Rapid identification of nonessential genes of herpes simplex virus type 1 by Tn5 mutagenesis.

The large genome of herpes simplex virus type of (HSV-1) encodes at least 80 polypeptides, the majority of which have no recognized function. A subgroup of these gene products appears to be nonessential for virus replication in cell culture, but contributes to the complex life cycle of the virus in the host. To identify such functions, a simple insertional mutagenesis method has been used for selective inactivation of individual HSV-1 genes. The bacterial transposon Tn5 was allowed to insert randomly into cloned restriction fragments representing the entire short unique (US) region of the HSV-1 genome. Of the 12 open reading frames that were mutagenized with Tn5, mutant derivatives of US2, US4, and US5 were recombined into the virus. These three genes proved to be nonessential for HSV-1 replication in Vero (African Green monkey kidney) cells and the US4 gene appeared to be involved in viral pathogenesis in the central nervous system of mice. This rapid mutagenesis procedure should prove useful in exploring the entire HSV-1 genome as well as the genomes of other complex animal viruses.

Molecular dynamics of a cytochrome c-cytochrome b5 electron transfer complex.

Cytochrome c and cytochrome b5 form an electrostatically associated electron transfer complex. Computer models of this and related complexes that were generated by docking the x-ray structures of the individual proteins have provided insight into the specificity and mechanism of electron transfer reactions. Previous static modeling studies were extended by molecular dynamics simulations of a cytochrome c-cytochrome b5 intermolecular complex. The simulations indicate that electrostatic interactions at the molecular interface results in a flexible association complex that samples alternative interheme geometries and molecular conformations. Many of these transient geometries appear to be more favorable for electron transfer than those formed in the initial model complex. Of particular interest is a conformational change that occurred in phenylalanine 82 of cytochrome c that allowed the phenyl side chain to bridge the two cytochrome heme groups.

Structural origins of high-affinity biotin binding to streptavidin.

The high affinity of the noncovalent interaction between biotin and streptavidin forms the basis for many diagnostic assays that require the formation of an irreversible and specific linkage between biological macromolecules. Comparison of the refined crystal structures of apo and a streptavidin:biotin complex shows that the high affinity results from several factors. These factors include the formation of multiple hydrogen bonds and van der Waals interactions between biotin and the protein, together with the ordering of surface polypeptide loops that bury the biotin in the protein interior. Structural alterations at the biotin binding site produce quaternary changes in the streptavidin tetramer. These changes apparently propagate through cooperative deformations in the twisted beta sheets that link tetramer subunits.

Protein crystal growth in microgravity.

The crystals of most proteins or other biological macromolecules are poorly ordered and diffract to lower resolutions than those observed for most crystals of simple organic and inorganic compounds. Crystallization in the microgravity environment of space may improve crystal quality by eliminating convection effects near growing crystal surfaces. A series of 11 different protein crystal growth experiments was performed on U.S. space shuttle flight STS-26 in September 1988. The microgravity-grown crystals of gamma-interferon D1, porcine elastase, and isocitrate lyase are larger, display more uniform morphologies, and yield diffraction data to significantly higher resolutions than the best crystals of these proteins grown on Earth.

Etiology-Specific Mineralization Patterns in Patients with Labyrinthitis Ossificans.

