Characterization of proteinases from the midgut of Rhipicephalus (Boophilus) microplus involved in the generation of antimicrobial peptides

Background: Hemoglobin is a rich source of biologically active peptides, some of which are potent antimicrobials (hemocidins). A few hemocidins have been purified from the midgut contents of ticks. Nonetheless, how antimicrobials are generated in the tick midgut and their role in immunity is still poorly understood. Here we report, for the first time, the contribution of two midgut proteinases to the generation of hemocidins. Results: An aspartic proteinase, designated BmAP, was isolated from the midgut of Rhipicephalus (Boophilus) microplus using three chromatographic steps. Reverse transcription-quantitative polymerase chain reaction revealed that BmAP is restricted to the midgut. The other enzyme is a previously characterized midgut cathepsin L-like cysteine proteinase designated BmCL1. Substrate specificities of native BmAP and recombinant BmCL1 were mapped using a synthetic combinatorial peptide library and bovine hemoglobin. BmCL1 preferred substrates containing non-polar residues at P2 subsite and polar residues at P1, whereas BmAP hydrolysed substrates containing non-polar amino acids at P1 and P1`. Conclusions: BmAP and BmCL1 generate hemocidins from hemoglobin alpha and beta chains in vitro. We postulate that hemocidins may be important for the control of tick pathogens and midgut flora.

Useful Strategies for Algal Volatile Analysis

The production of volatile organic compounds (VOC) by plants is well known. However, few scientific groups have studied VOC produced by green, brown and red algae. Headspace collection of volatiles and solid phase microextraction, as well as the traditional extraction by hydrodistillation combined with analytical chromatographic techniques (i.e., GC-MS), have significantly improved the investigation of VOC from plants and algae. The major volatile compounds found in seaweeds are hydrocarbons, terpenes, phenols, alcohols, aldehydes, ketones, esters, fatty acids and halogen or sulfur-containing compounds. This article presents an overview of VOC isolated from and identified in marine macro-algae. Focus is given to non-halogenated and non-sulfur volatile compounds, as well as strategies to analyze and identify algal VOC by GC-MS.