Square-wave cathodic adsorptive stripping voltammetry based on adsorptive accumulation is a very sensitive technique for the trace determination of xanthosine 5 0 -monophosphate (5 0 -XMP) and xantho-sine 5 0 -diphosphate (5 0 -XDP). The determination is based on the strong interaction of the adsorption of xanthosine phosphate compounds on a mercury elec-trode surface, forming Hg(II)-xanthate. The cathodic reduction of the accumulated Hg(II)-xanthate complex provides the basis for direct stripping measurements of the investigated biological compounds at sub-nanomolar concentration levels. Moreover, controlled adsorptive accumulation of the Cu(II) complex of xanthosine phosphate is also reported to assay trace amounts of xanthosine phosphate. The height of the sharp chelate peak of adsorbed Cu(II)-xanthate, coupled with the flat baseline, facilitates measurements at nanomolar and submicromolar concentration levels. The adsorption and the redox behaviour of the in-vestigated complexes are indicated by cyclic voltam-metry. Experimental and instrumental parameters for the quantitative determination were optimized. Statis-tical analysis of the calibration curve data is also included

Abstract.Cathodic adsorptive stripping voltammetry (CASV) was applied for the determination of rutin in pharmaceuticals, human urine, and blood serum. An electrochemical stripping procedure for trace measure-ments of rutin was developed based on the adsorption of the Cu 2þ -rutin complex on a hanging mercury drop electrode and applied to the quantification of the drug. Cyclic voltammetry was used to characterize the inter-facial and redox behavior of the adsorbed Cu 2þ -rutin complex. Experimental and instrumental parameters for quantitative determination were optimized, and a detection limit of 4.910 9 molL 1 in presence of Cu 2þ-ions for quantification of rutin under optimum conditions was derived. The sharp peak of the adsorbed Cu 2þ -rutin complex associated with an effective inter-facial accumulation of this compound facilitates the determination of rutin in biological fluids with good recoveries. The degree of interference from potentially present metal ions and organic compounds on the CASV signal for Cu 2þ -rutin complex was evaluated.

Abstract.Cathodic adsorptive stripping voltammetry (CASV) was applied for the determination of rutin in pharmaceuticals, human urine, and blood serum. An electrochemical stripping procedure for trace measure-ments of rutin was developed based on the adsorption of the Cu 2þ -rutin complex on a hanging mercury drop electrode and applied to the quantification of the drug. Cyclic voltammetry was used to characterize the inter-facial and redox behavior of the adsorbed Cu 2þ -rutin complex. Experimental and instrumental parameters for quantitative determination were optimized, and a detection limit of 4.910 9 molL 1 in presence of Cu 2þ-ions for quantification of rutin under optimum conditions was derived. The sharp peak of the adsorbed Cu 2þ -rutin complex associated with an effective inter-facial accumulation of this compound facilitates the determination of rutin in biological fluids with good recoveries. The degree of interference from potentially present metal ions and organic compounds on the CASV signal for Cu 2þ -rutin complex was evaluated.

Flutamide (Flu) is a chemotherapeutic drug, used for the treatment of prostate cancer. It functions by interfering DNA in fast growing cells and preventing them from reproducing. The present work is focused on the interaction of Flu with single and double stranded DNA at different temperatures and at physiological pH 7.4 (human blood pH). Cyclic voltammetry, square wave voltammetry and UV–visible spectroscopy were used to analyze the interaction of Flu with DNA. The voltammetric results indicate Flu gets intercalated between dsDNA bases and the strength of interaction is independent on the ionic strength. The hyperchromic effect in absorption spectra of Flu-dsDNA complex affirmed the intercalative mode of binding between Flu and dsDNA. Comparison of the mode interaction of Flu with dsDNA and ssDNA was discussed. The binding constants, stoichiometric coefficients and thermodynamic parameters of Flu-dsDNA and Flu-ssDNA complexes were evaluated. The association between Flu and dsDNA is maximum at 308 K which depicts the most stable complexes are formed at near human body tempera-ture. The decrease in the peak current of Flu resulting from its interaction with DNA was employed for determination of dsDNA and ssDNA concentration. The detection limits of dsDNA and ssDNA were found to be 4.2710 7 M and 1.8710 7 M, respectively

