Redox components and structure of the respiratory NADH:ubiquinone oxidoreductase (complex I)

Surgical ablation of extrinsic cardiac nerve fibers results in a chronically denervated state of the left ventricle of the heart. The present study was performed to elucidate the effect of a period of 5 weeks of chronic denervation on cardiac catecholamine levels in general and dopamine in particular. Moreover, the possible effect on cardiac beta-adrenoceptor subtypes was investigated. Experiments were performed on adult dogs. In addition to adrenaline and noradrenaline the tissue levels of dopamine were found to be severely depressed. A significant shift from beta1- to beta2-adrenoceptor subtype was observed, while the total beta-adrenoceptor density remained unaffected. The present findings indicate that catecholamine synthesis in chronically denervated hearts is impaired upstream of dopamine and that a shift in beta-adrenoceptor subtype occurs already within a relatively short period of five weeks of denervation, and suggest that the lack of endogenous catecholamines influence the relative expression levels of the two subtypes of beta-adrenoceptors present in cardiac tissue.     

Mitochondrial NADH:ubiquinone oxidoreductase (complex I): proximity of the subunits of the flavoprotein and the iron-sulfur protein subcomplexes

The proximities of the three subunits (51, 24, and 9 kDa) of the flavoprotein subcomplex (FP) and five subunits (75, 49, 30, 18, and 13) of the iron-sulfur protein subcomplex (IP) of the bovine NADH: ubiquinone oxidoreductase (complex I) were investigated by cross-linking studies. The cross-linking reagents used were disuccinimidyl tartrate and ethylene glycol bis(succinimidyl succinate). The cross-linked products were identified by sodium dodecyl sulfate gel electrophoresis and immunoblotting with antibodies specific for each subunit. Results showed that the three FP subunits are juxtaposed to one another, and only the 51 kDa subunit of FP is in close proximity to only the 75-kDa subunit of IP. The 75-kDa subunit cross-linked to the 30- and the 13-kDa subunits, the 49-kDa subunit cross-linked to the 30-, 18-, and 13-kDa subunits, and the 30-kDa subunit cross-linked to the 18- and the 13-kDa subunits. No cross-linked products of 75+49-, 75+18-, or 18+13-kDa subunits were detected. These results are consistent with the occurrence of potential electron carriers in FP and IP subunits. These electron carriers are FMN and one iron-sulfur cluster in the 51-kDa subunit, one iron-sulfur cluster in the 24-kDa subunit, and apparently two iron-sulfur clusters in the 75-kDa subunit.     

Steady-state kinetics of the reduction of coenzyme Q analogs by complex I (NADH:ubiquinone oxidoreductase) in bovine heart mitochondria and submitochondrial particles

The reduction kinetics of coenzyme Q (CoQ, ubiquinone) by NADH:ubiquinone oxidoreductase (complex I, EC 1.6.99.3) was investigated in bovine heart mitochondrial membranes using water-soluble homologs and analogs of the endogenous ubiquinone acceptor CoQ10 [the lower homologs from CoQ0 to CoQ3, the 6-pentyl (PB) and 6-decyl (DB) analogs, and duroquinone]. By far the best substrates in bovine heart submitochondrial particles are CoQ1 and PB. The kinetics of NADH-CoQ reductase was investigated in detail using CoQ1 and PB as acceptors. The kinetic pattern follows a ping-pong mechanism; the Km for CoQ1 is in the range of 20 microM but is reversibly increased to 60 microM by extraction of the endogenous CoQ10. The increased Km in CoQ10-depleted membranes indicates that endogenous ubiquinone not only does not exert significant product inhibition but rather is required for the appropriate structure of the acceptor site. The much lower Vmax with CoQ2 but not with DB as acceptor, associated with an almost identical Km, suggests that the sites for endogenous ubiquinone bind 6-isoprenyl- and 6-alkylubiquinones with similar affinity, but the mode of electron transfer is less efficient with CoQ2. The Kmin (kcat/Km) for CoQ1 is 4 orders of magnitude lower than the bimolecular collisional constant calculated from fluorescence quenching of membrane probes; moreover, the activation energy calculated from Arrhenius plots of kmin is much higher than that of the collisional quenching constants. These observations strongly suggest that the interaction of the exogenous quinones with the enzyme is not diffusion-controlled. Contrary to other systems, in bovine submitochondrial particles, CoQ1 usually appears to be able to support a rate approaching that of endogenous CoQ10, as shown by application of the "pool equation" [Kr?ger, A., & Klingenberg, M. (1973) Eur. J. Biochem. 39, 313-323] relating the rate of ubiquinone reduction to the rate of ubiquinol oxidation and the overall rate through the ubiquinone pool.     

