P-protein of Chandipura virus is an N-protein-specific chaperone that acts at the nucleation stage

The nucleocapsid protein N of Chandipura virus is prone to aggregation in vitro. We have shown that this aggregation occurs in two phases in a nucleation-dependent manner. Electron microscopy suggests that the aggregated state may have a ring-like structure. Using a GFP fusion, we have shown that the N-protein also aggregates in vivo. The P-protein suppresses the N-protein aggregation efficiently, both in vitro and in vivo. Increased lag phase in the presence of the P-protein suggests that chaperone-like action of the P-protein occurs before the nucleation event. The P-protein, however, does not exert any chaperone-like action against other proteins, suggesting that it binds to the N-protein specifically. Surface plasmon resonance and fluorescence enhancement indeed suggest that the P-protein binds tightly to the native N-protein. The P-protein is thus an N-protein-specific chaperone which inhibits the nucleation phase of N-protein aggregation, thus keeping a pool of encapsidation-competent N-protein for viral maturation.     

Effect of thidiazuron in germinating tamarind seedlings

Summary Tamarind, a multipurpose tropical tree species, is economically important for sustainable development of wasteland due to its hardy nature and adaptability to various agroclimatic ocnditions. Reports on in vitro morphogenesis in this species are limited, due to its recalcitrant and callogenic nature. To overcome these limitations, an attempt was made to induce meristematic activity in seedling explants. Seedlings were germinated in medium with or without thidiazuron (4.54, 9.08, 13.12, 18.16 μM). This growth regulator restricted the differentiation of the apical meristem to form shoots. It triggered proliferation of the meristematic tissue at the cotyledonary node and a large number of meristematic buds appeared in a ridial pattern around the node. The meristematic activity extended to the junction of the epicotyl and hypocotyl, giving rise to buds in the form of protuberances in all sides of the junction. These buds differentiated to form shoot primordia and subsequently to shoots in medium devoid of growth regulators. Plants developed by micrografting of these shoots on seedling-derived rootstocks survived in soil.     

The fusion core complex of the peste des petits ruminants virus is a six-helix bundle assembly

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 alpha-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-stranded coiled-coil structure and lies at the periphery of the structure. These results are discussed in the context of a common fusion mechanism among paramyxoviruses.     

Solution structure of the HIV-1 integrase-binding domain in LEDGF/p75

Lens epithelium-derived growth factor (LEDGF)/p75 is the dominant binding partner of HIV-1 integrase (IN) in human cells. We have determined the NMR structure of the integrase-binding domain (IBD) in LEDGF and identified amino acid residues essential for the interaction. The IBD is a compact right-handed bundle composed of five alpha-helices. Based on folding topology, the IBD is structurally related to a diverse family of alpha-helical proteins that includes eukaryotic translation initiation factor eIF4G and karyopherin-beta. LEDGF residues essential for the interaction with IN were localized to interhelical loop regions of the bundle structure. Interaction-defective IN mutants were previously shown to cripple replication although they retained catalytic function. The initial structure determination of a host cell factor that tightly binds to a retroviral enzyme lays the groundwork for understanding enzyme-host interactions important for viral replication.     

A CD8+ T cell clone specific for antigen also recognizes peptidomimics present in anti-idiotypic antibody: implications for T cell memory

The relay hypothesis [R. Nayak, S. Mitra-Kaushik, M.S. Shaila, Perpetuation of immunological memory: a relay hypothesis, Immunology 102 (2001) 387-395] was earlier proposed to explain perpetuation of immunological memory without requiring long lived memory cells or persisting antigen. This hypothesis envisaged cycles of interaction and proliferation of complementary idiotypic B cells (Burnet cells) and anti-idiotypic B cells (Jerne cells) as the primary reason for perpetuation of immunological memory. The presence of peptidomimics of antigen in anti-idiotypic antibody and their presentation to antigen specific T cells was postulated to be primary reason for perpetuation of T cell memory. Using a viral hemagglutinin as a model, in this work, we demonstrate the presence of peptidomimics in the variable region of an anti-idiotypic antibody capable of functionally mimicking the antigen derived peptides. A CD8+ CTL clone was generated against the hemagglutinin protein which specifically responds to either peptidomimic synthesizing cells or peptidomimic pulsed antigen presenting cells. Thus, it appears reasonable that a population of activated antigen specific T cells is maintained in the body by presentation of peptidomimic through Jerne cells and other antigen presenting cells long after immunization.     

