Quantification of two viruses in technical preparations of Orgyia pseudotsugata baculovirus by means of buoyant density centrifugation of viral deoxyribonucleic acid.

A reliable method was developed for the quantitative determination of two nuclear polyhcdrosis viruses present in commercially prepared viral insecticides used against Orgyia pseudotsugata. Deoxyribonucleic acids, from nuclear polyhedrosis bundle virus and nuclear polyhedrosis single-rod virus, were separated on CsCl gradients according to their respective buoyant densities, 1.715 and 1.704 g/ml. The proportions of the two viruses were quantified by measuring the relative absorbance at 254 nm of their deoxyribonucleic acid peaks.     

Mapping unprocessed epitopes using deletion mutagenesis of gene fusions.

To locate the antigenic determinant recognized by a monoclonal antibody directed against a baculovirus capsid protein, a series of overlapping deletions of a fusion protein were immunologically screened with the monoclonal antibody. The immunoreactive fusion protein was derived from a restriction fragment which contained a large portion of a baculovirus capsid protein open reading frame fused in-frame with a truncated trpE gene in a bacterial (pATH3) expression system. To map the epitope, nested sets of 5' and 3' deletion mutants were generated. Mutants were characterized by the DNA insert size or by the size of the expressed fusion protein. Selected N- and C-termini truncated fusion proteins were Western blotted and incubated with the monoclonal antibody to identify mutants which retained the epitope. Plasmid DNA from mutants which flank the 5' and 3' junction of the antigenic determinant were sequenced to determine the epitope junction. By screening forty 3' deletions and sixty-four 5' deletions, the antigenic determinant was localized to a region of seven amino acids.     

A 37-kilodalton glycoprotein from a baculovirus of Orgyia pseudotsugata is localized to cytoplasmic inclusion bodies.

The gene encoding a 37-kDa glycoprotein (gp37) of Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) was located and sequenced. gp37 of OpMNPV was found to have 62 and 37% amino acid sequence identity with gp37 of Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) and with a protein reported to be a component of occlusion bodies from Choristoneura biennis entomopoxvirus, respectively. The mRNA start site of the OpMNPV gp37 gene was mapped within a late promoter sequence (TTAAG). A TrpE fusion protein containing 55% of the OpMNPV gp37 gene amino acid sequence was used to generate a monospecific antiserum. Western immunoblot analysis of OpMNPV-infected Lymantria dispar cells detected gp37 beginning at 24 h postinfection. Immunoelectron microscopy indicated that the protein is concentrated in cytoplasmic inclusion bodies late in infection. In contrast to gp37 of AcMNPV which was present in the matrix of occlusion bodies, OpMNPV gp37 was not observed in this location. Neither OpMNPV nor AcMNPV gp37 was associated with the polyhedron envelope.     

Characterization of DNA from three nuclear polyhedrosis viruses pathogenic for Choristoneura sp

Three nuclear polyhedrosis viruses isolated from larvae of the insect genus Choristoneura showed polyhedrins of 28–30,000 daltons, genome sizes of 78–82 × 10⁶ daltons, and guanine plus cytosine contents of 47.9–49.4%. It was demonstrated by comparison of restriction endonuclease fragment patterns that two of the viruses are closely related genetically.     

Quantification of two viruses in technical preparations of Orgyia pseudotsugata baculovirus by means of buoyant density centrifugation of viral deoxyribonucleic acid.

A reliable method was developed for the quantitative determination of two nuclear polyhcdrosis viruses present in commercially prepared viral insecticides used against Orgyia pseudotsugata. Deoxyribonucleic acids, from nuclear polyhedrosis bundle virus and nuclear polyhedrosis single-rod virus, were separated on CsCl gradients according to their respective buoyant densities, 1.715 and 1.704 g/ml. The proportions of the two viruses were quantified by measuring the relative absorbance at 254 nm of their deoxyribonucleic acid peaks.     

Binding of the baculovirus very late expression factor 1 (VLF-1) to different DNA structures

Background

Baculovirus genomes encode a gene called very late expression factor 1 (VLF-1) that is a member of the integrase (Int) family of proteins. In this report we describe the binding properties of purified Autographa californica multiple capsid nucleopolyhedrovirus (AcMNPV) VLF-1 to a number of different DNA structures including homologous regions. In addition, its enzymatic activity was examined.

