Role of the sonchus yellow net virus N protein in formation of nuclear viroplasms

Sonchus yellow net virus is a plant nucleorhabdovirus whose nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins form large viroplasms in the nuclei of infected plants (C. R. F. Martins, J. A. Johnson, D. M. Lawrence, T. J. Choi, A. Pisi, S. L. Tobin, D. Lapidus, J. D. O. Wagner, S. Ruzin, K. McDonald, and A. O. Jackson, J. Virol. 72:5669-5679, 1998). When expressed alone, the N protein localizes to the nuclei of plant and yeast (Saccharomyces cerevisiae) cells and the P protein is distributed throughout the cells, but coexpression of N and P results in formation of subnuclear viroplasm-like foci (M. M. Goodin, J. Austin, R. Tobias, M. Fujita, C. Morales, and A. O. Jackson, J. Virol. 75:9393-9406, 2001; M. M. Goodin, R. G. Dietzgen, D. Schichnes, S. Ruzin, and A. O. Jackson, Plant J. 31:375-383, 2002). We now show that the N protein and various fluorescent derivatives form similar subnuclear foci in plant cells and that homologous interactions mediated by a helix-loop-helix region near the amino terminus are required for formation of the foci. Mutations within the helix-loop-helix region also interfere with N- and P-protein interactions that are required for N and P colocalization in the subnuclear foci. Affinity purification of N proteins harboring single mutations within the motif revealed that Tyr40 is critical for N-N and N-P interactions. Additional in vitro binding assays also indicated that the N protein binds to yeast and plant importin alpha homologues, whereas mutations in the carboxy-terminal nuclear localization signal abrogate importin alpha binding. The P protein did not bind to the importin alpha homologues, suggesting that the N and P proteins use different pathways for nuclear entry. Our results in toto support a model suggesting that during infection, the N and P proteins enter the nucleus independently, that viroplasm formation requires homologous N-protein interactions, and that P protein targeting to the viroplasm requires N-P protein interactions that occur after N and P protein import into the nucleus.     

Tracing the transition of methicillin resistance in sub-populations of Staphylococcus aureus, using SELDI-TOF Mass Spectrometry and Artificial Neural Network Analysis

Strains (n = 99) of Staphylococcus aureus isolated from a large number of clinical sources and tested for methicillin sensitivity were analysed by MALDI-TOF-MS using the Weak Cation Exchange (CM10) ProteinChip Array (designated SELDI-TOF-MS). The profile data generated was analysed using Artificial Neural Network (ANN) Analysis modelling techniques. Seven key ions identified by the ANNs that were predictive of MRSA and MSSA were validated by incorporation into a model. This model exhibited an area under the ROC curve value of 0.9147 indicating the potential application of this approach for rapidly characterising MRSA and MSSA isolates. Nearly all strains (n = 97) were correctly assigned to the correct group, with only two aberrant MSSA strains being misclassified. However, approximately 21% of the strains appeared to be in a process of transition as resistance to methicillin was being acquired.     

A nicardipine-sensitive Ca2+ entry contributes to the hypotonicity-induced increase in [Ca2+]i of principal cells in rat cortical collecting duct

We examined the mechanisms involved in the [Ca(2+)](i) response to the extracellular hypotonicity in the principal cells of freshly isolated rat cortical collecting duct (CCD), using Fura-2/AM fluorescence imaging. Reduction of extracellular osmolality from 305 (control) to 195 mosmol/kgH(2)O (hypotonic) evoked transient increase in [Ca(2+)](i) of principal cells of rat CCDs. The [Ca(2+)](i) increase was markedly attenuated by the removal of extracellular Ca(2+)(.) The application of a P(2) purinoceptor antagonist, suramin failed to inhibit the hypotonicity-induced [Ca(2+)](i) increase. The [Ca(2+)](i) increase in response to extracellular hypotonicity was not influenced by application of Gd(3+) and ruthenium red. On the other hand, a voltage-gated Ca(2+) channel inhibitor, nicardipine, significantly reduced the peak amplitude of [Ca(2+)](i) increase in the principal cells. In order to assess Ca(2+) entry during the hypotonic stimulation, we measured the quenching of Fura-2 fluorescence intensity by Mn(2+). The hypotonic stimulation enhanced quenching of Fura-2 fluorescence by Mn(2+), indicating that a Ca(2+)-permeable pathway was activated by the hypotonicity. The hypotonicity-mediated enhancement of Mn(2+) quenching was significantly inhibited by nicardipine. These results strongly suggested that a nicardipine-sensitive Ca(2+) entry pathway would contribute to the mechanisms underlying the hypotonicity-induced [Ca(2+)](i) elevation of principal cells in rat CCD.     