Population Genomic Analysis Reveals a Rich Speciation and Demographic History of Orang-utans (Pongo pygmaeus and Pongo abelii)

To gain insights into evolutionary forces that have shaped the history of Bornean and Sumatran populations of orang-utans, we compare patterns of variation across more than 11 million single nucleotide polymorphisms found by previous mitochondrial and autosomal genome sequencing of 10 wild-caught orang-utans. Our analysis of the mitochondrial data yields a far more ancient split time between the two populations (∼3.4 million years ago) than estimates based on autosomal data (0.4 million years ago), suggesting a complex speciation process with moderate levels of primarily male migration. We find that the distribution of selection coefficients consistent with the observed frequency spectrum of autosomal non-synonymous polymorphisms in orang-utans is similar to the distribution in humans. Our analysis indicates that 35% of genes have evolved under detectable negative selection. Overall, our findings suggest that purifying natural selection, genetic drift, and a complex demographic history are the dominant drivers of genome evolution for the two orang-utan populations.     

TollML: a database of toll-like receptor structural motifs

Toll-like receptors (TLRs) play a key role in the innate immune system. TLRs recognize pathogen-associated molecular patterns and initiate an intracellular kinase cascade to induce an immediate defensive response. During recent years TLRs have become the focus of tremendous research interest. A central repository for the growing amount of relevant TLR sequence information has been created. Nevertheless, structural motifs of most sequenced TLR proteins, such as leucine-rich repeats (LRRs), are poorly annotated in the established databases. A database that organizes the structural motifs of TLRs could be useful for developing pattern recognition programs, structural modeling and understanding functional mechanisms of TLRs. We describe TollML, a database that integrates all of the TLR sequencing data from the NCBI protein database. Entries were first divided into TLR families (TLR1-23) and then semi-automatically subdivided into three levels of structural motif categories: (1) signal peptide (SP), ectodomain (ECD), transmembrane domain (TD) and Toll/IL-1 receptor (TIR) domain of each TLR; (2) LRRs of each ECD; (3) highly conserved segment (HCS), variable segment (VS) and insertions of each LRR. These categories can be searched quickly using an easy-to-use web interface and dynamically displayed by graphics. Additionally, all entries have hyperlinks to various sources including NCBI, Swiss-Prot, PDB, LRRML and PubMed in order to provide broad external information for users. The TollML database is available at .     

Ratio determination of plasma wild-type and L159R apoA-I using mass spectrometry: tools for studying apoA-IFin

In this report, methods are described to isolate milligram quantities of a mutant apolipoprotein A-I (apoA-I) protein for use in structure-function studies. Expression of the L159R apoA-I mutation in humans reduces the concentration of plasma wild-type apoA-I, thus displaying a dominant negative phenotype in vivo. Earlier attempts to express and isolate this mutant protein resulted in extensive degradation and protein misfolding. Using an Escherichia coli expression system used predominantly for the isolation of soluble apoA-I mutant proteins, we describe the expression and purification of L159R apoA-I (apoA-I(Fin)) from inclusion bodies. In addition, we describe a mass spectrometric method for measuring the L159R-to-wild-type apoA-I ratio in a 1 microl plasma sample. These new methods will facilitate further studies into the mechanism behind the dominant negative phenotype associated with the expression of the L159R apoA-I protein in humans.     

A novel high throughput 96-well-based fluorimetric method to measure amikacin in pharmaceutical formulations: development using response surface methodology

An aminoglycoside antibiotic, amikacin, is used to treat severe and recurring bacterial infections. Due to the absence of a chromophore, however, amikacin must be extensively derivatized before being quantified, both in analytical and bioanalytical samples. In this study, for the first time, we developed a simple and sensitive method for measuring amikacin sulfate using spectrofluorimetry with a 96-well plate reader, based on the design of the experiment's approach. To develop a robust and reproducible spectrofluorimetric method, the influence of essential attributes, namely pH of the buffer, heating temperature, and concentration of reagents, were evaluated using univariate analysis followed by multivariate analysis (central composite design). International Conference of Harmonization guidelines were used to validate the optimized method. The developed technique is linear from 1.9 to 10 μg/ml with a regression coefficient of 0.9991. The detection and quantification limits were 0.649 μg/ml and 1.9 μg/ml, respectively. For the developed method, both intraday and interday precision (%RSD) were less than 5%. Using the method, amikacin concentrations were quantified in prepared amikacin liposomes and commercial formulations of Amicin®. The developed method greatly reduces sample volume and is a rapid, high throughput microplate-based fluorescence approach for the convenient and cost-effective measurement of amikacin in pharmaceutical formulations. In comparison with previously published approaches, the suggested method allowed for quick analysis of a high number of samples in a short amount of time (96 samples in 125 sec), resulting in an average duration of analysis of 1.3 sec per sample.