Interactions between Ethanol, syn-2,3-Hexanediol, 3-Hydroxyhexan-2-one, and 3-Hydroxyoctan-2-one Lures on Trap Catches of Hardwood Longhorn Beetles in Southeastern United States.

The effectiveness of a four-component "super lure" consisting of ethanol (E) and the cerambycid pheromones syn-2,3-hexanediol (D6), racemic 3-hydroxyhexan-2-one (K6), and racemic 3-hydroxyoctan-2-one (K8) on trap catches of Cerambycidae (Coleoptera) was determined in southeast United States with seven trapping experiments in 2011-2013. We captured 74 species of longhorn beetles in our three-year study. Ethanol significantly increased the mean catches of seven species and increased the number of cerambycid species detected. Traps with the "super lure" were effective for 8 of 13 species of Cerambycidae previously shown to be attracted to binary combinations of ethanol plus one of the three pheromones. However, the "super lure" was less effective for the remaining five species with catch reductions of 40-90% compared with combinations of ethanol and one or two of the pheromones. For example, K6 + K8 lures reduced catches of Anelaphus villosus (F.) in traps with E + D6 by 90%. Similarly, catches of Anelaphus pumilus (Newman) in traps with E + K6 + D6 were reduced by 50% with the addition of K8. Catches of Knulliana cincta (Drury) in traps with K6 + K8 lures were interrupted by D6, an effect negated by the addition of ethanol. Given the interruptive effects on trap catches of some species when lures are combined in a single trap, developing optimal lure blends to maximize detection efficacy will be a challenge for managers of detection programs for non-native invasive species of longhorn beetles.

Pine Sawyers (Coleoptera: Cerambycidae) Attracted to α-Pinene, Monochamol, and Ipsenol in North America.

Detection tools are needed for Monochamus species (Coleoptera: Cerambycidae) because they are known to introduce pine wilt disease by vectoring nematodes in Asia, Europe, and North America. In 2012-2014, we examined the effects of the semiochemicals monochamol and ipsenol on the flight responses of the sawyer beetles Monochamus carolinensis (Olivier), Monochamus clamator (LeConte), Monochamus mutator LeConte, Monochamus notatus (Drury), Monochamus obtusus Casey, Monochamus scutellatus (Say), and Monochamus titillator (F.) complex (Coleoptera: Cerambycidae) to traps baited with α-pinene. Experiments were set in pine forests in New Brunswick and Ontario (Canada), and Arizona, Georgia, Michigan, Montana, Oregon, South Carolina, Utah, and Washington (United States). In brief, 40 traps were placed in 10 blocks of 4 traps per block per location. Traps were baited with: 1) α-pinene; 2) α-pinene + monochamol; 3) α-pinene + ipsenol; and 4) α-pinene + monochamol + ipsenol. Monochamol increased catches of six species and one species complex of Monochamus with an additive effect of ipsenol for five species and one species complex. There was no evidence of synergy between monochamol and ipsenol on beetle catches. Monochamol had no effect on catches of other Cerambycidae or on any associated species of bark beetles, weevils, or bark beetle predators. We present a robust data set suggesting that the combination of α-pinene, ipsenol, and monochamol may be a useful lure for detecting Monochamus species.

The effects of organic nitrates on osteoporosis: a systematic review.

Current treatments for osteoporosis are limited by lack of effect on cortical bone, side effects, and, in some cases, cost. Organic nitrates, which act as nitric oxide donors, may be a potential alternative. This systematic review summarizes the clinical data that reports on the effects of organic nitrates and bone. Organic nitrates, which act as nitric oxide donors, are novel agents that have several advantages over the currently available treatments for osteoporosis. This systematic review summarizes the clinical data that reports on the effects of organic nitrates on bone. We searched Medline (1966 to November 2012), EMBASE (1980 to November 2012), and the Cochrane Central Register of Controlled Trials (Issue 11, 2012). Keywords included nitrates, osteoporosis, bone mineral density (BMD), and fractures. We identified 200 citations. Of these, a total of 29 were retrieved for more detailed evaluation and we excluded 19 manuscripts: 15 because they did not present original data and four because they did not provide data on the intervention or outcome of interest. As such, we included ten studies in literature review. Of these ten studies two were observational cohort studies reporting nitrate use was associated with increased BMD; two were case control studies reporting that use of nitrates were associated with lower risk of hip fracture; two were randomized controlled trials (RCT) comparing alendronate to organic nitrates for treatment of postmenopausal women and demonstrating that both agents increased lumbar spine BMD. The two largest RCT with the longest follow-up, both of which compared effects of organic nitrates to placebo on BMD in women without osteoporosis, reported conflicting results. Headaches were the most common adverse event among women taking nitrates. No studies have reported on fracture efficacy. Further research is needed before recommending organic nitrates for the treatment of postmenopausal osteoporosis.

