Widespread but small-scale changes in the structural and dynamic properties of vaccinia virus poly(A) polymerase upon association with its processivity factor in solution.

Vaccinia virus poly(A) polymerase (VP55) has been analyzed via hydrogen-deuterium exchange (HDX) mass spectrometry in the absence and presence of its processivity factor, VP39, to improve our understanding of the mechanism by which processivity is impressed on the polymerase. For 119 peptic peptides covering 74.1% of VP55, the extent of HDX at 900 s was interpreted in the context of parameters deduced from the VP55-VP39 X-ray crystal structure. While HDX exhibited a degree of correlation with the mean SASA of whole residues within each peptide segment, HDX was generally more active than expected from either the SASA or hydrogen bonding status of the exchangeable amide proton, indicating a significant molecular dynamics contribution to amide proton deprotection. Peptic peptides undergoing either more or less HDX than expected were distributed throughout VP55 and showed consistency between multiple overlapping peptides. VP39 had a net, marginal cooling effect on VP55, indicating a possible restriction of VP55's flexibility. VP39's cooling effect was most extensive within the central domain of VP55's three domains, while a patch within VP55's C-terminal domain showed an increased level of HDX in the presence of VP39. Langevin dynamics all-atom simulations of VP55 motions showed slower relaxation to equilibrium in the absence of VP39. At equilibrium, regions showing extremes of variation in simulated atomic fluctuation were localized within VP55's N- and C-terminal domains, and VP39 had a predominantly cooling effect on VP55. Broadly, across VP55's peptic peptides, a mild negative correlation was noted between the extent to which deuteration was more active than predicted from the structure and the amplitudes of the simulated atomic fluctuation and/or degree of disorder at equilibrium.

Two tools for applying chromatographic retention data to the mass-based identification of peptides during hydrogen/deuterium exchange experiments by nano-liquid chromatography/matrix-assisted laser desorption/ionization mass spectrometry.

Two tools are described for integrating LC elution position with mass-based data in hydrogen-deuterium exchange (HDX) experiments by nano-liquid chromatography/matrix-assisted laser desorption/ionization mass spectrometry (nanoLC/MALDI-MS, a novel approach to HDX-MS). The first of these, 'TOF2H-Z Comparator', highlights peptides in HDX experiments that are potentially misidentified on the basis of mass alone. The program first calculates normalized values for the organic solvent concentration responsible for the elution of ions in nanoLC/MALDI HDX experiments. It then allows the solvent gradients for the multiple experiments contributing to an MS/MS-confirmed peptic peptide library to be brought into mutual alignment by iteratively re-modeling variables among LC parameters such as gradient shape, solvent species, fraction duration and LC dead time. Finally, using the program, high-probability chromatographic outliers can be flagged within HDX experimental data. The role of the second tool, 'TOF2H-XIC Comparator', is to normalize the LC chromatograms corresponding to all deuteration timepoints of all HDX experiments of a project, to a common reference. Accurate normalization facilitates the verification of chromatographic consistency between all ions whose spectral segments contribute to particular deuterium uptake plots. Gradient normalization in this manner revealed chromatographic inconsistencies between ions whose masses were either indistinguishable or separated by precise isotopic increments.

Atomic force microscopy investigation of vaccinia virus structure.

Vaccinia virus was treated in a controlled manner with various combinations of nonionic detergents, reducing agents, and proteolytic enzymes, and successive products of the reactions were visualized using atomic force microscopy (AFM). Following removal of the outer lipid/protein membrane, a layer 20 to 40 nm in thickness was encountered that was composed of fibrous elements which, under reducing conditions, rapidly decomposed into individual monomers on the substrate. Beneath this layer was the virus core and its prominent lateral bodies, which could be dissociated or degraded with proteases. The core, in addition to the lateral bodies, was composed of a thick, multilayered shell of proteins of diverse sizes and shapes. The shell, which was readily etched with proteases, was thoroughly permeated with pores, or channels. Prolonged exposure to proteases and reductants produced disgorgement of the viral DNA from the remainders of the cores and also left residual, flattened, protease-resistant sacs on the imaging substrate. The DNA was readily visualized by AFM, which revealed some regions to be "soldered" by proteins, others to be heavily complexed with protein, and yet other parts to apparently exist as bundled, naked DNA. Prolonged exposure to proteases deproteinized the DNA, leaving masses of extended, free DNA. Estimates of the interior core volume suggest moderate but not extreme compaction of the genome.

