Determination of Francisella tularensis AcpB acid phosphatase substrate preferences

The Francisella species encode 4 main acid phosphatases (Acp) that are potentially involved in pathogenesis through currently unknown mechanisms. Only 2 of these enzymes, AcpA and AcpC, have been biochemically characterized to date. In this work we describe the catalytic properties of Francisella tularensis AcpB utilizing an array of 120 phosphorylated substrates. In contrast to most acid phosphatases, the purified enzyme showed a narrow range of substrate preferences, with the highest affinity towards thiamine phosphate (Km = 150 μM). Francisella species do not possess a thiamine biosynthetic pathway even though vitamin B1 is indispensable in numerous cellular functions. Consequently, thiamine should be incorporated from the environment, in this case, from the host cell. Our results suggested that AcpB could provide the hydrolytic activity necessary to transform the nontransportable phosphorylated vitamin B1 present in tissues to a form that can be absorbed by the intracellular pathogen.     

Sandwich ELISA for quantitative detection of human collagen prolyl 4-hydroxylase

Background  

We describe a method for specific, quantitative and quick detection of human collagen prolyl 4-hydroxylase (C-P4H), the key enzyme for collagen prolyl-4 hydroxylation, in crude samples based on a sandwich ELISA principle. The method is relevant to active C-P4H level monitoring during recombinant C-P4H and collagen production in different expression systems. The assay proves to be specific for the active C-P4H α₂β₂ tetramer due to the use of antibodies against its both subunits. Thus in keeping with the method C-P4H is captured by coupled to an anti-α subunit antibody magnetic beads and an anti-β subunit antibody binds to the PDI/β subunit of the protein. Then the following holoenzyme detection is accomplished by a goat anti-rabbit IgG labeled with alkaline phosphatase which AP catalyzes the reaction of a substrate transformation with fluorescent signal generation.     

Effect of double-stranded DNA on maturation of dendritic cells in vitro

A preparation of human genomic fragmented double-stranded DNA (dsDNA) was used as maturation stimulus in cultures of human dendritic cells (DCs) generated in compliance with the interferon protocol. Culturing of the DCs in medium with 5 μg/ml of the DNA preparation was associated with a decrease in the relative proportion of CD14 + cells and an increase in that of CD83 + cells. These changes are markers of DC maturation. The efficiency with which the DNA preparation was able to elicit DC maturation was commensurate with that of lypopolysaccharide from bacterial cell, the standard inducer of DC maturation. Generated ex vivo, matured in the presence of the human DNA preparation, pulsed with tumor antigens mouse DCs were used as a vaccine in biological tests for its antitumor activity. The experimental results demonstrate that reinfusion of mature pulsed with tumor antigens DCs cause a statistically significant suppression of tumor graft growth.     

Hormonal status of the pollen-pistil system at the progamic phase of fertilization after compatible and incompatible pollination in Petunia hybrida L.

The hormonal status of the pollen-pistil system in Petunia hybrida L. during the progamic phase of fertilization was investigated. The contents of indolyl-3-acetic acid (IAA), abscisic acid (ABA), and cytokinins, as well as the rate of ethylene production in the pistils and their parts (stigma, style, and ovary) were measured over an 8-h period following compatible and self-incompatible pollination. In both pollinations, the phytohormones were present in various proportions in the stigma, style and ovary: the stigma was the main site of ethylene synthesis and contained 90% of the ABA, while the style contained 80% of the total cytokinin content in the pollinated pistil. Relatively low levels of hormones in the ovary did not influence the hormonal status of the pollen-pistil system. The interaction of the male gametophyte with the stigmatic tissues was accompanied by a 7- to 10-fold increase in ethylene production and a 1.5- to 2.0-fold increase in IAA content in the pollen-pistil system over 0–4 h. Pollen tube growth after self-incompatible pollination, in contrast to compatible pollination, was accompanied by a 3-fold increase in the ABA content in the stigma and style and by a 5-fold higher cytokinin content in the stylar tissues. Thus, the ethylene/ABA status of the stigma may play a role in controlling the processes of adhesion, hydration, and germination of pollen grains during pollination while the auxin/cytokinin status of the style may be involved in controlling pollen tube growth.