7-ketocholesterol is an endogenous modulator for the arylhydrocarbon receptor

We have identified 7-ketocholesterol (7-KC) as an endogenous modulator that inhibits transactivation by the arylhydrocarbon receptor (AhR) through competitive binding against xenobiotic ligands. 7-KC binds AhR and displaces labeled dioxin (2,3,7,8-tetrachlorodibenzo(p)dioxin (TCDD)). IC(50) is 5 x 10(-7) m in vivo and 7 x 10(-6) m in vitro. These figures are consistent with its concentration in human blood plasma and tissues. Association with 7-KC prevents AhR binding to DNA. 7-KC blocks the TCDD-mediated transactivation of stably expressed reporter gene constructs in T47-D cells as well as the expression of the endogenous CYP 1A1 gene in HepG2 cells and in primary porcine aortic endothelial cells. Injection of 7-KC to rats blocks the induction of CYP 1A1 messenger RNA and protein in endothelial cells from myocardial blood vessels. The differential sensitivity of mammalian species to toxic effects of AhR ligands, especially dioxin (TCDD), correlates with the expression of 7-hydroxycholesterol dehydrogenase, which synthesizes 7-KC from 7-hydroxycholesterol. The documented involvement of AhR ligands in cardiovascular diseases through lipid peroxidation and endothelium dysfunction can now be examined in the context of displacement of this protective modulator.     

A comparison of bone strain measurements at anatomically relevant sites using surface gauges versus strain gauged bone staples

Instrumented bone staples were first introduced as an alternative to surface-mounted strain gauges for use in human in vivo bone strain measurements because their fixation to bone is secure and requires not only minimally invasive surgery. Bench-top bone bending models have shown that the output from strain gauged bone staples compares favorably to that of traditional mounted gauges. However their within- and across-subject performance at sites typically instrumented in vivo has never been examined. This study used seven human cadaver lower extremities with an age range of 23-81 years old and a dynamic gait simulator to examine and compare axial strains in the mid tibial diaphysis and on the dorsal surface of the second metatarsal as measured simultaneously with strain gauged bone staples and with traditional surface-mounted gauges. Rosette configurations were used at the tibial site for deriving principal compression and tension, and shear strains. Axial outputs from the two gauge types demonstrated strong linear relationships for the tibia (r(2)=0.78-0.94) and the second metatarsal (r(2)=0.96-0.99), but coefficients (slopes) for the relationship were variable (range 7-20), across subjects and across sites. The apparent low reliability of strain gauged staples may be explained by the fact that both strain gauged staples and surface strain gauges are inexact to some degree, do not measure strains from exactly the same areas and strain gauged staples reflect surface strains as well as deformations within the cortex. There were no relationships for the principal tibia compression, tension or shear strain measurements derived from the two rosette gauge types, reflecting the very different anatomical areas measured by each of the constructs in this study. Strain gauged bone staples may be most useful in comparing relative axial intra-subject differences between activities, but inter-subject variability may require larger sample sizes to detect differences between populations.     

The nuclear-bound form of the progesterone receptor is generated through a hormone-dependent phosphorylation

The solubilized ("cytosolic") receptor present in the rabbit uterus in the absence of hormone and the chromatin-bound ("nuclear") receptor obtained after injection of a progestin were compared. Crude cellular extracts were analyzed by immunoblotting and receptors were purified by immunoaffinity chromatography. With both methods it was observed that the electrophoretic mobility of the "nuclear" receptor was slower than that of the "cytosolic" receptor. This difference in mobility appeared to be due to the existence of variably phosphorylated forms of receptor. The phosphorylation reaction was examined in uterine slices. In the absence of hormone the cytosolic receptor was phosphorylated. When hormone was added the phosphorylation of receptor was markedly enhanced and the electrophoretic mobility of the "nuclear" receptor was decreased. These experiments thus show that the receptor in its "cytosolic" form is a phosphoprotein. Under the effect of the hormone the receptor is further phosphorylated on some supplementary site(s). This polyphosphoprotein is the chromatin-bound, putatively active, form of the receptor. In this respect the intracellular progesterone receptor is similar to various membrane receptors for hormones and growth factors which are phosphorylated upon binding of their ligand.     