Activation of RHOA-VAV1 signaling in angioimmunoblastic T-cell lymphoma.

Somatic G17V RHOA mutations were found in 50-70% of angioimmunoblastic T-cell lymphoma (AITL). The mutant RHOA lacks GTP binding capacity, suggesting defects in the classical RHOA signaling. Here, we discovered the novel function of the G17V RHOA: VAV1 was identified as a G17V RHOA-specific binding partner via high-throughput screening. We found that binding of G17V RHOA to VAV1 augmented its adaptor function through phosphorylation of 174Tyr, resulting in acceleration of T-cell receptor (TCR) signaling. Enrichment of cytokine and chemokine-related pathways was also evident by the expression of G17V RHOA. We further identified VAV1 mutations and a new translocation, VAV1-STAP2, in seven of the 85 RHOA mutation-negative samples (8.2%), whereas none of the 41 RHOA mutation-positive samples exhibited VAV1 mutations. Augmentation of 174Tyr phosphorylation was also demonstrated in VAV1-STAP2. Dasatinib, a multikinase inhibitor, efficiently blocked the accelerated VAV1 phosphorylation and the associating TCR signaling by both G17V RHOA and VAV1-STAP2 expression. Phospho-VAV1 staining was demonstrated in the clinical specimens harboring G17V RHOA and VAV1 mutations at a higher frequency than those without. Our findings indicate that the G17V RHOA-VAV1 axis may provide a new therapeutic target in AITL.

Identification of rpo30, a vaccinia virus RNA polymerase gene with structural similarity to a eucaryotic transcription elongation factor.

Eucaryotic transcription factors that stimulate RNA polymerase II by increasing the efficiency of elongation of specifically or randomly initiated RNA chains have been isolated and characterized. We have identified a 30-kilodalton (kDa) vaccinia virus-encoded protein with apparent homology to SII, a 34-kDa mammalian transcriptional elongation factor. In addition to amino acid sequence similarities, both proteins contain C-terminal putative zinc finger domains. Identification of the gene, rpo30, encoding the vaccinia virus protein was achieved by using antibody to the purified viral RNA polymerase for immunoprecipitation of the in vitro translation products of in vivo-synthesized early mRNA selected by hybridization to cloned DNA fragments of the viral genome. Western immunoblot analysis using antiserum made to the vaccinia rpo30 protein expressed in bacteria indicated that the 30-kDa protein remains associated with highly purified viral RNA polymerase. Thus, the vaccinia virus protein, unlike its eucaryotic homolog, is an integral RNA polymerase subunit rather than a readily separable transcription factor. Further studies showed that the expression of rpo30 is regulated by dual early and later promoters.

Structural basis of mRNA cap recognition by proteins.

Crystal structures have recently become available for two proteins (VP39 and eIF4E) complexed with their cognate ligand - the mRNA cap. Despite their total structural dissimilarity, both proteins bind N7-methylguanine between two parallel aromatic sidechains. The resulting stacked arrangement governs their high specificity for the alkylated form of the nucleobase.

Naturalistic assessment of the learner license period.

The purpose of this study was to describe the characteristics and progression of practice driving during the learner license period in a sample of teenagers. During the first and last 10h of practice driving, we examined (1) the amount, variety and complexity of conditions of practice; (2) the nature of parental instruction; and (3) errors that teens made while driving. Data were collected from 90 teens and 131 parents living in Virginia, USA, using in-vehicle cameras, audio recorders, GPS and trip recorders. Based on data collected from the instrumented vehicles, teens practiced for 46.6h on average, slightly higher than the GDL requirement for their jurisdiction, though half did not complete the required 45h of practice and only 17% completed the required 15h of night time driving. Exposure to diverse roadways increased over the practice driving period, which averaged 10.6 months. Most driving instruction occurred in reaction to specific driving situations, such as navigating and identifying hazards, and could be characterized as co-driving. Higher order instruction, which relates to the tactics or strategies for safe driving, was less frequent, but remained stable through the practice driving period. Instruction of all forms was more likely following an elevated gravitational force (g-force) event. Errors decreased over time, suggesting improvements in manual and judgment skills, but engagement in potentially distracting secondary tasks increased (when an adult was in the vehicle). A small percentage of trips occurred with no passenger in the front seat, and the g-force rate during these trips was almost 5 times higher than trips with an adult front-seat passenger. Taken collectively, these findings indicate (1) most teens got at least the required amount of supervised practice, but some did not; (2) instruction was mainly reactive and included some higher order instruction; (3) teens driving skills improved despite increased exposure to complex driving conditions, but secondary tasks also increased. Opportunities remained for improving the quality and variability in supervision and enhancing the development of skills during the lengthy period of practice.

