We investigated two strains of uricase (+) Enterobacter agglomerans, one isolated from the apple maggot fly (AMF) and one from the Mexican fruit fly (MFF), for 1) attractiveness to MFF, and 2) production of attractive chemicals. Regarding chemicals demonstrated attractive to the MFF, the MFF bacterial strain produced more 2,5-dimethylpyrazine, 2-phenylethanol, and indole than the AMF strain, whereas the AMF, but not the MFF strain, produced 3-hydroxybutanone. Cell types that predominated in plated subcultures varied from batch to batch resulting in variation in volatiles production, especially by the AMF strain where indole was sometimes a major component of the odor and at other times not detectable. Despite the greater production of attractive chemicals by the MFF strain, the AMF strain was consistently more attractive and the MFF strain was not different from uninoculated control plates. Statistical analyses indicated negative correlations of attractiveness with production of indole, 2,5-dimethylpyrazine, and 2-phenylethanol, and positive correlation with 3-hydroxybutanone. Results support previous findings with the Mexican fruit fly that showed combinations of attractive chemicals sometimes are not attractive.
A herpes simplex virus type 1 (HSV-1) Ori S analogue in which the A؉T sequence linking the box I and II elements was replaced by two single-stranded oligo(dT)s is unwound by the UL9 protein-ICP8 complex. Unwinding of wild-type Ori S by the UL9 protein-ICP8 complex was also observed under conditions which destabilize the A؉T sequence. These experiments support a model for the unwinding of Ori S in which destabilization of the A؉T sequence can generate a single-stranded DNA binding site for ICP8, which then associates with the UL9 protein bound to boxes I and II to promote the bidirectional unwinding of Ori S .Herpes simplex virus type 1 (HSV-1) encodes a 94-kDa origin binding protein, the product of the UL9 gene, which is essential for HSV-1 DNA replication (5, 10). The origin binding protein (UL9 protein) is a homodimer that binds the two inverted pentanucleotide repeats, boxes I and II of the HSV-1 origin of replication, Ori S . Boxes I and II are linked by an AϩT-rich sequence of 18 nucleotide residues (5). In addition to its origin binding activity, the UL9 protein possesses DNAdependent ATPase and 3Ј-5Ј helicase activities (3, 6). Despite its helicase activity, we have been unable to detect the unwinding of duplex DNA containing Ori S (9).Previous studies have shown that a complex of the UL9 protein and the HSV-1-encoded single-stranded DNA binding protein, ICP8, can efficiently unwind a duplex box I if it possesses a 3Ј single-stranded tail at least 18 nucleotides in length, positioned downstream of box I (8). These findings suggested a model for the unwinding of Ori S in which a complex of the UL9 protein bound to boxes I and II and ICP8 bound to single-stranded DNA generated at the AϩT-rich linker, possibly as a consequence of transcription (4), unwinds the origin of DNA replication to provide access to the replication machinery, thereby permitting the initiation of DNA replication.To test this model, we have investigated the ability of the UL9 protein-ICP8 complex to unwind an Ori S analogue in which the AϩT-rich linker has been replaced by two single strands each consisting of 18 deoxythymidylate residues. We have found this Ori S analogue to be unwound by the complex. In addition, we have found that at temperatures that would be expected to destabilize the AϩT-rich linker, some unwinding of wild-type Ori S can be observed. Ori S bearing mutations in boxes I and II which inhibit binding of UL9 protein could not be unwound under these conditions. These findings support the model in which destabilization of the AϩT-rich linker to provide a binding site for ICP8 represents the initial event in the UL9 protein-promoted unwinding of Ori S .The structure of the duplex oligonucleotides used are shown in Fig. 1. The single-stranded oligonucleotides were purchased from Operon Technologies, Inc. They were purified prior to use by 16% polyacrylamide electrophoresis under denaturing conditions. The concentration of single-stranded oligonucleotides was determined spectrophotometrically assuming that 1 absorbance uni...
Epoxy resin adhesives are widely used because of their strength, versatility, and ability to bond a variety of substrates. Furfurylamines represent a potential, new class of epoxy curing agents. Furfuryl amine (FA), tetrahydrofurfuryl amine (THFA), and 5,5′‐methylenebis‐2‐furanmethanamine (DFA) were studied as possible epoxy curing agents. The utility of FA and THFA are limited by their volatility at the temperatures needed to effect cure of diglycidyl‐ether of bisphenol A (DGEBA) based epoxy resins. DFA is a very effective epoxy curing agent with the ability to cure DGEBA at rates similar to that of standard epoxy curing agents such as liethylenetriamine.
Conventional methods to determine esterase activity from insects are composed of a three-step process where the enzyme is allowed to hydrolyze a 1-naphthyl acetate substrate, that reaction is quenched by a SDS detergent, and then a Fast Blue B dye complex is formed with 1-naphthol, the product of 1-naphthyl acetate hydrolysis. These methods measure dye-product complex rather than the product, 1-naphthol. A new assay is presented that continuously monitors the formation of 1-naphthol with the hydrolysis of an esterase substrate. The esterase activity was determined as the slope of the linear regression change in absorbance over time at 320 nm. The continuous assay provides a simple, rapid, and sensitive method for measuring esterases extracted from a single diamondback moth in 1-10 min. The detection limit of the assay is approximately 0.6 microM 1-naphthol. The 1-naphthol product from the esterase reaction was confirmed by HPLC analysis. According to the assay, the K(m) and V(max) values of the esterase were 28 +/- 2 microM and 6.0 +/- 0.1 microM/min, respectively, at 37 degrees C for 1-naphthyl acetate. The K(i) value was 9 +/- 2 microM using azadirachtin, an insecticide from neem tree, Azadirachta indica (A.Juss). Azadirachtin was a reversible competitive inhibitor of the esterase activity.
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