Subcellular Localization of the Barley Stripe Mosaic Virus Triple Gene Block Proteins

Barley stripe mosaic virus (BSMV) spreads from cell to cell through the coordinated actions of three triple gene block (TGB) proteins (TGB1, TGB2, and TGB3) arranged in overlapping open reading frames (ORFs). Our previous studies (D. M. Lawrence and A. O. Jackson, J. Virol. 75:8712-8723, 2001; D. M. Lawrence and A. O. Jackson, Mol. Plant Pathol. 2:65-75, 2001) have shown that each of these proteins is required for cell-to-cell movement in monocot and dicot hosts. We recently found (H.-S. Lim, J. N. Bragg, U. Ganesan, D. M. Lawrence, J. Yu, M. Isogai, J. Hammond, and A. O. Jackson, J. Virol. 82:4991-5006, 2008) that TGB1 engages in homologous interactions leading to the formation of a ribonucleoprotein complex containing viral genomic and messenger RNAs, and we have also demonstrated that TGB3 functions in heterologous interactions with TGB1 and TGB2. We have now used Agrobacterium tumefaciens-mediated protein expression in Nicotiana benthamiana leaf cells and site-specific mutagenesis to determine how TGB protein interactions influence their subcellular localization and virus spread. Confocal microscopy revealed that the TGB3 protein localizes at the cell wall (CW) in close association with plasmodesmata and that the deletion or mutagenesis of a single amino acid at the immediate C terminus can affect CW targeting. TGB3 also directed the localization of TGB2 from the endoplasmic reticulum to the CW, and this targeting was shown to be dependent on interactions between the TGB2 and TGB3 proteins. The optimal localization of the TGB1 protein at the CW also required TGB2 and TGB3 interactions, but in this context, site-specific TGB1 helicase motif mutants varied in their localization patterns. The results suggest that the ability of TGB1 to engage in homologous binding interactions is not essential for targeting to the CW. However, the relative expression levels of TGB2 and TGB3 influenced the cytosolic and CW distributions of TGB1 and TGB2. Moreover, in both cases, localization at the CW was optimal at the 10:1 TGB2-to-TGB3 ratios occurring in virus infections, and mutations reducing CW localization had corresponding effects on BSMV movement phenotypes. These data support a model whereby TGB protein interactions function in the subcellular targeting of movement protein complexes and the ability of BSMV to move from cell to cell.Plants use macromolecular trafficking pathways through plasmodesmata (PD) as a means to regulate developmental processes and physiological functions, and they also rely on these channels as avenues to communicate and mount defense responses to pathogen challenge (2, 37, 55). Local and systemic plant virus invasion depends on the abilities of viruses to use these pathways to spread from initially infected cells to the vascular tissue and distal regions of the plant. To this end, viruses infecting plants have evolved movement proteins (MPs) that coopt host trafficking pathways to target virus genomes to the PD and to facilitate the cell-to-cell transit of infectious entities (4, 13, 36, 48, 55). Virus MPs vary in size, number, and genome organization, but they share a number of functional characteristics including localization to PD, an ability to increase the size exclusion limits of PD, and RNA binding activities (3, 7, 8, 24, 27, 58).Viruses containing triple gene block (TGB) MPs have been the subjects of a number of investigations (4, 6, 39, 53, 54). Interestingly, viruses with a range of diverse genome structures encode MPs in a TGB, but these proteins fall into two major TGB classes that have substantial differences in protein structure and variations in their physical, functional, and cellular interactions (19, 30, 39, 45, 48). For example, the hordeivirus-like TGB1 proteins contain substantial N-terminal extensions that are lacking in the potexvirus-like TGB1 proteins, but the two classes of proteins share a conserved helicase domain at their C termini (39). The available evidence also indicates that hordeivirus-like and potexvirus-like TGB1 proteins share common biochemical features, including RNA binding abilities (3, 13, 23, 35, 44, 56), RNA helicase activities (22), associated NTPase activities (3, 13, 23, 33, 35, 44), and the capacity to form homologous interactions (29, 30, 45). However, the potexvirus-like TGB1 proteins localize at the CW when expressed autonomously and also facilitate increases in PD size exclusion limits, whereas the hordeivirus-like TGB1 proteins lack both these activities (39, 53). Major differences are also evident in the organizations of the potexvirus-like and hordeivirus-like TGB3 proteins, which share no