Nucleophilic substitution of the reactive chlorine atoms of mono- and dichlorine clathrochelate FeBd₂(HGmCl)(BF)₂ and FeBd₂(Cl₂Gm)(BF)₂ precursors (where Bd²⁻, HGmCl²⁻ and Cl₂Gm²⁻ are α-benzyldioxime, chloroglyoxime and dichloroglyoxine dianions, respectively) with HSCH₂CH₂SH and (HSCH₂CH₂)₂S in the presence of triethylamine afforded the FeBd₂(HGmSCH₂CH₂SH)(BF)₂ (1) and FeBd₂(HGm(SCH₂CH₂)₂SH)(BF)₂ clathrochelates with a sole thiol terminated spacer substituent and the FeBd₂((HS(CH₂CH₂S)₂)₂Gm)(BF)₂ clathrochelate with two longchain thiol-sulfide ribbed groups. The crystal and molecular structures of these complexes were obtained by X-ray crystallography. The clathrochelate molecules possess a geometry intermediate between a trigonal prism (TP) and a trigonal antiprism (TAP). The X-ray data are in a good agreement with the ⁵⁷Fe Mössbauer parameters for complexes studied. These parameters characterize them as the low-spin iron(II) complexes (the isomeric shift values) with TP-TAP geometry (the quadrupole splitting values). Application of clathrochelate self-assembled monolayer (SAM) as the redox mediators for the determination of hydrogen peroxide is discussed. The calibration curve for hydrogen peroxide concentrations was found to be in the range from 2.0 · 10⁻⁶ to 1.6 · 10⁻⁵ mol · L⁻¹ with limit of detection equal to 1.0 · 10⁻⁶ mol · L⁻¹. The clathrochelate SAM on gold electrode provides precise and sensitive amperometric determination of hydrogen peroxide.     

Diverse Metastable Structures Formed by Small Oligomers of α-Synuclein Probed by Force Spectroscopy

Oligomeric aggregates are widely suspected as toxic agents in diseases caused by protein aggregation, yet they remain poorly characterized, partly because they are challenging to isolate from a heterogeneous mixture of species. We developed an assay for characterizing structure, stability, and kinetics of individual oligomers at high resolution and sensitivity using single-molecule force spectroscopy, and applied it to observe the formation of transient structured aggregates within single oligomers of α-synuclein, an intrinsically-disordered protein linked to Parkinson’s disease. Measurements of the molecular extension as the proteins unfolded under tension in optical tweezers revealed that even small oligomers could form numerous metastable structures, with a surprisingly broad range of sizes. Comparing the structures formed in monomers, dimers and tetramers, we found that the average mechanical stability increased with oligomer size. Most structures formed within a minute, with size-dependent rates. These results provide a new window onto the complex α-synuclein aggregation landscape, characterizing the microscopic structural heterogeneity and kinetics of different pathways.     

Western blot analysis of bile or intestinal fluid from patients with septic shock or systemic inflammatory response syndrome, using antibodies to TNF-alpha, IL-1 alpha and IL-1 beta

Septic shock or systemic inflammatory response syndrome (SIRS) often develops in patients following burns, traumatic injury, surgery or biliary obstruction. Although the inflammatory cytokines TNF-alpha and IL-1 have been strongly implicated in the development of these syndromes, treatment of patients by the systemic administration of inhibitors of TNF-alpha or IL-1 has shown limited effectiveness. Recent reports suggest that septic shock may be perpetuated by inflammatory cytokines secreted by the liver in response to bacterial translocation resulting from cytokine-induced gastrointestinal damage. The present study sought to demonstrate the presence of high levels of inflammatory cytokines in the bile or small intestine of patients suffering from septic shock or SIRS, with a view to the development of strategies for the reduction of gastrointestinal damage through intraduodenal administration of cytokine inhibitors. Western blot analysis of human bile or intestinal fluid using anti-TNF-alpha antibodies resulted in the detection of a number of bands in samples from patients with septic shock or SIRS. However, these proteins differed antigenically from human recombinant TNF-alpha (rTNF-alpha) and showed no activity in a biological assay for TNF-alpha. Antibodies to IL-1 alpha and IL-1 beta detected several strong bands, some of which appeared to be identical to recombinant IL-1 alpha and IL-1 beta. It is concluded that proteins resembling several known inflammatory cytokines are present in the bile and intestine of septic shock patients, but it is suggested that further work is required to determine the nature and function of these molecules.     