Molecular flexibility and discontinuous translocation of a non-templated polymerase.

Little is known regarding the translocation of non-templated nucleic acid polymerases with respect to single-stranded primers. VP55, the vaccinia virus poly(A) polymerase, translocates as it processively adds a approximately 3-7 adenylate tail to primers possessing only three ribouridylate residues (as an (rU)(2)-N(15)-rU motif), and a approximately 25-30 adenylate tail to primers that are more U-rich. Here, three models were addressed for the translocation of VP55 with respect to its primer, namely: (a) rigid protein/rigid nucleic acid; (b) flexible protein/rigid nucleic acid; (c) rigid protein/flexible nucleic acid. Analysis of free and covalently VP55-attached primers favored either (b) or a version of (c) incorporating a passive steric block, and suggested two regions of relative motion between polymerase and primer. Inclusion of a 6nt uridylate-rich patch at the primer 3' end switched the polymerase from approximately 3-7 nt to approximately 25-30 nt tail addition without affecting initial binding affinity. By synthesizing this patch as a (rU/dC) pool, discontinuous polymerase movements could be detected.

A polyadenylylation-specific RNA-contact site on the surface of the bifunctional vaccinia virus RNA modifying protein VP39 that is distinct from the mRNA 5' end-binding "cleft".

VP39 is a bifunctional mRNA-modifying protein that acts as both an mRNA cap-specific 2'-O-methyltransferase and a processivity factor for VP55, the vaccinia poly(A) polymerase catalytic subunit. Although regions of the protein surface required for methyltransferase function are well defined, it has been unclear whether the protein polyadenylylation function requires direct RNA contact and, if so, where the contact site(s) might be located on the protein surface. Here, we show that the VP55-VP39 heterodimer forms a stable complex with a 50mer oligonucleotide bearing a U2-N25-U motif, as opposed to the U2-N15-U motif that is optimal for stable complex formation with VP55 alone. An oligonucleotide bearing a U2-N25-U motif in which the downstream U residue is replaced with 4thioU can be efficiently photocrosslinked to VP39, but only in the context of the VP55-VP39 heterodimer. By partial proteolysis of end-labeled VP39, the site of oligonucleotide photocrosslinking was localized to the region of VP39 between residues Lys90 and Arg122. Peptide microsequencing and confirmatory mutagenesis identified the side-chain of Arg107 as the photocrosslinking site. Substitution of this residue with lysine abolished photocrosslinking entirely, consistent with the established RNA binding role of arginine in other RNA-binding proteins. This study provides clear evidence for a polyadenylylation-specific RNA-contact site on the surface of VP39, which is distinct from the RNA-binding methyltransferase "cleft" characterized in recent crystallographic and biochemical studies.

Specificity of Tn5 insertions into a 36-bp DNA sequence repeated in tandem seven times.

The target junction sequences of six independent Tn5 insertions into a 36-bp tandemly repeated DNA segment have been determined. In all instances Tn5 preferentially inserts near one end of the tandem repeat, but in four out of six cases the insertion is between different nucleotides. The target sequence shares some similarity (8 out of 11 bp) with the ends of Tn5. All six insertions are accompanied by duplication of 9 bp of target DNA. The data imply that, even though Tn5 appears to insert randomly on a macro scale, at the nucleotide sequence level insertion into target DNA, which has limited similarity to the Tn5 end reactive sequences, may be a preferred event.

Stable chelating linkage for reversible immobilization of oligohistidine tagged proteins in the BIAcore surface plasmon resonance detector.

