Regulation of Hepatitis C Virion Production via Phosphorylation of the NS5A Protein

Hepatitis C virus (HCV) is a significant pathogen, infecting some 170 million people worldwide. Persistent virus infection often leads to cirrhosis and liver cancer. In the infected cell many RNA directed processes must occur to maintain and spread infection. Viral genomic RNA is constantly replicating, serving as template for translation, and being packaged into new virus particles; processes that cannot occur simultaneously. Little is known about the regulation of these events. The viral NS5A phosphoprotein has been proposed as a regulator of events in the HCV life cycle for years, but the details have remained enigmatic. NS5A is a three-domain protein and the requirement of domains I and II for RNA replication is well documented. NS5A domain III is not required for RNA replication, and the function of this region in the HCV lifecycle is unknown. We have identified a small deletion in domain III that disrupts the production of infectious virus particles without altering the efficiency of HCV RNA replication. This deletion disrupts virus production at an early stage of assembly, as no intracellular virus is generated and no viral RNA and nucleocapsid protein are released from cells. Genetic mapping has indicated a single serine residue within the deletion is responsible for the observed phenotype. This serine residue lies within a casein kinase II consensus motif, and mutations that mimic phosphorylation suggest that phosphorylation at this position regulates the production of infectious virus. We have shown by genetic silencing and chemical inhibition experiments that NS5A requires casein kinase II phosphorylation at this position for virion production. A mutation that mimics phosphorylation at this position is insensitive to these manipulations of casein kinase II activity. These data provide the first evidence for a function of the domain III of NS5A and implicate NS5A as an important regulator of the RNA replication and virion assembly of HCV. The ability to uncouple virus production from RNA replication, as described herein, may be useful in understanding HCV assembly and may be therapeutically important

DIGE Proteome Analysis Reveals Suitability of Ischemic Cardiac In Vitro Model for Studying Cellular Response to Acute Ischemia and Regeneration

Proteomic analysis of myocardial tissue from patient population is suited to yield insights into cellular and molecular mechanisms taking place in cardiovascular diseases. However, it has been limited by small sized biopsies and complicated by high variances between patients. Therefore, there is a high demand for suitable model systems with the capability to simulate ischemic and cardiotoxic effects in vitro, under defined conditions. In this context, we established an in vitro ischemia/reperfusion cardiac disease model based on the contractile HL-1 cell line. To identify pathways involved in the cellular alterations induced by ischemia and thereby defining disease-specific biomarkers and potential target structures for new drug candidates we used fluorescence 2D-difference gel electrophoresis. By comparing spot density changes in ischemic and reperfusion samples we detected several protein spots that were differentially abundant. Using MALDI-TOF/TOF-MS and ESI-MS the proteins were identified and subsequently grouped by functionality. Most prominent were changes in apoptosis signalling, cell structure and energy-metabolism. Alterations were confirmed by analysis of human biopsies from patients with ischemic cardiomyopathy

Mikrosystem zur Kontrolle der Wirkstoffabgabe aus einem Wirkstoffreservoir sowie Implantat mit Mikrosystem

DE 10063612 A UPAB: 20020919 NOVELTY - A microsystem for controlling delivery of an active material (I) from a reservoir comprises: (A) a thin, (I)-impermeable, flexible support comprising (I)-permeable hole(s) microducts and/or microcracks with electrodes on both sides; (B) several electrodes in the region of the holes and electronics integrated in (A) to control the electrodes; and (C) layer of electroporous material covering one side of (A). DETAILED DESCRIPTION - An INDEPENDENT CLAIM is also included for an implant with the microsystem with (I) reservoir(s) fixed to or in the surface of the implant and completely covered by the microsystem. USE - The implant is used as cardiac catheter or stent (claimed). ADVANTAGE - The microsystem allows event-controlled release of active materials, even in applications with very critical position conditions. Since it is flexible and requires only minimum space, it can be suited to many positions in the body

3D-Biohybrid systems: Applications in drug screening

Biotechnology demands powerful methods for the functional characterisation and monitoring of molecular alterations in tissues in response to various stimuli. Currently, cellular biosensors provide information about cell and tissue internal transduction pathways. In this article, recent biosensor systems are briefly described and the use of 3D tissue aggregates as recognition elements is discussed. An example of an innovative approach for drug testing using 3D heart muscle aggregates, as well as tumor models, positioned in capillary systems for electrical potential recording and impedance measurement is described. The effectiveness of drugs and therapies can be tested and monitored in a short time using such biohybrid sensors

Aryl acylamidase activity exhibited by butyrylcholinesterase is higher in chick than in horse, but much lower than in fetal calf serum.

Several side activities have been attributed to butyrylcholinesterase (BChE), including aryl acylamidase (AAA) activity, which is an amidase-like activity with unknown physiological function splitting the artificial substrate o-nitroacetanilide. For avians, extensive developmental data have pointed to neurogenetic functions of BChE, however, a possible AAA activity of BChE has not been studied. In this study, we first compare the relative levels of AAA exhibited by BChE in whole sera from chick, fetal calves (FCS) and horse. Remarkably, FCS exhibits a 400-fold higher ratio of AAA/BChE than horse and 80-fold higher than chick serum. We then show that an immunoisolated preparation of BChE from chicken serum presents significant activity for AAA. Both in sera and with the purified enzyme, the AAA activity is fully inhibited by anticholinesterase drugs, showing that AAA activity is exclusively conveyed by the BChE molecule. Noticeably, AAA inhibition even occurs at lower drug concentrations than that of BChE activity itself. Moreover, AAA is sensitive to serotonin. These data indicate that (1) AAA is a general feature of serum BChE of vertebrates including avians, (2) AAA is effectively inhibited by cholinergic and serotonergic agents, and (3) AAA may have a developmental role, since it is much pronounced in a serum from fetal animals. Functionally, deamination of neuropeptides, a link between cholinergic and serotonergic neurotransmitter systems, and roles in lipoprotein metabolism could be relevant

Transfection of reaggregating embryonic chicken retinal cells with an antisense 5'-DNA butyrylcholinesterase expression vector inhibits proliferation and alters morphogenesis.

The function of the enzyme butyrylcholinesterase (BChE) in the developing and mature brain is still unclear. We have inserted 577 bp of the 5' upstream region plus 106 bp of the exon 1 of the rabbit BChE gene in reverse orientation under control of an SV40 early promoter derivative in an expression vector. This vector was introduced by calcium phosphate-mediated transfection into embryonic chicken retina cells during the first days of reaggregation culture. Depending on the retinal origin, the transfected cell population forms histotypic retina-like spheres, so-called rosetted or stratified retinospheroids. We show that antisense 5'-BChE gene expression decreased the steady-state mRNA level of BChE and the translation of the BChE protein, inhibited proliferation, and accelerated histogenesis in both cellular systems. The pronounced effects of antisense 5'-BChE transfection of spheroids document a key role of BChE during the early reaggregation process of retinal cells, most likely by regulating their growth and differentiation