BACKGROUND AND PURPOSE: Our aim was to identify whether specific patterns of ossification in labyrinthitis ossificans are associated with the known risk factors. Labyrinthitis ossificans has been described as sequela of prior temporal bone trauma, prior infection, and other disorders including sickle cell disease. Specific patterns of mineralization in the membranous labyrinth associated with these risk factors has not been previously described. MATERIALS AND METHODS: This was a retrospective study evaluating temporal bone CT scans at our institution from November 2005 to May 2018 in patients with labyrinthitis ossificans. Membranous labyrinthine structures evaluated for ossification included the following: basal, middle, and apical cochlear turns; lateral, posterior, and superior semicircular canals; and the vestibule for both ears in all patients. These structures were assigned a severity score, 0-4, based on degree of mineralization. Clinical records were reviewed for potential labyrinthitis ossificans risk factors. Basic descriptive statistics and a mixed model were used to correlate the degree and patterns of ossification with clinical history. RESULTS: Forty-four patients (58 ears) with labyrinthitis ossificans were identified and evaluated. The most common risk factors were chronic otomastoiditis (n = 18), temporal bone surgery (n = 9), temporal bone trauma (n = 6), sickle cell disease (n = 5), and meningitis (n = 4). For all etiologies, the semicircular canals were most severely affected, and the vestibule was the least. In patients with prior temporal bone surgery, significantly greater mineralization was seen in the basal turn of the cochlea (P = .027), the vestibule (P = .001), and semicircular canals (P < .001-.008). No significant pattern was identified in patients with meningitis, sickle cell disease, or trauma. CONCLUSIONS: Significant patterns of mineralization in labyrinthitis ossificans were observed in patients with prior temporal bone surgery. For all etiologies, the semicircular canals were most severely affected. No significant mineralization pattern was observed in patients with chronic otomastoiditis, meningitis, sickle cell disease, or prior temporal bone trauma.

Metabolism and effects on platelet function of the purified eicosapentaenoic and docosahexaenoic acids in humans.

Metabolism and effects on platelet function of 6 g/d for 6 d of either eicosapentaenoic acid (EPA, C20:5 omega-3) or docosahexaenoic acid (DCHA, C22:6 omega-3) in volunteers were compared in a randomized crossover study. Incorporation kinetics revealed that EPA appeared in plasma free fatty acids and plasma phospholipids after 4 h, but was not incorporated into platelet phosphatidylcholine and -ethanolamine until day 6. This indicates that platelet fatty acid composition does not immediately reflect that of the surrounding plasma milieu, but rather may be determined during megakaryocyte maturation. Importantly, EPA was not incorporated into platelet phosphatidylinositol or -serine in vivo, thus reflecting selective biosynthesis of platelet phospholipids. After dietary EPA, C22:5 omega-3 increased in plasma and platelet phospholipids. In contrast, DCHA-levels were unaltered. After DCHA-ingestion, C20:5 omega-3 concentrations rose in plasma phospholipids, implying that retroconversion took place. These findings indicate that dietary DCHA can serve as a source of EPA. During this short-term study, ingestion of both EPA and DCHA resulted in reduced platelet aggregation in response to collagen. The response to ADP was lowered significantly only by DCHA. After either EPA or DCHA, thromboxane formation was unchanged in serum derived from clotted whole blood as was total in vivo synthesis measured by excretion of immunoreactive 2,3-dinor thromboxane B2/3. We conclude that DCHA reduces platelet responsiveness, contributing to the antithrombotic effects of omega-3 fatty acid-rich fish oil ingestion, of which DCHA is a major component.

Long-term effects of dietary marine omega-3 fatty acids upon plasma and cellular lipids, platelet function, and eicosanoid formation in humans.

We studied the incorporation and metabolism of eicosapentanoic (EPA) and docosahexaenoic acid in six human volunteers who supplemented their normal Western diet for 5 mo daily with 10-40 ml of cod liver oil, rich in omega-3 polyunsaturated fatty acids. EPA and docosahexaenoic acid were incorporated into the total phospholipids of plasma, platelets, and erythrocytes in a dose- and time-dependent manner. During omega-3 fatty acid ingestion serum triacylglycerols were lowered and platelet aggregation upon low doses of collagen was reduced. Concomitantly, formation and excretion of prostanoids showed a characteristic change. As measured in serum from whole clotted blood, thromboxane A3 was formed in small amounts, whereas thromboxane A2 formation was reduced to 50% of control values. Excretion of the main urinary thromboxane A metabolites was unaltered in subjects with low basal excretion rates, but decreased markedly in two subjects with high control values. As determined from the main urinary metabolite, prostaglandin I3 was formed from EPA at rates up to 50% of unaltered prostaglandin I2 formation. The biochemical and functional changes observed lasted for the entire supplementation period of 5 mo and were reversible within 12 wk after cessation of cod liver oil intake. Favorable changes induced by long-chain omega-3 fatty acids include a dose-related and sustained shift of the prostaglandin I/thromboxane A balance to a more antiaggregatory and vasodilatory state.