We describe a stable chelating linkage for the reversible immobilization of oligohistidine tagged proteins in the flow cell of the 'BIAcore' surface plasmon resonance (SPR) biosensor. The carboxymethylated dextran surface of the flow cell was covalently derivatized with N-(5-amino-1-carboxypentyl)iminodiacetic acid (NTA ligand) via its single primary amino group, and the derivatized surface charged with Ni2+. 6His-VP55, an N-terminally tagged derivative of the catalytic subunit of the heterodimeric vaccinia virus poly(A) polymerase, was immobilized to this surface in a manner that was dependent upon the immobilized NTA ligand, the prior injection of Ni2+ at a concentration of > 10(-5) M and the 6His tag, and which was reversible upon injection of EDTA. The stability of immobilization varied inversely with the amount of 6His-VP55 immobilized and was greatest in buffer of pH 8.0 or greater, containing NaCl at a concentration of 0.1 M. Utilizing these conditions, 6His-VP55 remained stably immobilized during 60 min of buffer flow at moderate flow rates. VP39, the stimulatory subunit of vaccinia poly(A) polymerase, interacted with the immobilized 6His-VP55. Approximately 99% of immobilized 6His-VP55 molecules were available for VP39 binding, in contrast to the approximately 40% availability for 6His-VP55 molecules immobilized covalently, via primary amino groups. Three additional proteins, tagged at either the N- or C-terminus with oligohistidine, were shown to be stably immobilized via the chelating linkage. This simple method permits immobilization of proteins in the BIAcore biosensor via a commonly employed affinity tag, in a stable and reversible manner, and requires only a single biosensor flow cell for the iterative generation of immobilized protein surfaces.

Mitochondrial protein synthesis in Plasmodium falciparum.

Protein synthesis in intact Plasmodium falciparum was 333 times more sensitive to cycloheximide than to chloramphenicol. The 50% inhibitory concentration (IC50) of cycloheximide in a 27-h assay in vitro was 6 X 10(-7) M but no constant cycloheximide-insensitive fraction of total protein synthesis was observed at concentrations of this inhibitor between 10(-7) and 10(-2) M. 0.24% of total protein synthesis occurred in the presence of 10(-3) M cycloheximide but the chloramphenicol sensitivity of this fraction was similar to that of overall protein synthesis (IC50 2 X 10(-4) M). The major fraction of protein synthesis by P. falciparum, therefore, is assumed to be cytoplasmic and to occur on 80S ribosomes. Cycloheximide-insensitive, chloramphenicol-sensitive (70S ribosomal) protein synthesis being undetectable by the methods employed, mitochondrial protein synthesis in P. falciparum is presumed to constitute a considerably smaller fraction of the total protein synthetic capacity than observed in other lower eukaryotes.

Use of vaccinia virus poly(A) polymerase for RNA 3'-end labeling with a chain-terminating nucleotide or a short 3' homopolymer tract.

Conditions are described for the 3'-end labeling of RNA with 32P 3'-dATP (3'-deoxyadenosine-5'-triphosphate), a chain-terminating nucleotide, using the poly(A) polymerase (PAP) encoded by vaccinia virus. Reaction time, divalent cation species and concentration, and the requirement for both subunits of the PAP were investigated. In the presence of Mn2+, vaccinia PAP is able to tail RNA primers with tracts of 3'-oligo(U), oligo(C) and oligo(G). Conditions for the addition of labeled 3'-homopolymer tracts were characterized. The use of low nucleotide concentrations in this study revealed an apparently fixed divalent cation concentration optimum of 0.1 mM, distinct from the previously noted requirement for a 1:1 divalent cation:NTP complex. This indicates a possible requirement for multiple divalent cations in nucleotidyl transfer by vaccinia PAP.

Effects of extended periods of reserpine and α-methyl-p-tyrosine treatment on the development of the putamen in fetal rabbits.

Developing nigrostriatal neuroblasts exhibit catecholamine-induced fluorescence before their axons have left the vicinity of the cell bodies. To evaluate possible developmental effects of dopamine, we have used reserpine and α-methyl-p-tyrosine to deplete dopamine chronically during the development of these axons. We found that dopamine-induced fluorescence was either absent or markedly decreased in the fetal putamens. To determine whether the absence of fluorescence was due to a reduction of dopamine terminals, the uptake of tritium-labeled dopamine was measured in the putamen. Uptake of labeled dopamine was significantly depressed in reserpine-treated fetuses to 70% of that of controls; however, no depression of labeled dopamine was found in the α-methyl-p-tyrosine-treated fetuses. After both drug treatments, the striatal perikarya were less mature than those of controls. Although we cannot rule out possible non-specific or toxic effects of the drugs, these observations support the conclusion that presynaptic dopamine may be important for development of target neurons in the neostriatum.

Synergy of four macrolide antibiotics with chloroquine against chloroquine-resistant Plasmodium falciparum in vitro.