Flutamide (Flu) is a chemotherapeutic drug, used for the treatment of prostate cancer. It functions by interfering DNA in fast growing cells and preventing them from reproducing. The present work is focused on the interaction of Flu with single and double stranded DNA at different temperatures and at physiological pH 7.4 (human blood pH). Cyclic voltammetry, square wave voltammetry and UV–visible spectroscopy were used to analyze the interaction of Flu with DNA. The voltammetric results indicate Flu gets intercalated between dsDNA bases and the strength of interaction is independent on the ionic strength. The hyperchromic effect in absorption spectra of Flu-dsDNA complex affirmed the intercalative mode of binding between Flu and dsDNA. Comparison of the mode interaction of Flu with dsDNA and ssDNA was discussed. The binding constants, stoichiometric coefficients and thermodynamic parameters of Flu-dsDNA and Flu-ssDNA complexes were evaluated. The association between Flu and dsDNA is maximum at 308 K which depicts the most stable complexes are formed at near human body tempera-ture. The decrease in the peak current of Flu resulting from its interaction with DNA was employed for determination of dsDNA and ssDNA concentration. The detection limits of dsDNA and ssDNA were found to be 4.2710 7 M and 1.8710 7 M, respectively

Bombyxin F1 gene, a new bombyxin family gene, has been identified. The F1 gene forms a pair with bombyxin B10 gene with an opposite transcriptional orientation and the gene pair F1/B10 is located between bombyxin gene pairs B9/C1 and A7/B7 in a bombyxin gene cluster. The nucleotide sequence of the F1 gene and its deduced amino acid sequence deviate moderately from those characterized previously for the family-A, family-B, family-C, family-D, and family-E bombyxin genes; the bombyxin F1 gene and preprobombyxin F1 share no more than 62% and 53% sequence identities with other bombyxin members, respectively. Harr-plot analysis indicated that the spacer of the F1/B10 gene pair has low sequence similarity with that of other bombyxin gene pairs characterized. The bombyxin F1 mRNA in Bombyx mori brain was shown to locate in four pairs of medial neurosecretory cells, which also produce other bombyxin family mRNAs. Genomic Southern hybridization indicated that the Bombyx haploid genome contains a single copy of the family-F bombyxin gene.

Here we describe a novel approach to isolate proteins involved in insect hemolymph coagulation. In order to avoid problems in purifying clot proteins after they had been crosslinked, we performed an in vitro coagulation reaction with cell-free hemolymph from the lepidopteran Galleria mellonella and used the resulting complexes to produce a specific antiserum. The antiserum reacted with a subset of hemolymph proteins as well as with granular cells, but not with other hemocyte types of Galleria. Screening expression libraries identified some positive clones, which turned out to code for some previously characterized components of immune cascades, as well as some novel candidates for clotting factors. Known components include members of both the coagulation system and the prophenol-activating cascade, lending support to the idea that both systems work together during the formation of a hemolymph clot. Novel candidates for insect clotting factors include a mucin-like protein, a glutathione-S-transferase, and a distant member of the alpha-crystallin/small heat shock protein family. Using assays measuring the activity of transglutaminase, a key enzyme in clotting reactions in both vertebrates and invertebrates, we found a partial overlap between transglutaminase substrates and proteins recognized by the antiserum against the in vitro-induced clot.

Components of the insect clot, an extremely rapid forming and critical part of insect immunity, are just beginning to be identified (1). Here we present a proteomic comparison of larval hemolymph before and after clotting to learn more about this process. This approach was supplemented by the identification of substrates for the enzyme transglutaminase, which plays a role in both vertebrate blood clotting (as factor XIIIa) and hemolymph coagulation in arthropods. Hemolymph proteins present in lower amounts after clotting include CG8502 (a protein with a mucin-type domain and a domain with similarity to cuticular components), CG11313 (a protein with similarity to prophenoloxidase-activating proteases), and two phenoloxidases, lipophorin, a secreted gelsolin, and CG15825, which had previously been isolated from clots (2). Proteins whose levels increase after clotting include a ferritin-subunit and two members of the immunoglobulin family with a high similarity to the small immunoglobulin-like molecules involved in mammalian innate immunity. Our results correlate with findings from another study of coagulation (2) that involved a different experimental approach. Proteomics allows the isolation of novel candidate clotting factors, leading to a more complete picture of clotting. In addition, our two-dimensional protein map of cell-free Drosophila hemolymph includes many additional proteins that were not found in studies performed on whole hemolymph.

Our studies on the developmental regulation of glycosylation in Drosophila melanogaster led us to identify and characterize gp150, an ecdysone-regulated mucin that is found in hemocytes, the gut (peritrophic membrane) and in the salivary glands. We are particularly interested in mucin immune functions and found that gp150 is released from larval hemocytes, becomes part of the clot and participates in the entrapment of bacteria. By RT-PCR and RNAi experiments, we identified gp150 as the previously described I71-7, an ecdysone-induced salivary glue protein. We discuss the evolutionary and biochemical implications of the dual use of salivary proteins for immune functions in insects. Further molecular characterization of such shared proteins may enable a better understanding of the properties of proteins involved in containment and elimination of microbes, as well as hemostasis and wound repair.

Elsevier