Lovastatin increases arachidonic acid levels and stimulates thromboxane synthesis in human liver and monocytic cell lines.

The effect of lovastatin (LOV), the inhibitor of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, on linoleic acid (LA, 18:2n-6) metabolism was examined in human monocytic Mono Mac 6 (MM6) and hepatoma Hep G2 cells. The desaturation of LA was examined after LOV (72 h, 10 microM) or dimethylsulfoxide (LOV carrier, < 0.1%) and [14C]LA (last 18 h, 0.3 microCi, 5 microM). In both cell lines, LOV reduced the percentage of 14C label associated with LA and increased the percentage of label in the 20:4n-6 and the 22:5n-6 fractions. In Hep G2 but not MM6 cells, this effect was fully reversible by means of coincubation with mevalonic acid (500 microM), but not with cholesterol or lipoproteins. In both cell lines, the LOV-mediated increase in LA desaturation resulted in dose-dependent reductions of LA and elevations of AA in cellular phospholipids. The lipids secreted by LOV-treated Hep G2 cells were also enriched in arachidonic acid (AA). In the MM6 cells, LOV increased release of thromboxane upon stimulation with the calcium ionophore A23187. In summary, our findings of higher LA desaturation and AA enrichment of lipids secreted by the Hep G2 cells suggest that LOV treatment may increase the delivery of AA from the liver to extrahepatic tissues. The changes in membrane fatty acid composition can influence a variety of cellular functions, such as eicosanoid synthesis in monocytic cells. The mechanism appears to be related to the reduced availability of intermediates of cholesterogenesis.

Molecular views of viral polyprotein processing revealed by the crystal structure of the hepatitis C virus bifunctional protease-helicase.

BACKGROUND: Hepatitis C virus (HCV) currently infects approximately 3% of the world's population. HCV RNA is translated into a polyprotein that during maturation is cleaved into functional components. One component, nonstructural protein 3 (NS3), is a 631-residue bifunctional enzyme with protease and helicase activities. The NS3 serine protease processes the HCV polyprotein by both cis and trans mechanisms. The structural aspects of cis processing, the autoproteolysis step whereby the protease releases itself from the polyprotein, have not been characterized. The structural basis for inclusion of protease and helicase activities in a single polypeptide is also unknown. RESULTS: We report here the 2.5 A resolution structure of an engineered molecule containing the complete NS3 sequence and the protease activation domain of nonstructural protein 4A (NS4A) in a single polypeptide chain (single chain or scNS3-NS4A). In the molecule, the helicase and protease domains are segregated and connected by a single strand. The helicase necleoside triphosphate and RNA interaction sites are exposed to solvent. The protease active site of scNS3-NS4A is occupied by the NS3 C terminus, which is part of the helicase domain. Thus, the intramolecular complex shows one product of NS3-mediated cleavage at the NS3-NS4A junction of the HCV polyprotein bound at the protease active site. CONCLUSIONS: The scNS3-NS4A structure provides the first atomic view of polyprotein cis processing. Both local and global structural rearrangements follow the cis cleavage reaction, and large segments of the polyprotein can be folded prior to proteolytic processing. That the product complex of the cis cleavage reaction exists in a stable molecular conformation suggests autoinhibition and substrate-induced activation mechanisms for regulation of NS3 protease activity.

Differences in arachidonic acid release, metabolism and leukotriene B4 synthesis in human polymorphonuclear leukocytes activated by different stimuli.