discernible relatedness, differ in the numbers of their transmembrane domains, and indeed appear to have a polyphyletic origin (39).In both TGB classes, the movement strategy employs the coordinated actions of all three proteins. However, the coat protein is dispensable for one or more phases of movement of benyvirus, hordeivirus, pecluvirus, and pomovirus, encoding hordeivirus-like (class I) MPs, but is absolutely required for cell-to-cell movement of potexvirus-like (class II) MPs encoded by allexivirus, carlavirus, foveavirus, and potexvirus (6, 19, 39, 54). These variations clearly demonstrate that the two classes of TGB proteins have profound differences in their functional properties and in their associations with other virus and host proteins. Hence, comparative analyses of the functional and biological properties of the two classes of proteins in their common hosts may reveal important activities relevant to viral pathogenesis. To provide more information about the hordeivirus-like movement mechanisms, we are investigating the TGB interactions of Barley stripe mosaic virus (BSMV).BSMV is the type member of the genus Hordeivirus, which includes Poa semilatent virus (PSLV), Lychnis ringspot virus, and Anthoxanthum latent blanching virus (6, 19). Hordeiviruses have positive-sense, single-stranded RNA genomes consisting of three segments, designated α, β, and γ. The RNAβ segment encodes the coat protein, which is translated directly from genomic RNAβ (gRNAβ), and the TGB proteins, which are expressed from two subgenomic RNAs (sgRNAs), designated sgRNAβ1 and sgRNAβ2 (60). The coat protein is dispensable for the systemic movement of BSMV (41), and mutational analyses indicate that the TGB1, TGB2, and TGB3 proteins are each essential for cell-to-cell movement in monocot and dicot hosts (28). The BSMV TGB1 (58-kDa) protein is expressed from sgRNAβ1 at higher levels than the smaller hydrophobic TGB2 (14-kDa) and TGB3 (17-kDa) proteins, which are coexpressed from the bicistronic sgRNAβ2 during replication (14, 60). BSMV TGB1 has binding activity for both single-stranded and double-stranded RNAs (13) and forms nucleoprotein complexes with each of the BSMV gRNAs and sgRNAs (30). The hordeivirus-like TGB1 proteins differ from the potexvirus-like TGB1 proteins in having longer N-terminal domains with positively charged amino acids, but both classes of proteins have conserved C-terminal NTPase/helicase domains (13, 39, 49). In BSMV, mutations of conserved amino acids within the TGB1 helicase motif abrogate cell-to-cell movement and alter subcellular localization in infected protoplasts (27). Plants infected with a BSMV β-green fluorescent protein-TGB1 (β-GFP-TGB1) reporter virus also exhibited paired foci on both sides of the CW, and the plasma membranes of infected protoplasts developed punctate foci (27). TGB1 and TGB2 are also essential for plasma membrane targeting because β-GFP-TGB1 reporter derivatives that were unable to express TGB2 or TGB3 fluoresce at perinuclear membranes of protoplasts (27). Particle bombardment studies with the related hordeivirus PSLV also suggested that the expression of TGB3 is required to shift the localization of TGB2 from the endoplasmic reticulum (ER) to the peripheral membranes (50), and transgenically expressed PSLV TGB3 appears to be associated with PD due to its colocalization with callose markers (17).We have recently shown that TGB2 and TGB3 interact physically and have identified single amino acids in each protein that are required for these interactions (19, 30). TGB3 also interacts with TGB1, and we have proposed that these interactions facilitate the transport of ribonucleoprotein (RNP) complexes to the PD (30). However, the effects of TGB protein interactions on subcellular localization have not been defined. Moreover, because of possible convergent evolution of the hordeivirus-like and potexvirus-like TGB-containing viruses (39), the mechanisms of action resulting in transport may differ among different genera or even among different virus species within a genus. To obtain more refined information about these processes, we have now expressed fluorescent TGB fusion proteins transiently in Nicotiana benthamiana leaf cells by Agrobacterium tumefaciens infiltration and have assessed the subcellular localization patterns of BSMV wild-type (wt) and mutant TGB derivatives that differ in their interactions. We also have carried out reverse genetic experiments with selected BSMV TGB mutants to provide a biological context for the localization patterns appearing during ectopic Agrobacterium expression. These findings are elaborated in a model for TGB interactions required for the cell-to-cell movement of BSMV.     