Rabbits immunized with mixtures of staphylococcal protein A and autologous IgG produce anti-human IgG antibodies

The results of this study provide evidence that protein A may render IgG immunogenic in the autologous host. Antibodies to human but not rabbit IgG were detected in sera of rabbits immunized with a mixture of autologous serum and protein A. Anti-human IgG antibodies appeared within 2 weeks at which time the antibodies were of the IgM class. Upon further immunization, both IgM and IgG antibodies were produced with the IgG class predominating. The antibodies elicited by a mixture of protein A with autologous IgG resembled those which arise in response to autologous IgG that has been denatured by physicochemical means, in that they react mainly with foreign species IgG and weakly, if at all, with IgG of rabbit origin.     

Characterization of a casein kinase which interacts with the rabbit progesterone receptor. Differences with the in vivo hormone-dependent phosphorylation

Previous in vivo studies have shown that the rabbit progesterone receptor undergoes two phosphorylation reactions: one basal and a second one which is hormone-dependent. We report here on the presence and characteristics of a kinase activity found in receptor preparations highly purified by immunoaffinity chromatography. 1. This kinase activity is not due to the receptor molecule itself since the two proteins may be separated by several chromatographic and immunological methods. 2. The presence of the kinase in receptor preparations is not an artefact of the purification procedure. The kinase binds to the receptor as shown by coelution in immunoaffinity experiments and during various chromatographies. This interaction probably takes place in vivo and is not artefactually formed during solubilization of the receptor since the kinase also copurifies with receptors isolated from the uterine nuclei of progestin-treated rabbits. 3. This enzyme may be classified as a casein kinase since it readily phosphorylates the latter substrate (Km approximately equal to 0.15 mg/ml) and is not regulated by cyclic nucleotides, Ca2+ and calmodulin or phospholipids. Its classification as a casein kinase I or II is difficult since on the one hand it is inhibited by heparin, activated by polyamines and may use both ATP and GTP, but on the other hand it modifies only serine residues, and is not inhibited by heparin when the receptor itself is employed as a substrate. 4. The kinase which copurifies with the receptor does not mimic in vitro the effects of the hormone-dependent phosphorylation of the receptor observed in vivo: there is no enhancement of kinase activity by the hormone, and the phosphorylated receptor does not exhibit the characteristic "upshift" in its electrophoretic mobility. Thus either this kinase is not the enzyme responsible for the hormone-dependent receptor phosphorylation or, during purification, a factor has been lost which is necessary for retaining the hormone dependency of the reaction.     

Electron-microscopic studies on location of SH-groups in mitochondrial F1-ATPase using a ferritin label

A new approach has been suggested for electron-microscopic study of the structure of mitochondrial F1-ATPase based on ferritin labeling. By means of sequential treatment with 2-iminothiolane and Nbs2 we obtained a modified ferritin (NbsSPrCNH-Ft) able to react with SH-groups of proteins and to form conjugates in which the protein and ferritin are bound by disulfide bonds. An electron-microscopic investigation of the negatively stained preparations of mitochondrial F1-ATPase, preincubated with modified ferritin, revealed such enzyme-ferritin conjugates. In case of modified ferritin, containing 360 mol SH-groups per mol protein, and F1-ATPase, pretreated with N-ethylmaleimide and then with dithiothreitol, conjugates were obtained in which ferritin molecules are bound to several (as many as four) of the six protein masses, comprising a bilayer molecule of the enzyme. Taking into consideration the biochemical data on the location of accessible SH-groups (only in alpha, gamma or epsilon subunits), it is inferred from the results obtained that one of the protein masses is a complex between beta subunit and at least one of the minor subunits located partially on the molecule's external side. This indicates the nonequivalence of different copies of the major subunits. Averaged images of the particles of the F1-F0 complex from bovine heart mitochondria and bacteria Micrococcus lysodeicticus were obtained. It was found that F0 component is bound to two adjacent protein masses of the F1-ATPase molecule. It is suggested that this binding may be due the nonequivalency of single-type major subunits.