Monoclonal antibodies (Mabs) are valuable reagents for the purification, characterization and immunolocalization of proteins. In this study, we raised Mabs against human peroxisome proliferator-activated receptors (PPARs) using baculovirus particles displaying surface glycoprotein gp64-fusion proteins as the immunizing agent. In this system, to display fusion proteins on the viral surface, the amino terminal sequences of human PPARd and PPARg2 are inserted in-frame between the signal sequence and the mature domain of the gp64 nucleotide sequence.Mabs were raised by immunization with whole virus without a purification of the target antigens. The Mabs generated by this novel method were shown to recognize not only the gp64-PPARs fusion protein, but also mature, expressed proteins by a wide variety of techniques, including immunohistochemistry, immunoblotting, and electrophoretic mobility shift assays (EMSAs). Transfection of the transfer vector containing a nucleotide sequence encoding less than 30 amino acids along with linearized baculovirus DNA allows for the production of a high affinity antibody against the corresponding mature form. This method is of potential utility in that it allows the production of valuable antibodies without the requirement of a protein purification step.
Toxin-antitoxin (TA) systems are implicated in prokaryotic stress adaptation. Previously, bioinformatics analysis predicted that such systems are abundant in some slowly growing chemolithotrophs; e.g., Nitrosomonas europaea. Nevertheless, the molecular functions of these stress-response modules remain largely unclear, limiting insight regarding their physiological roles. Herein, we show that one of the putative MazF family members, encoded at the ALW85_RS04820 locus, constitutes a functional toxin that engenders a TA pair with its cognate MazE antitoxin. The coordinate application of a specialised RNA-Seq and a fluorescence quenching technique clarified that a unique triplet, UGG, serves as the determinant for MazF cleavage. Notably, statistical analysis predicted that two transcripts, which are unique in the autotroph, comprise the prime targets of the MazF endoribonuclease: hydroxylamine dehydrogenase (hao), which is essential for ammonia oxidation, and a large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcL), which plays an important role in carbon assimilation. Given that N. europaea obtains energy and reductants via ammonia oxidation and the carbon for its growth from carbon dioxide, the chemolithotroph might use the MazF endoribonuclease to modulate its translation profile and subsequent biochemical reactions.
Histamine (Hm) mediates and modulates inflammations through a variety of immunoregulatory actions1 '2) and is generally accepted to be released from mast cells and basophil< upon activation of the cells with allergenreagin complex.3) In contrast we have pre
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