Products of the 5-lipoxygenase pathway were analyzed after different stimuli in human polymorphonuclear leukocytes prelabeled with 3H-arachidonic acid. Upon stimulation with the Ca2+ ionophore, A23187, polymorphonuclear leukocytes generate 118.2 +/- 18 pg [3H]dihydroxyeicosatetraenoic acids (diHETEs, including 3H-leukotriene B4 and its 6-trans-stereoisomers), after exposure to serum coated zymosan (35.8 +/- 9 pg) and N-fMet-Leu-Phe (39.5 +/- 9 pg). Conversion of 3H-arachidonic acid paralleled its release after A23187 and fMet-Leu-Phe exposure leaving only 13.8 +/- 7% and 13.6 +/- 3% of the released 3H-arachidonic acid unmetabolized, respectively. In contrast, after stimulation with serum-coated zymosan only a small fraction of the released 3H-arachidonate was converted to 5-lipoxygenase products leaving 73.0 +/- 5% of the released 3H-arachidonic acid unmetabolized. In parallel, leukotriene B4 synthesis was studied in unlabeled polymorphonuclear leukocytes, resulting in 40 +/- 15 ng upon A23187 stimulation, 4 +/- 0.9 ng upon stimulation with fMet-Leu-Phe and 1.8 +/- 0.9 ng after serum-coated zymosan, showing a different ratio of leukotriene B4 to 3H-diHETE for A23187 in contrast to serum-coated zymosan and fMet-Leu-Phe. These results indicate that the coupling between the release of the precursor fatty acid and the metabolism via the 5-lipoxygenase pathway differs greatly between different stimuli.

Prostacyclin metabolism in adults and neonates. Urinary profiles of 6-ketoprostaglandin F1 alpha and 2,3-dinor-6-ketoprostaglandin F1 alpha studied by gas chromatography-mass spectrometry.

Metabolism of endogenous prostacyclin was studied in adults and neonates by measuring urinary levels of 6-ketoprostaglandin F1 alpha (spontaneous hydrolysis product) and 2,3-dinor-6-ketoprostaglandin F1 alpha (enzymatically formed by beta-oxidation). Quantification of prostanoids was achieved by capillary gas chromatography-mass spectrometry using the stable isotope dilution technique. Purification of the urinary lipid extract included silicic acid column chromatography and reverse- and straight-phase high-pressure liquid chromatographies. Accuracy of the method was proven by recovery experiments for both metabolites. Partial mass spectra of endogenous 6-ketoprostaglandin F1 alpha and 2,3-dinor-6-ketoprostaglandin F1 alpha were obtained from urine samples. In neonates (third day of life, n - 5 pooled urines) levels of 2,3-dinor-6-ketoprostaglandin F1 alpha (0.28 +/- 0.18 ng/ml) were much lower than those of 6-ketoprostaglandin F1 alpha (2.13 +/- 1.10 ng/ml), indicating low beta-oxidation activity at high prostacyclin formation. In adults (n = 7), levels of 2,3-dinor-6-ketoprostaglandin F1 alpha (0.27 +/- 0.21 ng/ml) and levels of 6-ketoprostaglandin F1 alpha (0.20 +/- 0.11 ng/ml) were about the same, indicating relatively high beta-oxidation at low prostacyclin formation. Values are expressed as mean +/- S.D.

Prostacyclin metabolites, in urine or adults and neonates, studied by gas chromatography-mass spectrometry and radioimmunoassay.

