Biochemical and immunocytochemical analysis of three novel microtubule-associated proteins in Drosophila melanogaster and the rat brain

Microtubules are essential organelles found in almost all eukaryotic cells. Some of their diverse functions include intracellular transport, cell division and maintenance of cell shape. Microtubules are composed of highly conserved proteins, the $\alpha$- and $\beta$-tubulins. Many of the functions associated with microtubules are thought to be due to the proteins that associate with them and are collectively termed microtubule-associated proteins or MAPs. Microtubule proteins (MTP), isolated from Drosophila embryos by repetitive cycles of assembly and disassembly, have been analyzed during embryonic development. The yield of twice-cycled MTP increased an average of 15-fold from 8 to 12 hours of development. Analysis of MTP purified from 6 and 16 hours after fertilization reveal differences in MAP composition with the 6 hour MTP having an increased number of putative MAPs compared to the 16 hour fraction.MTP from 12-16 hour embryo collections were used as antigen for the production of monoclonal antibodies (Mabs). Indirect immunofluorescence on whole embryos identified three hybridoma lines that stained cytoskeletal structures in the embryo, designated Mab DMAP45, DMAP55 and DMAP66. Mab DMAP45 recognizes a protein species with a My of 45 kD on immunoblots and in the embryo stains an actin-like pattern in the cytoplasm. DMAP45 also stains the developing ventral nerve cord. Mab DMAP55 predominantly recognizes a major band at 55 kD on immunoblots. Mab DMAP55 staining reveals structures present in spindle microtubules and the surrounding cytoplasm. Mab DMAP66 recognizes a single band of 66 kD on immunoblots and generally stains the cortical cytoplasm and in the yolk regions of the embryo. The staining patterns in the embryo and high-resolution electrophoretic analysis suggests that DMAP45 is $\gamma$-actin. In addition, Mab DMAP45 recognizes an additional protein species with the same molecular weight but a different pI suggesting that it may also recognize a posttranslational modification of actin not previously identified, one of recently discovered family of ARPs (actin-related proteins). DMAP55, based on its molecular weight and isoelectric point, may be a tubulin isoform. Although similar in molecular weight to tau protein, DMAP66 is not recognized by polyclonal antibodies against tau suggesting that it is a novel MAP not previously identified.Analyses of DMAP45, DMAP55 and DMAP66 proteins in rat brain tissue extracts and purified rat brain MTP identified proteins of almost identical molecular weights and isoelectric points. The presence of proteins with common biochemical properties in these widely divergent animal species suggests that they are related proteins that have been evolutionarily conserved.U of I OnlyETDs are only available to UIUC Users without author permissio

The Fusion Core Complex of the Peste des Petits Ruminants Virus Is a Six-Helix Bundle Assembly †

ABSTRACT: We describe the properties of the two heptad repeats (HR1 and HR2) of the Peste des petits ruminants virus (PPRV) fusion protein (F) to obtain insights into the mechanism by which these repeats influence PPRV-mediated cell fusion. Both HR1 and HR2 inhibit PPRV-mediated syncytia formation in Vero cells in vitro. Of these, HR2 was found to be more effective than HR1. We studied the mechanism of fusion inhibition by these two repeats by using various biophysical and biochemical methods either separately or together. CD spectral analysis of these repeats revealed that the R-helical content of HR1 and HR2 when used together is higher than that of their simulated spectrum in the mixture, suggesting the formation of a highly structured complex by these repeats. Protease protection assays confirmed that such a complex is highly stable. Electrospray mass spectrometry of protease-digested products of the HR1-HR2 complex showed protection of fragments corresponding to both HR1 and HR2 sequences involved in complex formation. By employing size-exclusion chromatography and chemical cross-linking experiments, we show that three units each of HR1 and HR2 form a complex in which HR1 is a trimer and HR2 is a monomer. Homology-based three-dimensional modeling of this complex showed that HR1 and HR2 together form a six-helix and trimeric coiled-coil bundle. In this model, the HR1 trimer forms the core whereas HR2, while interacting with HR1 in an antiparallel orientation, forms a two-strande