Susceptibility to Laurel Wilt and Disease Incidence in Two Rare Plant Species, Pondberry and Pondspice.

Laurel wilt, caused by Raffaelea lauricola, has been responsible for extensive losses of redbay (Persea borbonia) in South Carolina and Georgia since 2003. Symptoms of the disease have been noted in other species of the Lauraceae such as the federally endangered pondberry (Lindera melissifolia) and the threatened pondspice (Litsea aestivalis). Pondberry and pondspice seedlings were inoculated with R. lauricola from redbay, and both species proved highly susceptible to laurel wilt. Field assessments found substantial mortality of pondberry and pondspice, but in many cases the losses were not attributable to laurel wilt. R. lauricola was isolated from only 4 of 29 symptomatic pondberry plants at one site, but the fungus was not recovered from three plants at another site. R. lauricola was isolated from one of two symptomatic pondspice plants at one site, and from five of 11 plants at another site, but not from any plant at a third site. Insect bore holes, similar to those produced by Xyleborus glabratus (the vector of laurel wilt), were found in some pondberry and pondspice stems, but adults were not found. Damage caused by Xylosandrus compactus was found in pondberry stems, but this ambrosia beetle does not appear to be a vector of R. lauricola. Xyleborinus saxeseni adults were found in a dying pondspice with laurel wilt, and R. lauricola was recovered from two of three adults. Isolates of R. lauricola from pondberry, pondspice, and X. saxeseni had rDNA sequences that were identical to previously characterized isolates, and inoculation tests confirmed that they were pathogenic to redbay. Because pondberry and pondspice tend to be shrubby plants with small stem diameters, these species may not be frequently attacked by X. glabratus unless in close proximity to larger diameter redbay.

Insertion into Aspergillus nidulans of functional UDP-GlcNAc: alpha 3-D- mannoside beta-1,2-N-acetylglucosaminyl-transferase I, the enzyme catalysing the first committed step from oligomannose to hybrid and complex N-glycans.

Filamentous fungi are capable of secreting relatively large amounts of heterologous recombinant proteins. Recombinant human glycoproteins expressed in this system, however, carry only carbohydrates of the oligomannose type limiting their potential use in humans. One approach to the problem is genetic engineering of the fungal host to permit production of complex and hybrid N-glycans. UDP-GlcNAc:alpha 3-D-mannoside beta- 1,2-N-acetylglucosaminyltransferase I (GnT I) is essential for the conversion of oligomannose to hybrid and complex N-glycans in higher eukaryotic cells. Since GnT I is not produced by fungi, we have introduced into the genome of Aspergillus nidulans the gene encoding full-length rabbit GnT I and demonstrated the expression of GnT I enzyme activity at levels appreciably higher than occurs in most mammalian tissues. All the GnT I activity in the Aspergillus transformants remains intracellular suggesting that the rabbit trans-membrane sequence may be capable of targeting GnT I to the fungal Golgi apparatus.

Design and synthesis of RNA miniduplexes via a synthetic linker approach.

Double-stranded oligodeoxyribonucleotides or single-stranded oligoribonucleotides with specific secondary structure have been proposed as potential antagonists to target nucleic acid-binding proteins (the sense approach). A major limitation of this strategy is that these derivatives are generally considered to be too large for pharmaceutical applications. We have developed a synthetic linker approach whereby nucleic acid duplexes of a much smaller size (miniduplexes) can be generated directly from a standard oligonucleotide synthesis. In this approach, four synthetic linkers (derivatized respectively from 1,9-nonanediol, triethylene glycol, 1,3-propanediol, and hexaethylene glycol) of different length and hydrophobicity were designed and incorporated into a model RNA molecule based on the TAR stem-loop structure of HIV-1. Their thermal stabilities were evaluated by measuring denaturation profiles (Tm measurements). These linker-derivatized RNA molecules were then assessed for their ability to bind to either a full-length protein (HIV-1 Tat protein) or a short peptide (Tat-derived peptide) through RNA mobility shift assays. Results from this study indicate that such modified miniduplex structures retain full binding activity relative to that of the wild-type sequence (Kd values), while Tm values were increased by 24-31 degrees C compared to an open duplex of the same length. This system provides a new direction in the use of nucleic acid miniduplexes as a novel class of oligonucleotide analogues for both fundamental research and possible therapeutic applications.

Conserved nucleotides in the TAR RNA stem of human immunodeficiency virus type 1 are critical for Tat binding and trans activation: model for TAR RNA tertiary structure.