Pterulinic acid and pterulone, two novel inhibitors of NADH:ubiquinone oxidoreductase (complex I) produced by a Pterula species. II. Physico-chemical properties and structure elucidation

The structures of two novel fungal antibiotics, isolated from a Pterula species, that interfere with the NADH:ubiquinone oxidoreductase and inhibit the respiration of eucaryotes, were determined by spectroscopic techniques. Both compounds, pterulinic acid (1a) and pterulone (2), contain a 1-benzoxepin ring system and are chlorinated. Pterulinic acid (1a), which was obtained as a 1:5 inseparable mixture of the two isomers (Z)-1a and (E)-1a, in addition contains a furan. Their structures were determined by mass spectrometry and NMR spectroscopy, and 2D heteronuclear correlation experiments permitted the assignment of all NMR signals.     

Indolequinone antitumor agents: correlation between quinone structure, rate of metabolism by recombinant human NAD(P)H:quinone oxidoreductase, and in vitro cytotoxicity

A series of indolequinones bearing various functional groups has been synthesized, and the effects of substituents on the metabolism of the quinones by recombinant human NAD(P)H:quinone oxidoreductase (NQO1) were studied. Thus 5-methoxyindolequinones were prepared by the Nenitzescu reaction, followed by functional group interconversions. The methoxy group was subsequently displaced by amine nucleophiles to give a series of amine-substituted quinones. Metabolism of the quinones by NQO1 revealed that, in general, compounds with electron-withdrawing groups at the indole 3-position were among the best substrates, whereas those with amine groups at the 5-position were poor substrates. Compounds with a leaving group at the 3-indolyl methyl position generally inactivated the enzyme. The toxicity toward non-small-cell lung cancer cells with either high NQO1 activity (H460) or no detectable activity (H596) was also studied in representative quinones. Compounds which were good substrates for NQO1 showed the highest selectivity between the two cell lines.     

Immunolocalization of latent transforming growth factor-beta binding protein-1 (LTBP1) during mouse development: possible roles in epithelial and mesenchymal cytodifferentiation

Twenty-five conformation-dependent monoclonal antibodies (MAbs) produced by immunization of mice with oligomeric forms of the human immunodeficiency virus type 1 (HIV-1) envelope (env) glycoprotein were used to map exposed, immunogenic regions on oligomeric env. Based on MAb cross-competition, reactivity with diverse env proteins, and reactivity with a panel of gp120 mutants, seven distinct epitope clusters were identified. These include the classic CD4 binding site, V1/V2, and V3. in addition, several novel epitope clusters, including one mapping to the N- and C-termini of gp120, were identified. The locations of the seven epitope clusters on the gp120 core structure are proposed.     

Distinct sites of insulin-like growth factor (IGF)-II expression and localization in lesioned rat brain: possible roles of IGF binding proteins (IGFBPs) in the mediation of IGF-II activity

Although expression of the IGF-II has been demonstrated within the central nervous system (CNS), past studies have failed to reveal its precise roles or responses subsequent to a traumatic injury. To demonstrate that IGF-II, IGFBP, and IGF receptor (-R) expression alters in response to a penetrating CNS injury, we used the techniques of ribonuclease protection assay, in situ hybridization, immunohistochemistry, Western blotting, and RIA. Under normal physiology, IGF-II expression is restricted to the mesenchymal support structures of the brain, including the choroid plexus, where its expression is coincident with that of IGFBP-2. Between 1-7 days post lesion (dpl), in the acute phase following a penetrant wound to the CNS, IGF-II and IGF-IIR protein, but not messenger RNA, were colocalized, with IGF-I, IGF-IR, and IGFBP-1, -2, -3, and -6, to neurons, macrophages, astrocytes, and microglia within the damaged tissue. Within the cerebrospinal fluid (CSF), levels of IGF-II peptide increased to peak at 7 dpl. IGFBP-2, -3, and -6 were also observed within the CSF, with IGFBP-2 predominating and exhibiting an increase in binding efficiency from 7-10 dpl. In the chronic phase of injury (7-14 dpl), an increase in both IGF-II, IGF-IIR and IGFBP-5 messenger RNA and protein was observed specifically and focally in the marginal astrocytes forming the limiting glial membrane of the wound. Thus, our evidence suggests that there are two mechanisms of action for IGF-II within the injured rat brain. During the acute phase, the secretion of IGF-II from the choroid plexus into the CSF is up-regulated, resulting in increased transport of the peptide to the wound. In the CSF, transported IGF-II is complexed to IGFBP-2 and essentially demonstrates an endocrine mode of action with a balance of locally produced IGFBPs modulating its bioactivity in the wound. Later in the wounding response, levels of IGF-II decline in the CSF and the wound neuropil, possibly with the aid of increased IGFBP-5 levels that may help to locally sequester and down-regulate IGF-II activity. Hence, in the chronic phase of the injury response, IGF-II reasserts itself to a predominantly autocrine/paracrine role restricted to the mesenchymal support structures, including the glia limitans, which may help reestablish and maintain tissue homeostasis.     

