Prostaglandin profiles in nervous tissue and blood vessels of the brain of various animals

The endogenous formation of prostaglandin (PG) D2, E2, F2 alpha, and 6-keto-PGF1 alpha was determined in homogenates of mouse, rat, and rabbit brain, and of rat cerebral blood vessels, using gas chromatography mass spectrometry. In all species tested, 6-keto-PGF1 alpha could be identified in the brain homogenates, but was a minor component in relation to other PGs. In contrast 6-keto-PGF1 alpha was the most abundant PG in the blood vessels, being present in about 40-fold higher levels than in the brain tissue. PGD2 was the most abundant PG in rat and mouse brains, but was below detection limits in the analyzed blood vessels. These studies indicating differential metabolism of PG endoperoxides in nervous and vascular tissue, provide a biochemical basis for further studies on the role of the PGs in brain circulation and neuronal activity.     

Enzymic determination of branched-chain amino acids and 2-oxoacids in rat tissues. Transfer of 2-oxoacids from skeletal muscle to liver in vivo.

This study consists of (1) the extraction of proteoglycan from the human meniscus under dissociative conditions, (2) an investigation of the changes that occur in the abundance and structure of this proteoglycan with age and (3) a comparison of these findings with those for human articular-cartilage proteoglycan. Adult meniscus was found to possess proteoglycan molecules of similar size and glycosaminoglycan content to those present in cartilage, although tissue concentrations were considerably lower. In addition, age-related changes, with respect to the occurrence of keratan sulphate and the sulphation of chondroitin sulphate chains, were common to both tissues. The presence of aggregated proteoglycan was demonstrated, although specific interaction with hyaluronic acid was not conclusively shown biochemically. Differences were, however, noted in the structure of the proteoglycan between the two tissues: dermatan sulphate was found in the meniscus proteoglycan preparation and the core proteins exhibited some dissimilarities. A proteoglycan structure of this type would be compatible with its participation in meniscus elasticity, especially as the material is localized in a specific area.     

Human Fc gamma RI and Fc gamma RII interact with distinct but overlapping sites on human IgG.

Cellular receptors for IgG (Fc gamma R) mediate important protective functions. By using site-specific mutants of a chimeric antibody (mouse V H domain and L chain; human IgG3 C H domains), we have demonstrated that human Fc gamma RI interacts with a site in the lower hinge of human IgG (residues 234 to 237) and that this interaction dictates Fc gamma RI-mediated superoxide generation. Mutations at position 235 resulted in the most profound reductions in Fc gamma RI recognition. We have also mapped an interaction site for Fc gamma RII to the same region; however, mutations at position 234 and 237 resulted in the greatest reductions in Fc gamma RII recognition. The two receptors appear to recognize overlapping but nonidentical sites on the lower hinge of IgG. Deviations from the optimal motif 234-Leu-Leu-Gly-Gly-237 may then explain the human IgG subclass specificity profile for human Fc gamma RI and Fc gamma RII.     

Mechanism of intestinal 7 alpha-dehydroxylation of cholic acid: evidence that allo-deoxycholic acid is an inducible side-product

We previously reported that the 7 alpha-dehydroxylation of cholic acid appears to be carried out by a multi-step pathway in intestinal anaerobic bacteria both in vitro and in vivo. The pathway is hypothesized to involve an initial oxidation of the 3 alpha-hydroxy group and the introduction of a double bond at C4-C5 generating a 3-oxo-4-cholenoic bile acid intermediate. The loss of water generates a 3-oxo-4,6-choldienoic bile acid which is reduced (three steps) yielding deoxycholic acid. We synthesized, in radiolabel, the following putative bile acid intermediates of this pathway 7 alpha,12 alpha-dihydroxy-3-oxo-4-cholenoic acid, 7 alpha,12 alpha-dihydroxy-3-oxo-5 beta-cholanoic acid, 12 alpha-dihydroxy-3-oxo-4,6-choldienoic acid, and 12 alpha-hydroxy-3-oxo-4-cholenoic acid and showed that they could be converted to 3 alpha,12 alpha-dihydroxy-5 beta-cholanoic acid (deoxycholic acid) by whole cells or cell extracts of Eubacterium sp. VPI 12708. During studies of this pathway, we discovered the accumulation of two unidentified bile acid intermediates formed from cholic acid. These bile acids were purified by thin-layer chromatography and identified by gas-liquid chromatography-mass spectrometry as 12 alpha-hydroxy-3-oxo-5 alpha-cholanoic acid and 3 alpha,12 alpha-dihydroxy-5 alpha-cholanoic (allo-deoxycholic acid). Allo-deoxycholic acid was formed only in cell extracts prepared from bacteria induced by cholic acid, suggesting that their formation may be a branch of the cholic acid 7 alpha-dehydroxylation pathway in this bacterium.     

Reactivity of Hg(II) with superoxide: Evidence for the catalytic dismutation of superoxide by HG(II)

Mercuric ion, a well-known nephrotoxin, promotes oxidative tissue damage to kidney cells. One principal toxic action of Hg(II) is the disruption of mitochondrial functions, although the exact significance of this effect with regard to Hg(II) toxicity is poorly understood. In studies of the effects of Hg(II) on superoxide (O) and hydrogen peroxide (H₂O₂) production by rat kidney mitochondria, Hg(II) (1–6 μM), in the presence of antimycin A, caused a concentration-dependent increase (up to fivefold) in mitochondrial H₂O₂ production but an apparent decrease in mitochondrial O production. Hg(II) also inhibited O-dependent cytochrome c reduction (IC₅₀ ≈?2–3 μM) when O was produced from xanthine oxidase. In contrast, Hg(I) did not react with O in either system, suggesting little involvement of Hg(I) in the apparent dismutation of O by Hg(II). Hg(II) also inhibited the reactions of KO₂ (i.e., O) with hemin or horseradish peroxidase dissolved in dimethyl sulfoxide (DMSO). Finally, a combination of Hg(II) and KO₂ in DMSO resulted in a stable UV absorbance spectrum [currently assigned Hg(II)-peroxide] distinct from either Hg(II) or KO₂. These results suggest that Hg(II), despite possessing little redox activity, enhances the rate of O dismutation, leading to increased production of H₂O₂ by renal mitochondria. This property of Hg(II) may contribute to the oxidative tissue-damaging properties of mercury compounds.     