Endogenous levels of two metabolites of prostacyclin, 6-keto-prostaglandin F1 alpha (spontaneous hydrolysis product) and 6,15-diketo-13,14-dihydroprostaglandin F1 alpha (enzymatic degradation product) were measured in urine of adults and neonates by gas chromatography-mass spectrometry, using deuterated internal standards. The method comprised extraction of prostanoids by reverse-phase cartridges and purification by silicic acid column chromatography and reverse-and straight -phase high-performance liquid chromatographies. Exogenous 6-keto-prostaglandin F1 alpha and 6,15-diketo-13,14-dihydroprostaglandin F1 alpha added to urine were recovered quantitatively by gas chromatography-mass spectrometry. Endogenous levels of 6-keto-prostaglandin F1 alpha in urine of adults were 0.11 +/- 0.05 (S.D.) ng/ml (n = 12), whereas in urine of neonates the levels were much higher: 1.41 +/- 0.36 (S.D.) ng/ml (n = 5) on the 3rd day of life declining to 0.51 +/- 0.21 (S.D.) ng/ml (n = 5) on the 5th day. 6-keto-prostaglandin F1 alpha was also estimated in both age groups by radioimmunoassay. Urinary levels of 6,15-diketo-13, 14-dihydroprostaglandin F1 alpha in neonates on the 3rd day of life were 2.12 +/- 0.70 (S.D.) ng/ml (n = 4) and declined until the 5th day. In adult urine this metabolite was below the limit of detection (0.20 ng/ml).

1,25-(OH)2 vitamin D-3 enhances Paf-stimulated calcium mobilization in U937 cells by expression of specific Paf binding sites.

The effect of 1,25-(OH)2 vitamin D-3 (10 nM, 72 h) on cytosolic free Ca2+ concentration ([Ca2+]i) in U937 cells before and after stimulation with Paf, LTD4 and ADP was investigated. 1,25-(OH)2 vitamin D-3 increased basal [Ca2+]i from 98 +/- 1 nM to 121 +/- 5 nM (P less than 0.01) and the Paf (10 nM) stimulated increase in [Ca2+]i from 143 +/- 15 to 406 +/- 44 nM (P less than 0.01). These vitamin D-3 effects were time-related and occurred after 24 h (basal [Ca2+]i) and 12 h (Paf stimulated Ca2(+)-mobilization) but not after 3 h. In comparison, vitamin D-3 failed to modulate Ca2(+)-mobilization in response to ADP (1-40 microM) and increased it only in response to low leukotriene D4 concentrations (0.1-1 nM). The total binding of [3H]Paf (2.8 nM) was not significantly different in untreated vs. vitamin D-3-treated cells. However, the Paf receptor antagonist Web 2086 (1 microM) inhibited [3H]Paf binding only in vitamin D-3-treated cells. The specific binding reached a plateau of 28 +/- 3 fmol per 2.5.10(6) cells between 1.4 and 2.8 nM [3H]Paf. The Paf receptor antagonist Web 2086 (1-1000 nM) also inhibited the Paf-mediated Ca2(+)-mobilization in vitamin D-3-treated cells (IC50 = 191 +/- 55 M). These data suggest that the enhanced Ca2(+)-mobilization in vitamin D-3-treated U937 cells in response to Paf is mediated by an expression of putative Paf receptors.

Cholesterol modulates PAF-stimulated Ca2(+)-mobilization in monocytic U937 cells.

We investigated the effect of cellular cholesterol content on platelet activating factor (PAF)-stimulated Ca2+ mobilization in the human monocytic cell line U937. When cholesterol auxotroph U937 cells were depleted of cellular cholesterol by a 48-h incubation in delipidated medium, a 40% reduction in the PAF (100 nM)-stimulated increase in cytosolic Ca2+ concentration was seen. Ca2+ mobilization following stimulation with LTD4 (10 nM) or ATP (10 microM) was not affected. Addition of LDL (100 micrograms/ml, 24 h) to the delipidated medium completely recovered cellular cholesterol content and PAF-induced Ca2+ mobilization. These two LDL effects had very similar time- and dose-dependences. Partial recoveries of PAF-induced Ca2+ mobilization, seen after addition of pure cholesterol dissolved in ethanol (30 micrograms/ml, 24 h) or acetyl-LDL (100 micrograms/ml, 24 h), were associated with partial recoveries of cellular cholesterol content. Our results indicate that cellular cholesterol influences PAF-stimulated events in monocytic cells.