The antimalarial activity of four macrolide antibiotics was investigated against the multidrug resistant K1 strain of Plasmodium falciparum in vitro. ID50 (50% inhibitory concentration) values for erythromycin, spiramycin, tylosin tartrate and oleandomycin phosphate in 48-hour assays were 1.6 X 10(-4)M, 2.5 X 10(-5)M, 1.2 X 10(-5)M and 9 X 10(-6)M respectively, and in 96 hour assays were 10(-5)M, 2.6 X 10(-6)M, 2.6 X 10(-6) and 3 X 10(-6)M, respectively. Comparable values were obtained in assays in which drug effect was quantified from either parasite counts or 14C isoleucine incorporation. Each of the four macrolides displayed synergy with chloroquine at the IC90 (90% inhibitory concentration) level, but at the IC50 level synergy was either less pronounced or absent. For each combination this difference in the degree of synergy was significant at the 95% level of confidence. In replicate assays in which 3H hypoxanthine was the marker of drug effect, synergy between chloroquine and either erythromycin or spiramycin could not be detected.

Effects of chronic reserpine treatment on development of maturity of the putamen in fetal rabbits.

Developing nigrostriatal axons and their perikarya have substantial quantities of dopamine (DA) before the axons reach their postsynaptic target. In order to investigate possible developmental effects of these stores of DA, we have depleted DA chronically during critical periods in the ontogeny of the nigrostriatal system. Reserpine (0.04-0.14 mg/kg/day) was given repeatedly to maternal rabbits for various periods starting before neuroblasts of the substantia nigra first exhibit fluorescence until 2 days before term when the fetuses were sacrificed. Reserpine crossed the placenta and depleted DA in the fetal putamens. Control fetuses had widespread fluorescent axons and terminals. Counts of mature axonal boutons in electron micrographs of the putamen of reserpine-treated fetuses showed that there were 4.3 +/- 0.6 SE/100 microns2, which is less than 1/2 the control value of 10.2 +/- 0.6 SE/100 microns2 (p less than 0.001). The neuropil of the putamen of the reserpine-treated fetus was also less mature; the relative volume occupied by growth cones (40.5% +/- 5.7 SE) was twice that of controls (20.6% +/- 2.4 SE) (p less than 0.005). Although it remains to be shown that the delayed development of both pre- and postsynaptic elements of the nigrostriatal system is specifically related to the known ability of reserpine to deplete DA, the results are consistent with the hypothesis that early stores of DA may be important in developing dopaminergic systems.

Helping young adults understand their learning disabilities.

This study evaluated the effectiveness of an Understanding Learning Disabilities (ULD) course designed to promote self-understanding and self-advocacy skills in young adults with learning disabilities (LD) functioning in the low-average range intellectually. Nineteen first-year students with LD at the Threshold Program at Lesley College received 15 hours of training throughout one semester. Seventeen first-year students with LD at the Para-Educator Center at New York University served as the control group. Both a paper-and-pencil questionnaire and a role-play interview were used as pre- and postassessments. Results of ANOVAs performed on posttest questionnaire and interview scores support the effectiveness of the ULD course in expanding students' knowledge base regarding their learning disabilities and teaching them to apply their self-understanding in a social context. Moreover, students' performance on the questionnaire was an effective predictor of future work adjustment 1 year later. Significant correlates of ULD performance include IQ, academic achievement, and vocational functioning.

Static and dynamic protein phosphorylation in the Vaccinia virion.

To the best of our knowledge, two phosphorylation sites have been reported previously, among 11 known Vaccinia virus phosphoproteins. Here, via phosphopeptide mass spectrometry, up to 189 phosphorylation sites were identified among 48 proteins in preparations of purified Vaccinia mature virus (MV). 8.5% of phospho-residues were pTyr. Viral phosphoproteins were found in diverse functional classes, including structural proteins, membrane proteins and RNA polymerase subunits. Among the nine identified membrane phosphoproteins, the sites in just one, namely A14L, were deduced to be internal with respect to the accompanying membrane. Examination of sites in known substrates of the Vaccinia-encoded protein kinase VPK2, indicated VPK2 to be a proline-dependent kinase. The MV phosphoproteome was enriched in potential substrates of cellular kinases belonging to the CDK2/CDK3, CK2, and p38 groups. Quantitative mass spectrometry identified several sites that became phosphorylated during intravirion kinase activation in vitro, each showing one of two distinct pH-dependency profiles.