Fragment analogs as better mimics of obestatin

Obestatin is a twenty three amino acid peptide produced in the stomach by post translational modification of the preproghrelin gene. Since its discovery in 2005, many studies have shown that obestatin reduces feed intake and gain in body weight in rodents. Studies from our laboratory have shown the N-terminal thirteen residues mimic obestatin the best and residues 6–18 reduce epididymal fat significantly in adult male mice. In this study we have tried to increase the efficacy of these fragments. As an initial step, we have substituted G(8) with α-aminoisobutyricacid(Aib,U) and F(5) with cyclohexylalanine(Cha) in the N-terminal peptide to obtain two modified peptides and modified the middle fragment (residues 6–18) by substituting both the glycine residues at position 3 and 8 with α-aminoisobutyricacid(U). The rationale being, unusual amino acids could protect the peptides from immediate degradation and Aib would also induce secondary structure in these unstructured peptides. The N-terminal fragment with the G(8)U substitution fared the best. It reduced food intake, gain in body weight, levels of cholesterol and triglycerides in the blood, epididymal and perirenal fat in adult male mice similar to that of obestatin. The middle fragment with G(3,8)U double substitution was the second best.     

Discovery of betrixaban (PRT054021), N-(5-chloropyridin-2-yl)-2-(4-(N,N-dimethylcarbamimidoyl)benzamido)-5-methoxybenzamide,a highly potent,selective, and orally efficacious factor Xa inhibitor

Systematic SAR studies of in vitro factor Xa inhibitory activity around compound 1 were performed by modifying each of the three phenyl rings. A class of highly potent, selective, efficacious and orally bioavailable direct factor Xa inhibitors was discovered. These compounds were screened in hERG binding assays to examine the effects of substitution groups on the hERG channel affinity. From the leading compounds, betrixaban (compound 11, PRT054021) has been selected as the clinical candidate for development.     

Identification of phosphoproteins in Arabidopsis thaliana leaves using polyethylene glycol fractionation, immobilized metal-ion affinity chromatography, two-dimensional gel electrophoresis and mass spectrometry

Reversible protein phosphorylation is a key regulatory mechanism in cells. Identification and characterization of phosphoproteins requires specialized enrichment methods, due to the relatively low abundance of these proteins, and is further complicated in plants by the high abundance of Rubisco in green tissues. We present a novel method for plant phosphoproteome analysis that depletes Rubisco using polyethylene glycol fractionation and utilizes immobilized metal-ion affinity chromatography to enrich phosphoproteins. Subsequent protein separation by one- and two-dimensional gel electrophoresis is further improved by extracting the PEG-fractionated protein samples with SDS/phenol and methanol/chloroform to remove interfering compounds. Using this approach, we identified 132 phosphorylated proteins in a partial Arabidopsis leaf extract. These proteins are involved in a range of biological processes, including CO(2) fixation, protein assembly and folding, stress response, redox regulation, and cellular metabolism. Both large and small subunits of Rubisco were phosphorylated at multiple sites, and depletion of Rubisco enhanced detection of less abundant phosphoproteins, including those associated with state transitions between photosystems I and II. The discovery of a phosphorylated form of AtGRP7, a self-regulating RNA-binding protein that affects floral transition, as well as several previously uncharacterized ribosomal proteins confirm the utility of this approach for phosphoproteome analysis and its potential to increase our understanding of growth and development in plants.     

Preparation and <Emphasis Type="Italic">In Vitro</Emphasis>/<Emphasis Type="Italic">In Vivo</Emphasis> Evaluation of Microparticle Formulations Containing Meloxicam

In this study, we have formulated chitosan-coated sodium alginate microparticles containing meloxicam (MLX) and aimed to investigate the correlation between in vitro release and in vivo absorbed percentages of meloxicam. The microparticle formulations were prepared by orifice ionic gelation method with two different sodium alginate concentrations, as 1% and 2% (w/v), in order to provide different release rates. Additionally, an oral solution containing 15 mg of meloxicam was administered as the reference solution for evaluation of in vitro/in vivo correlation (ivivc). Following in vitro characterization, plasma levels of MLX and pharmacokinetic parameters [elimination half-life (t _1/2), maximum plasma concentration (C _max), time for C _max (t _max)] after oral administration to New Zealand rabbits were determined. Area under plasma concentration–time curve (AUC_0–∞) was calculated by using trapezoidal method. A linear regression was investigated between released% (in vitro) and absorbed% (in vivo) with a model-independent deconvolution approach. As a result, increase in sodium alginate content lengthened in vitro release time and in vivo t _max value. In addition, for ivivc, linear regression equations with r ² values of 0.8563 and 0.9402 were obtained for microparticles containing 1% and 2% (w/v) sodium alginate, respectively. Lower prediction error for 2% sodium alginate formulations (7.419 ± 4.068) compared to 1% sodium alginate formulations (9.458 ± 5.106) indicated a more precise ivivc for 2% sodium alginate formulation.     