Smoking alters thromboxane metabolism in man.

Chronic smoking is a major risk factor of atherosclerosis and coronary heart disease. The measurement of three major thromboxane A2 metabolites, 11-dehydrothromboxane B2, 2,3-dinorthromboxane B2 and thromboxane B2, in the urines of 13 apparently healthy smokers (average 39 years, range 27-56 years) showed significantly elevated excretion rates for all thromboxane A2 metabolites as compared to 10 apparently healthy age-matched non-smokers (average 37 years, range 26-56 years). Importantly, characteristic alterations in the thromboxane A2 metabolite pattern were found in the urines of smokers. The contribution of 2,3-dinorthromboxane B2 to total measured excretion of thromboxane A2 metabolites was 59.2% in smokers (404.0 +/- 53.0 pg/mg creatinine) versus 19.4% in non-smokers (85.2 +/- 8.3 pg/mg creatinine), that of 11-dehydrothromboxane B2 35.7% in smokers (673.2 +/- 88.9 pg/mg creatinine) as compared to 75.5% in non-smokers (332.6 +/- 30.9 pg/mg creatinine). The contribution of thromboxane B2 (57.5 +/- 7.7 pg/mg creatinine in smokers versus 21.9 +/- 1.5 pg/mg creatinine in non-smokers) was similar at 5.1%. The excretion of cotinine, the major urinary metabolite of nicotine that correlates well with the reported daily cigarette consumption (r = 0.97, P less than 0.0001), showed a good correlation to thromboxane A2 metabolite excretion (2,3-dinorthromboxane B2: r = 0.92, P less than 0.0001; 11-dehydrothromboxane B2; r = 0.87, P less than 0.0001).

Docosahexaenoic acid inhibits PAF and LTD4 stimulated [Ca2+]i-increase in differentiated monocytic U937 cells.

We investigated the effects of different polyunsaturated fatty acids (PUFAs) of the n-6 and n-3 family on the PAF and LTD4 stimulated increase in cytosolic free Ca(2+)-concentration [Ca2+]i in retinoic acid (RA) differentiated, human monocytic U937 cells. Docosahexaenoic acid (10 microM DHA) reduced the PAF induced increase in [Ca2+]i from 455 +/- 25 nM to 319 +/- 24 nM (P less than 0.01). DHA also significantly attenuated the LTD4 induced increase in [Ca2+]. However [Ca2+]i-increase stimulated by f-MLP, ATP, or ionophore A 23187 was not affected by DHA. Other PUFAs like eicosapentaenoic acid (EPA), alpha-linolenic acid (LnA), arachidonic acid (AA) or gamma-linoleic acid (LA) were ineffective. Cellular differentiation as assessed by nitrobluetetrazolium reduction and enhanced expression of specific PAF binding sites in RA treated cells were not altered by DHA. Fatty acid composition in cellular phospholipids revealed effective incorporation of each PUFA. The DHA-effect on [Ca2+]i was time dependent and occurred at 48 h, whereas the DHA-content in phospholipids reached a plateau already at 24 h. The antioxidant vitamin E, the lipoxygenase inhibitor NDGA and the cytochrome P-450 inhibitor SKF 525A completely prevented the DHA induced reduction of PAF stimulated [Ca2+]i-increase. In contrast, the cyclooxygenase inhibitor indomethacin had no effect. Our results indicate that DHA selectively reduces intracellular [Ca2+]i-increases induced by PAF and LTD4 in RA-treated U937 cells, presumably involving an oxidative modification of DHA.

Formation of prostacyclin and thromboxane in man as measured by the main urinary metabolites.