Results

VLF-1 was expressed in a recombinant baculovirus as a fusion with both HA and HIS₆ tags and its binding activity to different DNA structures was tested. No binding was evident to single and double strand structures, very low binding was observed to Y-forks, more binding was observed to three-way junctions, whereas cruciform structures showed high levels of binding. VLF-1 binding was affected by divalent cations; optimal binding to three-way junctions and cruciforms was 2 and 0 mM MgCl₂, respectively. Homologous region (hr) sequences was also examined including oligomers designed to expose the hr palindrome as a hairpin, linear double strand, or H-shaped structure. Efficient binding was observed to the hairpin and H-shaped structure. No topoisomerase or endonuclease activity was detected. Sedimentation analysis indicated that *VLF-1 is present as a monomer.

Conclusions

An HA- and HIS-tagged version of AcMNPV VLF-1 showed structure-dependent binding to DNA substrates with the highest binding affinity to cruciform DNA. These results are consistent with the involvement of VLF-1 in the processing of branched DNA molecules at the late stages of viral genome replication. We were unable to detect enzymatic activity associated with these complexes.

     

Relationship of Serum Vitamin D Concentrations and Allostatic Load as a Measure of Cumulative Biological Risk among the US Population: A Cross-Sectional Study

IntroductionThe allostatic load (AL) index is a multi-systemic measure of physiologic dysregulation known to be associated with chronic exposure to stress and adverse health outcomes. We examined the relationship between AL and serum 25-hydroxyvitamin D (25(OH)D) concentration in non-institutionalized US adults.MethodsData from the Third National Health and Nutrition Examination Survey (NHANES III, 1988–94) were used to calculate two versions of AL including 9 biomarkers and another two with 14 biomarkers (systolic and diastolic blood pressure, pulse rate, serum cholesterol, serum HDL-cholesterol, glycated hemoglobin, sex-specific waist-to-hip ratio, serum albumin, and serum C-reactive protein for AL1, and, additionally body mass index, serum triglyceride, serum creatinine, and serum herpes I & II antibodies for AL2), each set defined by predefined cut-offs or by quartiles. Serum vitamin D concentration was ranked into quartiles. Logistic regression, Poisson regression and linear regression were used to examine the association of serum 25(OH)D concentrations on AL, after adjusting for biological, physiological, socioeconomic, lifestyle, and health variables.ResultsOdds Ratios (OR) for high AL of the lowest 25(OH)D serum quartile were between 1.45 (95% CI: 1.28, 1.67) and 1.79 (95% CI: 1.39, 2.32) for the fully adjusted model, depending on AL version. Inverse relationships between vitamin D serum concentrations were observed for all AL versions and every adjustment. This relationship was consistent after stratification by sex, age or ethnic background. Sensitivity to low 25(OH)D concentrations was highest among the youngest group (20–39 years) with an OR of 2.11 (95% CI: 1.63, 2.73) for the lowest vitamin D quartile Q1.ConclusionsVitamin D had a consistent and statistically significant inverse association with all tested models of high AL, which remained consistent after adjusting for biological, socioeconomic, lifestyle and health variables. Our study adds evidence linking low 25(OH)D concentrations with poorer health, further-reaching than bone health.     

The redox state of the baculovirus single-stranded DNA-binding protein LEF-3 regulates its DNA binding, unwinding, and annealing activities

The single-stranded (ss) DNA-binding protein LEF-3 of Autographa californica multinucleocapsid nucleopolyhedrovirus promoted Mg(2+)-independent unwinding of DNA duplexes and annealing of complementary DNA strands. The unwinding and annealing activities of LEF-3 appeared to act in a competitive manner and were determined by the ratio of protein to DNA. At subsaturating and saturating concentrations, LEF-3 promoted annealing, whereas it promoted unwinding at oversaturation of DNA substrates. The LEF-3 binding to ssDNA and unwinding activity were sensitive to redox agents and were inhibited by oxidation of thiol groups in LEF-3 with 1,1'-azobis(N,N-dimethylformamide) (diamide) or by modification with the thiol-conjugating agent N-ethylmaleimide. Both oxidation and alkylation increased the dissociation constant of the interaction with model oligonucleotides indicating a decrease in an intrinsic affinity of LEF-3 for ssDNA. These results proved that free thiol groups are essential both for LEF-3 interaction with ssDNA and for DNA unwinding. In contrast, oxidation or modification of thiol groups stimulated the annealing activity of LEF-3 partially due to suppression of its unwinding activity. Treatment of LEF-3 with the reducing agent dithiothreitol inhibited annealing, indicating association of this activity with the oxidized protein. Thus, the balance between annealing and unwinding activities of LEF-3 was determined by the redox state of protein with the oxidized state favoring annealing and the reduced state favoring unwinding. An LEF-3 mutant in which the conservative cysteine Cys(214) was replaced with serine showed both a decreased binding to DNA and a reduced unwinding activity, thus indicating that this residue might participate in the regulation of LEF-3 activities.     

