Screening of banana bunchy top virus through multiplex PCR approach

Bunchy top disease caused by the banana bunchy top virus (BBTV) is a serious disease in hill banana. Detection of the BBTV infection in the planting material could help in the effective management of the disease. An attempt was made to develop a sensitive polymerase chain reaction (PCR) and multiplex PCR-based method for detection of BBTV in hill banana. DNA was isolated from the experimental plants at third and sixth months after planting. Multiplex PCR was done with Coat Protein (CP) and Replicase (Rep) gene-specific primer, and banana ethylene insensitive like protein (EISL) primer as internal control to identify failure in PCR reaction. This study revealed that multiplex PCR is effective for BBTV screening in hill banana with the advantage of overcoming the false positive in PCR amplification.     

F1-ATPase of Escherichia coli: THE ?-INHIBITED STATE FORMS AFTER ATP HYDROLYSIS,IS DISTINCT FROM THE ADP-INHIBITED STATE,AND RESPONDS DYNAMICALLY TO CATALYTIC SITE LIGANDS*

F₁-ATPase is the catalytic complex of rotary nanomotor ATP synthases. Bacterial ATP synthases can be autoinhibited by the C-terminal domain of subunit ϵ, which partially inserts into the enzyme''s central rotor cavity to block functional subunit rotation. Using a kinetic, optical assay of F₁·ϵ binding and dissociation, we show that formation of the extended, inhibitory conformation of ϵ (ϵ_X) initiates after ATP hydrolysis at the catalytic dwell step. Prehydrolysis conditions prevent formation of the ϵ_X state, and post-hydrolysis conditions stabilize it. We also show that ϵ inhibition and ADP inhibition are distinct, competing processes that can follow the catalytic dwell. We show that the N-terminal domain of ϵ is responsible for initial binding to F₁ and provides most of the binding energy. Without the C-terminal domain, partial inhibition by the ϵ N-terminal domain is due to enhanced ADP inhibition. The rapid effects of catalytic site ligands on conformational changes of F₁-bound ϵ suggest dynamic conformational and rotational mobility in F₁ that is paused near the catalytic dwell position.     

Heterochromatin Protein 1a (HP1a) Partner Specificity Is Determined by Critical Amino Acids in the Chromo Shadow Domain and C-terminal Extension

Drosophila melanogaster Heterochromatin Protein 1a (HP1a) is an essential protein critical for heterochromatin assembly and regulation. Its chromo shadow domain (CSD) homodimerizes, a requirement for binding protein partners that contain a PXVXL motif. How does HP1a select among its many different PXVXL-containing partners? HP1a binds tightly to Heterochromatin Protein 2 (HP2), but weakly to PIWI. We investigated differences in homodimerization and the impact of the C-terminal extension (CTE) by contrasting HP1a to its paralogue, HP1b. HP1a and HP1b differ in the dimerization interface, with HP1a having an Arg at position 188 rather than Glu. We find that while this substitution reduces the dimerization constant, it does not impact the binding surface as demonstrated by unchanged partner binding affinities. However, the CTE (only 4 residues in HP1a as compared with 87 residues in HP1b) is critical; the charged residues in HP1a are necessary for tight peptide binding. Examining a panel of amino acid substitutions in the HP1a CSD, we find that Leu-165 in HP1a interacts with HP2 but not PIWI, supporting the conclusion that different sites in the binding surface provide discrimination for partner selection. Partner sequence is also critical for affinity, as the remaining difference in binding between HP2 and PIWI polypeptides is eliminated by swapping the PXVXL motifs between the two. Taken together, these studies indicate that the binding surface of the HP1a CSD plus its short CTE provide the needed discrimination among HP1a''s partners, and that the CTE is important for differentiating the interactions of the Drosophila HP1 paralogs.     

Sorting Nexin 1 Loss Results in D5 Dopamine Receptor Dysfunction in Human Renal Proximal Tubule Cells and Hypertension in Mice

The peripheral dopaminergic system plays a crucial role in blood pressure regulation through its actions on renal hemodynamics and epithelial ion transport. The dopamine D5 receptor (D₅R) interacts with sorting nexin 1 (SNX1), a protein involved in receptor retrieval from the trans-Golgi network. In this report, we elucidated the spatial, temporal, and functional significance of this interaction in human renal proximal tubule cells and HEK293 cells stably expressing human D₅R and in mice. Silencing of SNX1 expression via RNAi resulted in the failure of D₅R to internalize and bind GTP, blunting of the agonist-induced increase in cAMP production and decrease in sodium transport, and up-regulation of angiotensin II receptor expression, of which expression was previously shown to be negatively regulated by D₅R. Moreover, siRNA-mediated depletion of renal SNX1 in C57BL/6J and BALB/cJ mice resulted in increased blood pressure and blunted natriuretic response to agonist in salt-loaded BALB/cJ mice. These data demonstrate a crucial role for SNX1 in D₅R trafficking and that SNX1 depletion results in D₅R dysfunction and thus may represent a novel mechanism for the pathogenesis of essential hypertension.     