Excretion of 2,3-dinor-6-ketoprostaglandin F1 alpha and 2,3-dinorthromboxane B2, the main urinary metabolites of prostacyclin and thromboxane, was evaluated by gas chromatography-mass spectroscopy and radioimmunoassay, respectively, at various conditions in man. In healthy young males excretion of 2,3-dinor-6-ketoprostaglandin F1 alpha was of little variability, whereas urinary 2,3-dinorthromboxane B2 showed marked interindividual but moderate intraindividual variations. The ratio of urinary 2,3-dinorthromboxane B2 to thromboxane B2 in young males was about 15. Excretion of 2,3-dinor-6-ketoprostaglandin F1 alpha in women of reproductive age was higher (155 +/- 23 ng/g creatinine, P less than 0.005) than in postmenopausal women (97 +/- 24 ng/g creatinine) and in men (78 +/- 7.6 ng/g creatinine) and increased significantly during pregnancy (1st trimester 230 +/- 50 ng/g creatinine; 3rd trimester 522 +/- 53 ng/g creatinine). Urinary 2,3-dinorthromboxane B2 showed no gender differences and no directed change was observed during pregnancy. In neonates urinary 2,3-dinorthromboxane B2 (6.328 +/- 1.210 ng/g creatinine) was high in their 3rd day of life and decreased rapidly thereafter. This pattern paralleled the behavior of 6-ketoprostaglandin F1 alpha. In young male smokers and non-smokers excretion of 2,3-dinor-6-ketoprostaglandin F1 alpha was not significantly different, whereas urinary 2,3-dinorthromboxane B2 was elevated in smokers (609 +/- 61 versus 351 +/- 41 ng/g creatinine, P less than 0.001). Values are mean +/- S.E.

Linoleic acid esterified in low density lipoprotein serves as substrate for increased arachidonic acid synthesis in differentiating monocytic cells.

The cellular metabolism of albumin- and lipoprotein-bound 18:2(n - 6) during monocytic differentiation was examined in the human premonocytic U937 and Mono Mac 6 cells. Differentiation for 72 h of U937 cells with retinoic acid (RA, 1 microM) or 1,25-(OH)2-vitamin D3 (1,25-D3, 10 nM) and of Mono Mac 6 cells with RA (1 microM) or lipopolysaccharide (LPS, 10 ng/ml) increased the desaturation and elongation of [1-14C]18:2(n - 6) to [1-14C]20:4(n - 6). In undifferentiated U937 and Mono Mac 6 cells, incubations with human LDL (100 micrograms/ml, 18 h) resulted in a 2.5-fold increase in 18:2(n - 6) levels in the cellular phospholipids. Differentiation of U937 cells with RA or or of Mono Mac 6 cells with LPS prior to LDL addition. Significantly reduced 18:2(n - 6) and elevated 20:4(n - 6) levels in cellular phospholipids. This increase in 20:4(n - 6) was likely not due to an increased incorporation of preformed 20:4(n - 6) esterified in LDL, as the receptor-specific degradation of [125I]LDL was reduced in both the RA-treated U937 and LPS-treated Mono Mac 6 cells. In U937 cells incubated with [1-14C]18:2(n - 6), the synthesis of TXB2, PGE2 and HHT could be detected after differentiation with RA. suggesting the availability of [1-14C]20:4(n - 6), derived from [1-14C]18:2(n - 6), for cyclooxygenase metabolism. Our results show that the conversion of 18:2(n - 6) to 20:4(n - 6) increases during monocyte differentiation. The 18:2(n - 6) supplied to the cells via the receptor-mediated uptake of LDL was utilized as substrate for the increased 20:4(n - 6) synthesis.

Cooperative ligand reorientations in cytochrome c3: a molecular dynamics simulation.

Molecular dynamics simulations of a tetraheme cytochrome c3 were performed to investigate dynamic aspects of the motion of the axial heme iron ligands. It was found that persistent transitions between alternate axial imidazole orientations of the histidine incorporated in the CXXCH heme binding sequence occurred via correlated motions. The correlated motions involved virtually all of the atoms comprising the polypeptide backbone of the heme binding sequence as well as the histidine imidazole side-chain.