Phosphoprotein of the Rinderpest Virus Forms a Tetramer through a Coiled Coil Region Important for Biological Function

Phosphoprotein (P) of negative sense RNA viruses functions as a transcriptional transactivator of the viral polymerase (L). We report here the characterization of oligomeric P protein of rinderpest virus (RPV) and provide a structural basis for its multimerization. By size exclusion chromatography and dynamic light scattering analyses we show that bacterially expressed P protein exists as an oligomer, thus excluding the role of phosphorylation in P protein oligomerization. Gel filtration analyses of various parts of the P protein, also expressed in Escherichia coli, revealed that the predicted coiled coil region in the C-terminal domain is responsible for P protein oligomerization. Dynamic light scattering analysis confirmed the oligomeric nature of the coiled coil region of P. Chemical cross-linking analysis suggested that the C-terminal coiled coil region exists as a tetramer. The tetramer is formed by coiled coil interaction as shown by circular dichroism spectral analysis. Based on sequence homology, we propose a three-dimensional structure of the multimerization domain of RPV P using the crystal structure for multimerization domain of sendai virus (SeV) P as a template. Four-stranded coiled coil structure of the model is stabilized by a series of interactions predominantly between short nonpolar side chains emerging from different strands. In an in vivo replication/transcription system using a synthetic minigenome of RPV, we show that multimerization is essential for P protein function(s), and the multimerization domain is highly conserved between two morbilliviruses namely RPV and peste de petits ruminants virus. These results are discussed in the context of biological functions of P protein among various negative- stranded RNA viruses

Query processing over distributed heterogeneous sensor networks in Future Internet : Scalable architecture and challenges

The wireless networked sensors embedded with everyday objects will become an integral part of Future Internet, where the interaction among people, computer and those objects will shift the current Internet to a new paradigm, namely the Internet of Things. The terabyte torrent of data generated by billions of sensors belonging to a large number of distributed heterogeneous sensor networks in Future Internet will only be valuable if they can be effectively used on purpose, which leads to the necessity of an Internet scale query processing framework. In this paper, firstly, we focus on the distinct challenges present in Internet scale query processing over distributed sensor networks. Then, we propose a flexible and scalable system architecture capable of handling the complex scenario that might arise from the integration of a large number of such networks in Future Internet. Finally, we discuss the overall query processing methodology over such system and present some directions on the possible solutions to a number of identified research challenges. The outcome of this paper would foster the sensor network research in Future Internet domai

Identification of a novel epitope in the C terminus of hepatitis C virus-E2 protein that induces potent and cross-reactive neutralizing antibodies

Hepatitis C virus (HCV) is a leading cause of chronic viral hepatitis, but an effective vaccine is still not available to prevent infection. Use of neutralizing antibodies could be a potential therapeutic option. In this study, the presence of anti-HCV antibodies in HCV-infected patients was assessed from 50 patients and the presence of neutralizing antibodies was examined using `hepatitis C virus-like particles'. Antibodies from two samples exhibited significant inhibitory activity, suggesting that these may neutralize viral infection. Antigenic determinants generating the neutralizing antibodies from these two samples were delineated by epitope mapping using the core, E1 and E2 regions and a stretch of 45 amino acid peptide (E2C45) derived from the C-terminal region of HCV-E2 protein (aa 634-679) was designed. Results suggest that this hitherto uncharacterized region has the potential to generate neutralizing antibodies against HCV and thus be effective in preventing virus entry into liver cells. Computational analysis of the structure of the modelled peptide (E2C45) suggested high conformational entropy for this region. Furthermore, E2C45 peptide-generated antibodies could block virus entry and monoclonal antibodies generated against this peptide could also significantly reduce virus replication in a cell culture system. It is possible that the inhibition could be partly due to a conformational alteration of the CD81-binding region, preventing virus attachment to liver cells. In conclusion, this work focused on the discovery of a novel epitope at the C terminus of E2 that induces potent neutralizing antibodies in HCV-infected patients