Interaction between the human immunodeficiency virus type 1 (HIV-1) trans-activator Tat and its cis-acting responsive RNA element TAR is necessary for activation of HIV-1 gene expression. We investigated the hypothesis that the essential uridine residue at position 23 in the bulge of TAR RNA is involved in intramolecular hydrogen bonding to stabilize an unique RNA structure required for recognition by Tat. Nucleotide substitutions in the two base pairs of the TAR stem directly above the essential trinucleotide bulge that maintain base pairing but change sequence prevent complex formation with Tat in vitro. Corresponding mutations tested in a trans-activation assay strongly affect the biological activity of TAR in vivo, suggesting an important role for these nucleotides in the Tat-TAR interaction. On the basis of these data, a model is proposed which implicates uridine 23 in a stable tertiary interaction with the GC pair directly above the bulge. This interaction would cause widening of the major groove of the RNA, thereby exposing its hydrogen-bonding surfaces for possible interaction with Tat. The model also predicts a gap between uridine 23 and the first base pair in the stem above, which would require one or more unpaired nucleotides to close, but does not predict any other role for such nucleotides. In accordance with this prediction, synthetic propyl phosphate linkers of equivalent length to 1 or 2 nucleotides, were found to be fully acceptable substitutes in the bulge above uridine 23, demonstrating that neither the bases nor the ribose moieties at these positions are implicated in the recognition of TAR RNA by Tat.

Critical chemical features in trans-acting-responsive RNA are required for interaction with human immunodeficiency virus type 1 Tat protein.

The human immunodeficiency virus type 1 Tat protein binds to an RNA stem-loop structure called TAR which is present at the 5' end of all human immunodeficiency virus type 1 transcripts. This binding is centered on a bulge within the stem of TAR and is an essential step in the trans-activation process which results in a dramatic increase in viral gene expression. By analysis of a series of TAR derivatives produced by transcription or direct chemical synthesis, we determined the structural and chemical requirements for Tat binding. Tat binds well to structures which have a bulge of two to at least five unpaired bases bounded on both sides by a double-stranded RNA stem. This apparent flexibility in bulge size is in contrast to an absolute requirement for an unpaired uridine (U) in the 5'-most position of the bulge (+23). Substitution of the U with either natural bases or chemical analogs demonstrated that the imido group at the N-3 position and, possibly, the carbonyl group at the C-4 position of U are critical for Tat binding. Cytosine (C), which differs from U at only these positions, is not an acceptable substitute. Furthermore, methylation at N-3 abolishes binding. While methylation of U at the C-5 position has little effect on binding, fluorination reduces it, possibly because of its effects on relative tautomer stability at the N-3 and C-4 positions. Thus, we have identified key moieties in the U residue that are of importance for the binding of Tat to TAR RNA. We hypothesize that the invariant U is involved in hydrogen bond interactions with either another part of TAR or the TAR-binding domain in Tat.

Rebuilding flavodoxin from C alpha coordinates: a test study.

The tertiary structure of flavodoxin has been model built from only the X-ray crystallographic alpha-carbon coordinates. Main-chain atoms were generated from a dictionary of backbone structures. Side-chain conformations were initially set according to observed statistical distributions, clashes were resolved with reference to other knowledge-based parameters, and finally, energy minimization was applied. The RMSD of the model was 1.7 A across all atoms to the native structure. Regular secondary structural elements were modeled more accurately than other regions. About 40% of the chi 1 torsional angles were modeled correctly. Packing of side chains in the core was energetically stable but diverged significantly from the native structure in some regions. The modeling of protein structures is increasing in popularity but relatively few checks have been applied to determine the accuracy of the approach. In this work a variety of parameters have been examined. It was found that close contacts, and hydrogen-bonding patterns could identify poorly packed residues. These tests, however, did not indicate which residues had a conformation different from the native structure or how to move such residues to bring them into agreement. To assist in the modeling of interacting side chains a database of known interactions has been prepared.

Electron transfer from cytochrome b5 to iron and copper complexes.

The rates of electron transfer from the tryptic fragment of bovine liver cytochrome b5 to FeIIINTA, FeIIIATP, CuIINTA, CuIIATP, and CuIIHis have been measured by anaerobic stopped-flow techniques. The rates of reduction of the Fe(III) complexes are independent of ionic strength, enhanced at low pH, and slightly inhibited by ZnIINTA. Saturation kinetics are observed with CuIINTA (kappa et = 0.05 s-1, K = 8.6 M-1), CuIIHis (kappa et = 0.2 s-1, K = 2.6 X 10(3) M-1), and CuIIATP (kappa et = 0.6 s-1, K = 4.5 X 10(3) M-1), thereby indicating that binding of Cu(II) to the protein occurs prior to electron transfer. 1H NMR resonances of the three surface histidines and some neighboring residues have been assigned by two-dimensional NMR techniques. NMR titration experiments show unequivocally that CuIINTA binds preferentially at a site near His-26 and Tyr-27.

