Expression and characterization of anticoagulant activity of salivary protein alALP from Asian tiger mosquito Aedes albopictus

@#Several bioactive molecules isolated from the saliva of blood-sucking arthropods, such as mosquitoes, have been shown to exhibit potential anticoagulant function. We have previously identified a 30kDa allergen named Aegyptin-like protein (alALP), which is highly homologous to Aegyptin, from the salivary glands of female Aedes albopictus (Asian tiger mosquito). In this study, we identified the conserved functional domain of alALP by using bioinformatic tools, and expressed the His-tagged alALP recombinant protein in sf9 insect cells by generation and transfection of a baculoviral expression plasmid carrying the fulllength cDNA of alALP. We purified this recombinant protein and examined its function on the inhibition of blood coagulation. The results showed that the purified His-alALP prolonged the Activated Partial Thromboplastin Time (APTT), Prothrombin Time (PT) and Thrombin Time (TT) in vitro as well as the Bleeding Time (BT) in vivo, which suggest that alALP could be a novel anticoagulant.

Expression and characterization of anticoagulant activity of salivary protein alALP from Asian tiger mosquito Aedes albopictus.

Several bioactive molecules isolated from the saliva of blood-sucking arthropods, such as mosquitoes, have been shown to exhibit potential anticoagulant function. We have previously identified a 30kDa allergen named Aegyptin-like protein (alALP), which is highly homologous to Aegyptin, from the salivary glands of female Aedes albopictus (Asian tiger mosquito). In this study, we identified the conserved functional domain of alALP by using bioinformatic tools, and expressed the His-tagged alALP recombinant protein in sf9 insect cells by generation and transfection of a baculoviral expression plasmid carrying the fulllength cDNA of alALP. We purified this recombinant protein and examined its function on the inhibition of blood coagulation. The results showed that the purified His-alALP prolonged the Activated Partial Thromboplastin Time (APTT), Prothrombin Time (PT) and Thrombin Time (TT) in vitro as well as the Bleeding Time (BT) in vivo, which suggest that alALP could be a novel anticoagulant.

Immunosuppressive effects of dihydroetorphine, a potent narcotic analgesic, in dihydroetorphine-dependent mice.

The immunomodulatory effects of dihydroetorphine were systematically investigated in subchronically treated mice. In a dose-dependent fashion, dihydroetorphine (total doses at 444.5, 889 and 1778 microg/kg) lowered the increase of body weight, decreased the weight of the spleen and thymus, weakened the delayed-type hypersensitivity, reduced the generation of antibody-forming cells, inhibited splenic lymphocyte proliferation induced by concanavalin A and lipopolysaccharide, suppressed the production of interleukin-2 in the supernatant of splenocytes induced by concanavalin A, and depleted the ratio of CD4+ and CD8+ subpopulations. Moreover, the physical dependence on dihydroetorphine was also evaluated to confirm that the immunosuppression was concomitant with the addiction to the drug. These results demonstrate that subchronic treatment with dihydroetorphine dose dependently suppresses both humoral and cell-mediated immune function, and that the immunosuppressive effects of dihydroetorphine are much more potent than those of morphine.

Involvement of mu-opioid receptors and alpha-adrenoceptors in the immunomodulatory effects of dihydroetorphine.

The present study investigated the effects of acutely administered dihydroetorphine on mitogen-stimulated lymphocyte proliferation and lymphokine production in mice. These immune functions were significantly suppressed by dihydroetorphine at 24 microg/kg and 128 microg/kg in a dose-dependent fashion. This study further examined the involvement of micro-opioid receptors and alpha-adrenoceptors in the immunomodulatory effects of dihydroetorphine. The micro-opioid receptor antagonist, naloxone (4 mg/kg), and alpha-adrenoceptor antagonist, phentolamine (10 mg/kg), but not the beta-adrenoceptor antagonist, propranolol (10 mg/kg), effectively blocked dihydroetorphine-induced suppression of splenic lymphocyte proliferation and lymphokine production. These results demonstrate that dihydroetorphine has significant immunosuppressive effects in mice and the mechanisms of these effects may lie in its interactions with opioid receptors and adrenergic pathways.

Characterization of plasmids with antimicrobial resistant genes in Pasteurella haemolytica A1.

Two R plasmids, pYFC1 and pYFC2, from Pasteurella haemolytica A1 encoding sulfonamide, streptomycin (pYFC1), and ampicillin (pYFC2) resistances have been characterized by restriction endonuclease digestions, subcloning or DNA sequencing. pYFC1 consists of 4225 bp and is 51.9% in AT content. Physical mapping indicated a highly conserved region of restriction sites among pYFC1, RSF1010, pGS05, pFM739, pHD148 and pGS03B. pYFC1 encoded a dihydropteroate synthase (29.8 kDa), and streptomycin kinase (29.6 kDa) which is homologous in nucleotide sequences or deduced amino acid sequence to that encoded by a broad-host range IncQ plasmid RSF1010. Based on the primary structure of pYFC1, the sulfonamide and streptomycin genes are derived from the same ancestor of RSF1010. pYFC2 is similar to the plasmid from P. haemolytica LNPB51 isolated in France by partial restriction enzyme mapping. pYFC1 and pYFC2 have the same size of 4.2 kbp.