Exposure limits: the underestimation of absorbed cell phone radiation, especially in children

The existing cell phone certification process uses a plastic model of the head called the Specific Anthropomorphic Mannequin (SAM), representing the top 10% of U.S. military recruits in 1989 and greatly underestimating the Specific Absorption Rate (SAR) for typical mobile phone users, especially children. A superior computer simulation certification process has been approved by the Federal Communications Commission (FCC) but is not employed to certify cell phones. In the United States, the FCC determines maximum allowed exposures. Many countries, especially European Union members, use the "guidelines" of International Commission on Non-Ionizing Radiation Protection (ICNIRP), a non governmental agency. Radiofrequency (RF) exposure to a head smaller than SAM will absorb a relatively higher SAR. Also, SAM uses a fluid having the average electrical properties of the head that cannot indicate differential absorption of specific brain tissue, nor absorption in children or smaller adults. The SAR for a 10-year old is up to 153% higher than the SAR for the SAM model. When electrical properties are considered, a child's head's absorption can be over two times greater, and absorption of the skull's bone marrow can be ten times greater than adults. Therefore, a new certification process is needed that incorporates different modes of use, head sizes, and tissue properties. Anatomically based models should be employed in revising safety standards for these ubiquitous modern devices and standards should be set by accountable, independent groups.     

Therapeutic Helminth Infection of Macaques with Idiopathic Chronic Diarrhea Alters the Inflammatory Signature and Mucosal Microbiota of the Colon

Idiopathic chronic diarrhea (ICD) is a leading cause of morbidity amongst rhesus monkeys kept in captivity. Here, we show that exposure of affected animals to the whipworm Trichuris trichiura led to clinical improvement in fecal consistency, accompanied by weight gain, in four out of the five treated monkeys. By flow cytometry analysis of pinch biopsies collected during colonoscopies before and after treatment, we found an induction of a mucosal T_H2 response following helminth treatment that was associated with a decrease in activated CD4⁺ Ki67+ cells. In parallel, expression profiling with oligonucleotide microarrays and real-time PCR analysis revealed reductions in T_H1-type inflammatory gene expression and increased expression of genes associated with IgE signaling, mast cell activation, eosinophil recruitment, alternative activation of macrophages, and worm expulsion. By quantifying bacterial 16S rRNA in pinch biopsies using real-time PCR analysis, we found reduced bacterial attachment to the intestinal mucosa post-treatment. Finally, deep sequencing of bacterial 16S rRNA revealed changes to the composition of microbial communities attached to the intestinal mucosa following helminth treatment. Thus, the genus Streptophyta of the phylum Cyanobacteria was vastly increased in abundance in three out of five ICD monkeys relative to healthy controls, but was reduced to control levels post-treatment; by contrast, the phylum Tenericutes was expanded post-treatment. These findings suggest that helminth treatment in primates can ameliorate colitis by restoring mucosal barrier functions and reducing overall bacterial attachment, and also by altering the communities of attached bacteria. These results also define ICD in monkeys as a tractable preclinical model for ulcerative colitis in which these effects can be further investigated.     

Bioactive styryllactones,two new naphthoquinones and one new styryllactone,and other constituents from Goniothalamus scortechinii

Two new naphthoquinones, goniothalaminone A (1) and B (2), and a new styryllactone, (?)-8-epi-9-deoxygoniopypyrone acetate (12) together with one known naphthoquinone (3), one known indolequinone (4), one known 1-azaanthraquinone (5), six known styryllactones (6–11) and one known sesquiterpene (13) were isolated from the roots and leaves of Goniothalamus scortechinii. The structures of the new compounds were elucidated by spectroscopic analysis and of the known compounds by comparison of their physical, UV, IR, ¹H and ¹³C NMR data with those of published compounds. Antiplasmodial, antimycobacterial and cytotoxic activities of the styryllactones were evaluated. Compounds 6–10 exhibited cytotoxic against human cancer cell lines, KB, BC and NCI-H187 with IC₅₀ values ranging from 0.13 to 11.7 μg/ml.     

Assembling neurospheres: dynamics of neural progenitor/stem cell aggregation probed using an optical trap

Optical trapping (tweezing) has been used in conjunction with fluid flow technology to dissect the mechanics and spatio-temporal dynamics of how neural progenitor/stem cells (NSCs) adhere and aggregate. Hitherto unavailable information has been obtained on the most probable minimum time (～5 s) and most probable minimum distance of approach (4-6 μm) required for irreversible adhesion of proximate cells to occur. Our experiments also allow us to study and quantify the spatial characteristics of filopodial- and membrane-mediated adhesion, and to probe the functional dynamics of NSCs to quantify a lower limit of the adhesive force by which NSCs aggregate (～18 pN). Our findings, which we also validate by computational modeling, have important implications for the neurosphere assay: once aggregated, neurospheres cannot disassemble merely by being subjected to shaking or by thermal effects. Our findings provide quantitative affirmation to the notion that the neurosphere assay may not be a valid measure of clonality and "stemness". Post-adhesion dynamics were also studied and oscillatory motion in filopodia-mediated adhesion was observed. Furthermore, we have also explored the effect of the removal of calcium ions: both filopodia-mediated as well as membrane-membrane adhesion were inhibited. On the other hand, F-actin disrupted the dynamics of such adhesion events such that filopodia-mediated adhesion was inhibited but not membrane-membrane adhesion.