Qualitative composition of carotenoids, catalase and superoxide dismutase activities in tissues of the bivalve mollusc Anadara inaequivalvis (Bruguiere, 1789)

Using high performance liquid chromatography, UV-VIS spectra and mass-spectra (FAB MS), 7 carotenoid species were identified in tissues of the bivalve mollusc Anadara inaequivalvis (Bruguiere, 1789): trans- and cis-pectenolon, alloxanthin, pectenol A, β-carotene, zeaxanthin, and diatoxanthin. Their quantitative ratio in hepatopancreas, gills and foot of animals were determined. A negative correlation (R ² is about 0.9) was revealed between tissues content of a series of carotenoids (trans- and cis-pectenolon, zeaxanthin, alloxanthin, and diatoxanthin) and activity of antioxidant enzymes (catalase and superoxide dismutase). The existence of competitive interrelations between these molecular systems is proposed and underlying causes are discussed.     

Quantitative and Qualitative Involvement of P3N-PIPO in Overcoming Recessive Resistance against Clover Yellow Vein Virus in Pea Carrying the cyv1 Gene

In pea carrying cyv1, a recessive gene for resistance to Clover yellow vein virus (ClYVV), ClYVV isolate Cl-no30 was restricted to the initially infected cells, whereas isolate 90-1 Br2 overcame this resistance. We mapped the region responsible for breaking of cyv1-mediated resistance by examining infection of cyv1 pea with chimeric viruses constructed from parts of Cl-no30 and 90-1 Br2. The breaking of resistance was attributed to the P3 cistron, which is known to produce two proteins: P3, from the main open reading frame (ORF), and P3N-PIPO, which has the N-terminal part of P3 fused to amino acids encoded by a small open reading frame (ORF) called PIPO in the +2 reading frame. We introduced point mutations that were synonymous with respect to the P3 protein but nonsynonymous with respect to the P3N-PIPO protein, and vice versa, into the chimeric viruses. Infection of plants with these mutant viruses revealed that both P3 and P3N-PIPO were involved in overcoming cyv1-mediated resistance. Moreover, P3N-PIPO quantitatively affected the virulence of Cl-no30 in cyv1 pea. Additional expression in trans of the P3N-PIPO derived from Cl-no30, using White clover mosaic virus as a vector, enabled Cl-no30 to move to systemic leaves in cyv1 pea. Susceptible pea plants infected with chimeric ClYVV possessing the P3 cistron of 90-1 Br2, and which were therefore virulent toward cyv1 pea, accumulated more P3N-PIPO than did those infected with Cl-no30, suggesting that the higher level of P3N-PIPO in infected cells contributed to the breaking of resistance by 90-1 Br2. This is the first report showing that P3N-PIPO is a virulence determinant in plants resistant to a potyvirus.     

Detection of Proton Movement Directly across Viral Membranes To Identify Novel Influenza Virus M2 Inhibitors

The influenza virus M2 protein is a well-validated yet underexploited proton-selective ion channel essential for influenza virus infectivity. Because M2 is a toxic viral ion channel, existing M2 inhibitors have been discovered through live virus inhibition or medicinal chemistry rather than M2-targeted high-throughput screening (HTS), and direct measurement of its activity has been limited to live cells or reconstituted lipid bilayers. Here, we describe a cell-free ion channel assay in which M2 ion channels are incorporated into virus-like particles (VLPs) and proton conductance is measured directly across the viral lipid bilayer, detecting changes in membrane potential, ion permeability, and ion channel function. Using this approach in high-throughput screening of over 100,000 compounds, we identified 19 M2-specific inhibitors, including two novel chemical scaffolds that inhibit both M2 function and influenza virus infectivity. Counterscreening for nonspecific disruption of viral bilayer ion permeability also identified a broad-spectrum antiviral compound that acts by disrupting the integrity of the viral membrane. In addition to its application to M2 and potentially other ion channels, this technology enables direct measurement of the electrochemical and biophysical characteristics of viral membranes.     

Human T Lymphotropic Virus Type 1 SU Residue 195 Plays a Role in Determining the Preferential CD4+ T Cell Immortalization/Transformation Tropism

Human T lymphotropic virus type 1 (HTLV-1) mainly causes adult T cell leukemia and predominantly immortalizes/transforms CD4⁺ T cells in culture. HTLV-2 is aleukemic and predominantly immortalizes/transforms CD8⁺ T cells in culture. We have shown previously that the viral envelope is the genetic determinant of the differential T cell tropism in culture. The surface component (SU) of the HTLV-1 envelope is responsible for binding to the cellular receptors for entry. Here, we dissect the HTLV-1 SU further to identify key domains that are involved in determining the immortalization tropism. We generated HTLV-1 envelope recombinant virus containing the HTLV-2 SU domain. HTLV-1/SU2 was capable of infecting and immortalizing freshly isolated peripheral blood mononuclear cells in culture. HTLV-1/SU2 shifted the CD4⁺ T cell immortalization tropism of wild-type HTLV-1 (wtHTLV-1) to a CD8⁺ T cell preference. Furthermore, a single amino acid substitution, N195D, in HTLV-1 SU (Ach.195) resulted in a shift to a CD8⁺ T cell immortalization tropism preference. Longitudinal phenotyping analyses of the in vitro transformation process revealed that CD4⁺ T cells emerged as the predominant population by week 5 in wtHTLV-1 cultures, while CD8⁺ T cells emerged as the predominant population by weeks 4 and 7 in wtHTLV-2 and Ach.195 cultures, respectively. Our results indicate that SU domain independently influences the preferential T cell immortalization tropism irrespective of the envelope counterpart transmembrane (TM) domain. We further showed that asparagine at position 195 in HTLV-1 SU is involved in determining this CD4⁺ T cell immortalization tropism. The slower emergence of the CD8⁺ T cell predominance in Ach.195-infected cultures suggests that other residues/domains contribute to this tropism preference.