Differential mitochondrial calcium responses in different cell types detected with a mitochondrial calcium fluorescent indicator, mito-GCaMP2

Mitochondrial calcium plays a crucial role in mitochondrial metabolism, cell calcium handling, and cell death. However, some mechanisms concerning mitochondrial calcium regulation are still unknown, especially how mitochondrial calcium couples with cytosolic calcium. In this work, we constructed a novel mitochondrial calcium fluorescent indicator (mito-GCaMP2) by genetic manipulation. Mito-GCaMP2 was imported into mitochondria with high efficiency and the fluorescent signals co-localized with that of tetramethyl rhodamine methyl ester, a mitochondrial membrane potential indicator. The mitochondrial inhibitors specifically decreased the signals of mito-GCaMP2. The apparent K(d) of mito-GCaMP2 was 195.0 nmol/L at pH 8.0 in adult rat cardiomyocytes. Furthermore, we observed that mito-GCaMP2 preferred the alkaline pH surrounding of mitochondria. In HeLa cells, we found that mitochondrial calcium ([Ca(2+)](mito)) responded to the changes of cytosolic calcium ([Ca(2+)](cyto)) induced by histamine or thapasigargin. Moreover, external Ca(2+) (100 μmol/L) directly induced an increase of [Ca(2+)](mito) in permeabilized HeLa cells. However, in rat cardiomyocytes [Ca(2+)](mito) did not respond to cytosolic calcium transients stimulated by electric pacing or caffeine. In permeabilized cardiomyocytes, 600 nmol/L free Ca(2+) repeatedly increased the fluorescent signals of mito-GCaMP2, which excluded the possibility that mito-GCaMP2 lost its function in cardiomyocytes mitochondria. These results showed that the response of mitochondrial calcium is diverse in different cell lineages and suggested that mitochondria in cardiomyocytes may have a special defense mechanism to control calcium flux.     

Syntheses, crystal structures, and magnetic properties of Ni(mnt)2-based molecular solids containing substituted isoquinolinium

Two novel ion-pair complexes, [RBzIQl]⁺[Ni(mnt)₂]⁻ (mnt²⁻ = maleonitriledithiolate, [RBzIQl]⁺ = 4-R-benzylisoquinolinium; R = H (1), Cl (2)) have been characterized structurally and magnetically. The anions and [BzIQl]⁺ cations of 1 form 1D column of alternating between cations and anions via π?π stacking interaction between Ni(mnt)₂ plane and isoquinoline ring, and the Ni(mnt)₂ anions between adjacent columns exist C?N, C?N, and N?N interaction. The anions and cations of 2 stack into well-segregated columns in the solid state; and the Ni(III) ions form a 1D zigzag chain in a Ni(mnt)₂ column through intermolecular Ni?S, S?S, Ni?Ni or π?π interactions. The chain is uniform in 2 with the Ni?Ni distances of 3.784 Å. Magnetic susceptibility measurements for these complexes in the temperature range 1.8-300 K show that 1 exhibits antiferromagnetic coupling behavior, and 2 exhibits unusual magnetic phase transitions around 45 K. The overall magnetic behavior for 2 indicates the presence of antiferromagnetic interaction in the high-temperature phase (HT) and spin gap in the low-temperature phase (LT).     

Incorporation of quantum dots on virus in polycationic solution

Developing methods to label viruses with fluorescent moieties has its merits in elucidating viral infection mechanisms and exploring novel antiviral therapeutics. Fluorescent quantum dots (QDs), an emerging probe for biological imaging and medical diagnostics, were employed in this study to tag retrovirus encoding enhanced green fluorescent protein (EGFP) genes. Electrostatic repulsion forces generated from both negatively charged retrovirus and QDs were neutralized by cationic Polybrene, forming colloidal complexes of QDs-virus. By examining the level of EGFP expression in 3T3 fibroblast cells treated with QDs-tagged retroviruses for 24 hours, the infectivity of retrovirus incorporated with QDs was shown to be only slightly decreased. Moreover, the imaging of QDs can be detected in the cellular milieu. In summary, the mild method developed here makes QDs-tagged virus a potential imaging probe for direct tracking the infection process and monitoring distribution of viral particles in infected cells.     

A New Secoiridoidal Glucosides from Gentiana rigescens (Gentianaceae)

A new acylated secoiridoidal glucoside, named gentiorigenoside A (1 ), was isolated from the root of Gentiana rigescens (Gentianaceae) , together with eight known compounds, gentiopicroside ( 2) , 6′- O-β- D-glucopyranosyl gentiopicroside (3) , loganic acid (4 ) , 6′- O-β- D-glucopyranosyl loganic acid ( 5), sweroside ( 6 ), 2′-( o, m-dihydoxybenzyl) -sweroside (7), swertiamarin (8) and secologanoside (9) . heir structures were elucidated on the basis of detailed spectroscopic analysis. Iridoidal glucosides 3, 5 and 9 were isolated for the first time from the title plant.