Prolonged morphine application modulates Bax and Hsp70 levels in primary rat neurons

Morphine has been used for pain treatment with a long history. Some data suggest that morphine is toxic to neurons and induces apoptosis, while other evidence shows that morphine could have beneficial effects against cell death. To determine how morphine affects pro-apoptotic protein Bax and molecular chaperone Hsp70, different concentrations of morphine were examined. Our results show that prolonged morphine administration for 5 days at 1microM concentration protects against serum deprivation induced cell death in rat primary neurons. Morphine treatment decreases Bax and Hsp70 levels in cultured rat primary neurons, suggesting morphine may have a protective role in staurosporine and serum deprivation induced cytotoxicity.     

Detection of inhibition of antimicrobial activity by mycobacterial lysates in human monocytes infected with Legionella pneumophila

Antimicrobial activity in human monocytes infected with Mycobacterium tuberculosis has been difficult to demonstrate in vitro, and the molecular mechanisms allowing the bacteria to survive intracellularly are unknown. As a means to test the influence of bacterial products in the microbicidal activity of monocytes we have developed an infection model with Legionella pneumophila, which is killed by interferon gamma activated cells. We demonstrate that this model is useful because M. tuberculosis lysates inhibit one hundred fold the interferon gamma induced activity against L. pneumophila. Comparable degrees of inhibition are also detected when we use lysates from the less pathogenic Mycobacterium gordonae and the pathogenic Staphylococcus aureus, suggesting the participation of a common mechanism. This hypothesis is supported by the fact that the pattern of cytokine secretion is similar in all cases. A significant difference is, however, observed when we used lysates from the non-pathogenic Escherichia coli, which resulted in the recovery of low numbers of bacteria, probably because they induce the cell death of infected monocytes.     

Phage-display derived single-chain fragment variable (scFv) antibodies recognizing conformational epitopes of Escherichia coli heat-labile enterotoxin B-subunit

Previously we have described studies on in vitro pentamer assembly of Escherichia coli (E. coli) derived heat-labile enterotoxin B subunit (EtxB) using conventional monoclonal antibodies (Amin et al., JBC 1995: 270, 20143-50 and Chung et al., JBC 2006: 281, 39465-70). To extend these studies further we have used phage-display to select single-chain Fragment variable (scFv) antibodies against different forms of the B-subunit. Two clones exhibiting strong and differential binding were chosen for detailed characterization. A comprehensive sequence analysis was performed to assign the framework and complementary-determining regions and a nonsense mutation present in one of these (scFv-B1.3.9) was corrected. Binding analysis showed that scFv-B1.3.9 bound in ELISA to both heat-denatured monomeric B-subunits (EtxB₁) and also displayed cross-reactivity towards pentameric EtxB (EtxB₅), although there was no reactivity towards monoganglioside (G_M1) captured EtxB₅. Another antibody (scFv-B5.2.14) had a different reactivity profile and, in ELISA, bound only to EtxB₅ but not to EtxB₁ or to EtxB₅ captured via G_M1. Surprisingly, in competition experiments, the assembled pentameric B-subunit inhibited binding of scFv-B5.2.14 to immobilized EtxB₅ only weakly, whereas reduced, but not oxidized, monomeric EtxB₁ was an efficient competitor. These results clearly demonstrate that B1.3.9 and B5.2.14 have different specificities for cryptic epitopes not accessible in the fully assembled G_M1 bound pentameric form of EtxB.Taken together our results show that we were able to successfully isolate and characterize recombinant scFvs that differentially recognize diverse denatured forms or assembly intermediates of the heat-labile enterotoxin B subunit of E. coli.     

TNT detection using llama antibodies and a two-step competitive fluid array immunoassay

Llamas possess unique subclasses of antibodies that lack light chains, and thus are made by the pairing of two heavy chains. IgG was purified from two llamas which had been immunized with trinitrobenzene-keyhole limpet hemocyanin. Conventional IgG1 and heavy chain IgG2 and IgG3 subclasses were fractionated using affinity chromatography. The effectiveness of heavy chain antibodies for the detection of trinitrotoluene (TNT) using a competitive fluid array immunoassay was evaluated and compared to both the llama IgG1 as well as a murine monoclonal anti-TNT antibody. It was found that heavy chain antibody bound TNT with selectivity similar to conventional antibodies, yet the heavy chain antibodies possessed greater thermal stability. The titer of the heavy chain antibodies however was found to be 10-fold lower than the IgG1; thus analytical assays were best demonstrated using the llama IgG1 conventional antibody. The TNT competitive immunoassay on the Luminex fluid analyzer had a dynamic range from ∼ 100 ng/mL to 10 μg/mL. Utilizing the same two-step competitive assay format the dynamic range of the monoclonal antibody was found to have a broad range (1 ng/mL to 1 μg/mL). This method was demonstrated on TNT contaminated soil extracts using both the llama IgG1 and the mouse monoclonal validating the utility of method for analysis of field samples.