Decreasing the Mitochondrial Synthesis of Malate in Potato Tubers Does Not Affect Plastidial Starch Synthesis, Suggesting That the Physiological Regulation of ADPglucose Pyrophosphorylase Is Context Dependent

Modulation of the malate content of tomato (Solanum lycopersicum) fruit by altering the expression of mitochondrially localized enzymes of the tricarboxylic acid cycle resulted in enhanced transitory starch accumulation and subsequent effects on postharvest fruit physiology. In this study, we assessed whether such a manipulation would similarly affect starch biosynthesis in an organ that displays a linear, as opposed to a transient, kinetic of starch accumulation. For this purpose, we used RNA interference to down-regulate the expression of fumarase in potato (Solanum tuberosum) under the control of the tuber-specific B33 promoter. Despite displaying similar reductions in both fumarase activity and malate content as observed in tomato fruit expressing the same construct, the resultant transformants were neither characterized by an increased flux to, or accumulation of, starch, nor by alteration in yield parameters. Since the effect in tomato was mechanistically linked to derepression of the reaction catalyzed by ADP-glucose pyrophosphorylase, we evaluated whether the lack of effect on starch biosynthesis was due to differences in enzymatic properties of the enzyme from potato and tomato or rather due to differential subcellular compartmentation of reductant in the different organs. The results are discussed in the context both of current models of metabolic compartmentation and engineering.Starch is the most important carbohydrate used for food and feed purposes and represents the major resource for our diet (Smith, 2008). The total yield of starch in rice (Oryza sativa), corn (Zea mays), wheat (Triticum aestivum), and potato (Solanum tuberosum) exceeds 10⁹ tons per year (Kossmann and Lloyd, 2000; Slattery et al., 2000). In addition to its use in a nonprocessed form, extracted starch is processed in many different ways, for instance as a high-Fru syrup, as a food additive, or for various technical purposes. As a result of this considerable importance, increasing the starch content of plant tissues has been a major goal for many years, with both classical breeding and biotechnological approaches being taken extensively over the last few decades (Martin and Smith, 1995; Regierer et al., 2002).The pathway by which carbon is converted from Suc to starch in the potato tuber is well established (Kruger, 1997; Fernie et al., 2002; Geigenberger et al., 2004; Geigenberger, 2011). Imported Suc is cleaved in the cytosol by Suc synthase, resulting in the formation of UDP-Glc and Fru; the UDP-Glc is subsequently converted to Glc-1-P by UDP-Glc pyrophosphorylase. The second product of the Suc synthase reaction, Fru, is efficiently phosphorylated to Fru-6-P by fructokinase (Renz et al., 1993; Davies et al., 2005). Fru-6-P is freely converted to Glc-6-P, in which form it normally enters the amyloplast (Kammerer et al., 1998; Tauberger et al., 2000; Zhang et al., 2008), and once in the plastid, it is converted to starch via the concerted action of plastidial phosphoglucomutase, ADP-Glc pyrophosphorylase (AGPase), and the various isoforms of starch synthase (Martin and Smith, 1995; Geigenberger, 2011). Of these reactions, although some of the control of starch synthesis resides in the plastidial phosphoglucomutase reaction (Fernie et al., 2001b), the AGPase reaction harbors the highest proportion of control within the linear pathway (Sweetlove et al., 1999; Geigenberger et al., 1999, 2004). In addition, considerable control resides in both the Glc-6-P phosphate antiporter (Zhang et al., 2008) and the amyloplastidial adenylate transporter (Tjaden et al., 1998; Zhang et al., 2008) as well as in reactions external to the pathways, such as the amyloplastidial adenylate kinase (Regierer et al., 2002), cytosolic UMP synthase (Geigenberger et al., 2005), and mitochondrial NAD-malic enzyme (Jenner et al., 2001).As part of our ongoing study of the constituent enzymes of the tricarboxylic acid (TCA) cycle, we made an initially surprising observation that increasing or decreasing the content of malate via a fruit-specific expression of antisense constructs targeted against the mitochondrial malate dehydrogenase or fumarase, respectively, resulted in opposing changes in the levels of starch (Centeno et al., 2011). We were able to demonstrate that these plants were characterized by an altered cellular redox balance and that this led to changes in the activation state of the AGPase reaction. Given that starch only accumulates transiently in tomato (Solanum lycopersicum; Beckles et al., 2001) as a consequence of this activation, the fruits were characterized by altered sugar content at ripening, a fact that dramatically altered their postharvest characteristics (Centeno et al., 2011). Here, we chose to express the antisense fumarase construct in potato in order to ascertain the effect of the manipulation in an organ that linearly accumulates starch across its development. The results obtained are compared and contrasted with those of the tomato fruit and within the context of current models of subcellular redox regulation.