Antibodies targeted against hypervariable and constant regions of cytochromes P450IIB1 and P450IIB2

Fusion proteins constructed between beta-galactosidase and six different segments of either cytochrome P450IIB1 or cytochrome P450IIB2 (ranging from 18 to 33 amino acids in length) were expressed in Escherichia coli. Rabbit antibodies raised against these fusion proteins were first adsorbed through a beta-galactosidase column and then immunopurified on a second column containing the corresponding fusion protein. With the exception of the antibodies directed against the hydrophobic amino-terminal segment of cytochrome P450IIB1, all the antipeptide antibodies recognized the major phenobarbital-inducible cytochromes P450IIB1 and -IIB2 on immunoblots of liver microsomal proteins. Two of the antibodies were raised against regions where cytochromes P450IIB1 and -IIB2 differ in primary structure, and were differentially reactive toward these two highly homologous cytochromes. Several of the antipeptide antibodies were also reactive with a third phenobarbital-inducible microsomal protein expressed in livers of some individual Sprague-Dawley rats which was shown to be more highly related to P450IIB1 than P450IIB2. This P450IIB1-related P450, designated P450IIB1*, was purified to apparent homogeneity and shown to hydroxylate the steroid hormones testosterone and androstenedione with the well-defined regiospecificity and high catalytic activity characteristic of P450IIB1. A fourth microsomal protein detected using the antipeptide antibodies appeared to be more highly related to P450IIB2. Because the segments on the P450 molecules recognized by these antipeptide antibodies are known, it is possible to predict where P450IIB1* and the P450IIB2-related protein differ from cytochromes P450IIB2 and -IIB1, respectively. These studies demonstrate the utility of site-specific anti-P450 antibodies raised to fusion peptides for studies on the expression of structurally related P450s and polymorphic variants within the cytochrome P450 gene superfamily.     

Targeting microRNA-mediated gene repression limits adipogenic conversion of skeletal muscle mesenchymal stromal cells

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Growth inhibition of Francisella tularensis live vaccine strain by IFN-gamma-activated macrophages is mediated by reactive nitrogen intermediates derived from L-arginine metabolism.

We have examined the abilities of the recombinant murine lymphokines IFN-gamma, granulocyte-macrophage (GM)-CSF, and IL-4 to stimulate the in vitro antimicrobial activity of macrophages against the live vaccine strain (LVS) of Francisella tularensis. Resident peritoneal macrophages from C57BL/6 strain mice were cultured overnight with IFN-gamma, GM-CSF, or IL-4, and then infected with LVS. In macrophages treated with IFN-gamma, the growth of LVS was suppressed by a factor of 100- to 1000-fold in comparison with untreated cells. This effect was dose-dependent and was enhanced by the addition of LPS. In contrast, macrophages treated with either GM-CSF or IL-4 exhibited no such enhanced antitularemic activity, even in the presence of LPS. Because reactive nitrogen intermediates derived from L-arginine metabolism have been implicated in the killing of various infectious organisms, we evaluated the possibility that such a mechanism might contribute to the antitularemic activity of IFN-gamma-stimulated macrophages. Macrophages were treated with NG-monomethyl-L-arginine (NMMA), an inhibitor of L-arginine metabolism in mammalian cells, during the activation procedure and throughout the course of infection. NMMA had no effect on the growth of LVS in unstimulated macrophages. In macrophages activated with IFN-gamma, however, NMMA suppressed their capacity to inhibit LVS growth. This effect was proportional to the dose of NMMA added and reversible by supplementing the medium with additional L-arginine, and there was a direct correlation between the production of nitrite by activated macrophages and their ability to inhibit LVS growth. Furthermore, the growth of LVS was inhibited by nitrogen metabolites in a cellfree system. The results of this study indicate that the mechanism of action of IFN-gamma on the resistance of macrophages to LVS growth is related, at least in part, to the production of reactive nitrogen metabolites.     

Studies on myofibrillar protein synthesis in chicken muscular dystrophy

Myofibrillar proteins synthesized $\text{in vitro}$ by normal and dystrophic chicken muscle polysomes were purified and analyzed by SDS gel electrophoresis. No substantial difference in the synthesis of myofibrillar proteins could be detected. These observations suggest that the loss of muscle mass that is observed in muscular dystrophy is not related to a translational defect in the dystrophic polysomes.     

Chromium-51 labeling of sheep red blood cells.

The failure of sheep red blood cells (RBCs) labeled with Chromium-51 (Cr-51) using the ascorbic acid technique to act as a suitable intravascular marker of blood volume in a septic sheep model prompted us to investigate the technique of radiolabeling sheep erythrocytes with this isotope. Consequently, we studied thirteen sheep in which the labeling efficiency of Cr-51 as sodium chromate and hemoglobin typing was determined for each animal. Mean Cr-51 labeling efficiency of sheep RBCs was 67.5% (n = 13). Although 5 of the 13 sheep were discovered to have two types of hemoglobin (Hb) as determined by electrophoresis, overall labeling efficiency of sheep RBCs was determined to be independent of the type of hemoglobin present. However, when two types of Hb were present (Hb-A and Hb-B), Cr-51 had a higher affinity for Hb-B (80%) than Hb-A (20%) even though both Hb types are present in similar proportions (Hb-A = 53%, Hb-B = 46%). The results of this study indicate that sheep RBCs express a lower labeling efficiency for Cr-51 than do human RBCs and that Cr-51 has a higher affinity for Hb-B than for Hb-A when both hemoglobin types are present. This difference is noteworthy when interpreting Cr-51 RBC data in experimental sheep models. Furthermore, caution should be exercised when extrapolating established human protocols to animal models.