Kinetics of hemoprotein reduction and interprotein heme transfer.

The transfer of hemin from one protein to another is an event biologically important for the conservation of heme iron. Hemin entering the circulation (or added to serum) is mainly bound by albumin and then transferred to hemopexin [Morgan, W.T., Liem, H.H., Sutor, R.P., & Muller-Eberhard, U. (1976) Biochim. Biophys. Acta 444, 435-445], and we are now investigating which mechanisms may be operative in enhancing this process. The presence of imidazole has been demonstrated to accelerate hemin transfer from albumin to hemopexin [Pasternack, R.F., Gibbs, E.J., Hoeflin, E., Kosar, W.P., Kubera, G., Skowronek, C. A., Wong, N.M., & Muller-Eberhard, U. (1983) Biochemistry 22, 1753-1758]. The present work is an examination of the effect of the reduction of albumin-bound hemin on the rate of its transfer to hemopexin. Hemin (HmIII., ferriprotoporphyrin IX) was reduced to HmII (ferroprotoporphyrin IX) by the addition of sodium dithionite under argon. The reduction kinetics of HmIII to HmII were studied separately in the two complexes: with human serum albumin (HSA), which binds up to 20 mol of heme/mol (the first mole with K congruent to 10(8)), and with hemopexin (HHx), which binds heme equimolarly (K congruent to 10(13)). The rate of reduction of HmIII to HmII on HSA was first order over several half-lives and linearly dependent on [S2O4(2-)]1/2. At [HSA]0/[HmIII] = 3, the kobsd was (5 X 10(-3) + 0.75[S2O4(2-)]1/2, and with [HSA]/[HmIII] approximately 25, the kobsd was (2 X 10(-3)) + 0.25[S2O4(2-)]1/2. The reduction of HmIII to HmII on human hemopexin (HHx) is much more rapid with kobsd = (2.5 X 10(3))[S2O4(2-)]1/2.(ABSTRACT TRUNCATED AT 250 WORDS)

Conversion of oxyhaemoglobin into methaemoglobin by ferricytochrome b5.

Ferricytochrome b5 was found to convert oxyhaemoglobin into methaemoglobin under conditions previously found to be optimal for complex-formation between ferricytochrome b5 and methaemoglobin [Mauk & Mauk (1982) Biochemistry 21, 4730-4734]. As this reaction is completely inhibited by CO, it is proposed that oxyhaemoglobin is oxidized after O2 dissociation, as has been suggested for the oxidation of oxyhaemoglobin by inorganic complexes. From the present analysis, ferricytochrome b5 seems unlikely to contribute significantly to methaemoglobin formation in vivo. Nevertheless, this observation provides a relatively convenient means of investigating the mechanism by which these two proteins interact.

Spectrophotometric analysis of the interaction between cytochrome b5 and cytochrome c.

The interaction between cytochrome c and the tryptic fragment of cytochrome b5 has been found to produce a difference spectrum in the Soret region with a maximum absorbance at 416 nm. The intensity of this difference has been used to determine the stoichiometry of complex formation and the stability of the complex formed. At pH 7.0 [25 degrees C (phosphate), mu = 0.01 M], the two proteins were found to form a 1:1 complex with an association constant, KA, of 8(3) x 10(4) M-1. The stability of the complex was found to be strongly dependent on ionic strength with KA increasing to 4(3) x 10(6) M-1 at mu = 0.001 M [25 degrees C, pH 7.0 (phosphate)]. Analysis of the dependence of KA on pH from pH 6.5 to 8 demonstrated that this complex is maximally stable between pH 7 and 8 or about midway between the isoelectric points of the two proteins. Analysis of the temperature dependence of KA revealed that formation of the complex between the two proteins is largely entropic in origin with delta Ho = 1 +/- 3 kcal/mol and delta So = 33 +/- 11 eu [pH 7.0 (phosphate), mu = 0.001 M]. This result may be explained either by the model of Clothia and Janin [Clothia, C., & Janin, J. (1975) Nature (London) 256, 705] in terms of extensive solvent reorganization upon complexation or by the model of Ross and Subramanian [Ross, P. D., & Subramanian, S. (1981) Biochemistry 20, 3096] in which the negative enthalpic and entropic contributions of short-range protein-protein interactions are offset by proton release.