Excitation of guided ELF-VLF waves through modification of the F<Subscript>2</Subscript> ionospheric layer by high-power radio waves

The possibility of controlled excitation of ELF-VLF electromagnetic waves through modification of the F₂ ionospheric layer by high-power high-frequency emission is demonstrated in a natural experiment by using the Sura midlatitude heating facility. The excited low-frequency waves can be used to explore the near-Earth space and stimulate the excitation of a magnetospheric maser.     

Efficiency of dolphin auditory perception in relation to time characteristics of the signal]

Experimental data are presented on evaluating the efficiency of auditory perception of the dolphin Tursiops truncatus of tonal signals of different duration and of the fixed energy on the background of Gauss obstacle. The dependence of percepting properties on time characteristics of the signal are stated. A comparative analysis of the efficiency of dolphin auditory system with that of the coordinated filter is carried out. Biological receiving systems are shown to be adaptive. The self-tuning linits of the dolphin auditory system at a given level of the possibility of correct detection are determined.     

CNS penetration of intrathecal-lumbar idursulfase in the monkey, dog and mouse: implications for neurological outcomes of lysosomal storage disorder

A major challenge for the treatment of many central nervous system (CNS) disorders is the lack of convenient and effective methods for delivering biological agents to the brain. Mucopolysaccharidosis II (Hunter syndrome) is a rare inherited lysosomal storage disorder resulting from a deficiency of iduronate-2-sulfatase (I2S). I2S is a large, highly glycosylated enzyme. Intravenous administration is not likely to be an effective therapy for disease-related neurological outcomes that require enzyme access to the brain cells, in particular neurons and oligodendrocytes. We demonstrate that intracerebroventricular and lumbar intrathecal administration of recombinant I2S in dogs and nonhuman primates resulted in widespread enzyme distribution in the brain parenchyma, including remarkable deposition in the lysosomes of both neurons and oligodendrocytes. Lumbar intrathecal administration also resulted in enzyme delivery to the spinal cord, whereas little enzyme was detected there after intraventricular administration. Mucopolysaccharidosis II model is available in mice. Lumbar administration of recombinant I2S to enzyme deficient animals reduced the storage of glycosaminoglycans in both superficial and deep brain tissues, with concurrent morphological improvements. The observed patterns of enzyme transport from cerebrospinal fluid to the CNS tissues and the resultant biological activity (a) warrant further investigation of intrathecal delivery of I2S via lumbar catheter as an experimental treatment for the neurological symptoms of Hunter syndrome and (b) may have broader implications for CNS treatment with biopharmaceuticals.     

Molecular genetic determinants of intraspecific polymorphism of the phytopathogenic fungus Cryphonectria parasitica

The review summarizes the current evidence on the phytopathogenic fungus Cryphonectria parasitica, which is a classic object for studying hypovirulence. Phenotypic manifestations of hypovirulence and themolecular mechanisms of action of the mycovirus Cryphonectria hypovirus (CHV) infecting the fungus are described in detail. Genetic determinants of vegetative incompatibility in C. parasitica (a phenomenon increasing polymorphism of the fungus and preventing CHV expansion) are considered. The data on C. parasitica polymorphism are correlated with the data on the distribution of different CHV species in the European, American, and Asian populations of the fungus.     

Synthesis, characterization and ethylene oligomerization studies of nickel(II) based new formazane derivatives

A series of nickel(II) complexes based novel mono- and bis-formazan ligands has been synthesized and characterized with IR, UV, NMR spectroscopies, CHN analysis, mass spectrometry, and single crystal X-ray diffraction analysis. The prepared complexes have been tested for catalytic activity in ethylene oligomerization.     

Preparation of isolated cells by alkaline dissociation of formalin fixed tissue]

A procedure is described for preparation of isolated cells by treating formaldehyde fixed tissues with a 50%-solution of KOH. This results in complete yield of cells from a variety of organs (liver, kidney, heart, spleen, etc.). The alkali-treated cells entirely retain their morphological and tinctorial peculiarities. It was shown that preparations derived from alkali-treated tissues were useful for a series of quantitative cytological and cytochemical techniques: cytofluorometric estimation of nuclear DNA; interferometric determination of dry cell mass; autoradiographic studies of nuclear DNA synthesis; cell number counts; evaluation of cell distribution according to the number of their nuclei; estimation of the mitotic index.