Binding of a chaperonin to the folding intermediates of lactate dehydrogenase.

When Bacillus stearothermophilus LDH dimer is incubated with increasing concentrations of the denaturant guanidinium chloride, three distinct unfolded states of the molecule are observed at equilibrium [Smith, C. J., et al. (1991) Biochemistry 30, 1028-1036]. The kinetics of LDH refolding are consistent with an unbranched progression through these states. The Escherichia coli chaperonin, GroEL, binds with high affinity to the completely denatured form and more weakly to the earliest folding intermediate, thus retarding the refolding process. A later structurally defined folding intermediate, corresponding to a molten globule form, is not bound by GroEL; neither is the inactive monomer. The complex between GroEL and denatured LDH is destabilized by the binding of magnesium/ATP (Mg/ATP) or by the nonhydrolyzable analogue adenylyl imidodiphosphate (AMP-PNP). From our initial kinetic data, we propose that GroEL exists in two interconvertible forms, one of which is stabilized by the binding of Mg/ATP but associates weakly with the unfolded protein. The other is destabilized by Mg/ATP and associates strongly with unfolded LDH. The relevance of these findings to the role of GroEL in vivo is discussed.     

Cloning, structure, and expression of a rat binding protein for polychlorinated biphenyls. Homology to the hormonally regulated progesterone-binding protein uteroglobin

Certain metabolites of polychlorinated biphenyls (PCBs) are retained in the Clara cells and in the airway lumen of rodent lung due to their interaction with a secretory 13-kDa protein. Here, we report the isolation of a cDNA encoding the rat lung PCB-binding protein. The identity of the PCB-binding protein is supported by expression of the cDNA in Cos-1 cells where the homogenates from transfected cells show specific binding of 4,4'-bis([ 3H]methylsulfonyl)-2,2',5,5'-tetrachlorobiphenyl, a high affinity ligand for the PCB-binding protein. Also a monospecific antiserum to the PCB-binding protein recognizes a 13-kDa protein in the homogenates of transfected cells but not in the corresponding fraction of mock-transfected cells. Northern blot analysis of total RNA from different rat tissues demonstrates that the cDNA detects a approximately 600-base pair mRNA which appears to be solely expressed in lung. Interestingly, DNA sequence analysis and prediction of the amino acid sequence reveals that the PCB-binding protein shares 53% positional amino acid identity with uteroglobin, a progesterone-binding protein found in rabbit uterus and lung. Furthermore, amino acids shown by x-ray crystallography to delineate the central cavity of uteroglobin, which fits progesterone, are highly conserved in the two proteins.     

Formation of C21 bile acids from plant sterols in the rat

Formation of bile acids from sitosterol in bile-fistulated female Wistar rats was studied with use of 4-14C-labeled sitosterol and sitosterol labeled with 3H in specific positions. The major part (about 75%) of the 14C radioactivity recovered as bile acids in bile after intravenous administration of [4-14C]sitosterol was found to be considerably more polar than cholic acid, and only trace amounts of radioactivity had chromatographic properties similar to those of cholic acid and chenodeoxycholic acid. It was shown that polar metabolites were formed by intermediate oxidation of the 3 beta-hydroxyl group (loss of 3H from 3 alpha-3H-labeled sitosterol) and that the most polar fraction did not contain a hydroxyl group at C7 (retention of 3H in 7 alpha,7 beta-3H2-labeled sitosterol). Furthermore, the polar metabolites had lost at least the terminal 6 or 7 carbon atoms of the side chain (loss of 3H from 22,23-3H2- and 24,28-3H2-labeled sitosterol). Experiments with 3H-labeled 7 alpha-hydroxysitosterol and 4-14C-labeled 26-hydroxysitosterol showed that none of these compounds was an efficient precursor to the polar metabolites. By analysis of purified most polar products of [4-14C] sitosterol by radio-gas chromatography and the same products of 7 alpha,7 beta-[2H2]sitosterol by combined gas chromatography-mass spectrometry, two major metabolites could be identified as C21 bile acids. One metabolite had three hydroxyl groups (3 alpha, 15, and unknown), and one had two hydroxyl groups (3 alpha, 15) and one keto group. Considerably less C21 bile acids were formed from [4-14C]sitosterol in male than in female Wistar rats. The C21 bile acids formed in male rats did not contain a 15-hydroxyl group. Conversion of a [4-14C]sitosterol into C21 bile acids did also occur in adrenalectomized and ovariectomized rats, indicating that endocrine tissues are not involved. Experiments with isolated perfused liver gave direct evidence that the overall conversion of sitosterol into C21 bile acids occurs in this organ. Intravenously injected 7 alpha,7 beta-3H-labeled campesterol gave a product pattern identical to that of 4-14C-labeled sitosterol. Possible mechanisms for hepatic conversion of sitosterol and campesterol into C21 bile acids are discussed.