The effects of test temperature,temper, and alloyed copper on the hydrogen-controlled crack growth rate of an Al-Zn-Mg-(Cu) alloy

The hydrogen-environment embrittlement (HEE)-controlled stage II crack growth rate of AA 7050 (6.09 wt pct Zn, 2.14 wt pct Mg, and 2.19 wt pct Cu) was investigated as a function of temper and alloyed copper level in a humid air environment at various temperatures. Three tempers representing the underaged (UA), peak-aged (PA), and overaged (OA) conditions were tested in 90 pct relative humidity (RH) air at temperatures between 25 °C and 90 °C. At all test temperatures, an increased degree of aging (from UA to OA) produced slower stage II crack growth rates. The stage II crack growth rate of each alloy and temper displayed an Arrhenius-type temperature dependence, with activation energies between 58 and 99 kJ/mol. For both the normal-copper and low-copper alloys, the fracture path was predominately intergranular at all test temperatures (25 °C to 90 °C) in each temper investigated. Comparison of the stage II HEE crack growth rates for normal- (2.19 wt pct) and low- (0.06 wt pct) copper alloys in the peak PA aged and OA tempers showed a beneficial effect of copper additions on the stage II crack growth rate in humid air. In the 2.19 wt pct copper alloy, the significant decrease (∼10 times at 25 °C) in the stage II crack growth rate upon overaging is attributed to an increase in the apparent activation energy for crack growth. In the 0.06 wt pct copper alloy, overaging did not increase the activation energy for crack growth but did lower the pre-exponential factor (v ₀), resulting in a modest (∼2.5 times at 25 °C) decrease in the crack growth rate. These results indicate that alloyed copper and thermal aging affect the kinetic factors that govern stage II HEE crack growth rates. The OA, copper-bearing alloys are not intrinsically immune to hydrogen-environment-assisted cracking, but are more resistant due to an increased apparent activation energy for stage II crack growth. An erratum to this article is available at .     

The effects of test temperature,temper, and alloyed copper on the hydrogen-controlled crack growth rate of an Al-Zn-Mg-(Cu) alloy

The hydrogen-environment embrittlement (HEE)-controlled stage II crack growth rate of AA 7050 (6.09 wt pct Zn, 2.14 wt pct Mg, and 2.19 wt pct Cu) was investigated as a function of temper and alloyed copper level in a humid air environment at various temperatures. Three tempers representing the underaged (UA), peak-aged (PA), and overaged (OA) conditions were tested in 90 pct relative humidity (RH) air at temperatures between 25 °C and 90 °C. At all test temperatures, an increased degree of aging (from UA to OA) produced slower stage II crack growth rates. The stage II crack growth rate of each alloy and temper displayed an Arrhenius-type temperature dependence, with activation energies between 58 and 99 kJ/mol. For both the normal-copper and low-copper alloys, the fracture path was predominately intergranular at all test temperatures (25 °C to 90 °C) in each temper investigated. Comparison of the stage II HEE crack growth rates for normal- (2.19 wt pct) and low- (0.06 wt pct) copper alloys in the peak PA aged and OA tempers showed a beneficial effect of copper additions on the stage II crack growth rate in humid air. In the 2.19 wt pct copper alloy, the significant decrease (∼10 times at 25 °C) in the stage II crack growth rate upon overaging is attributed to an increase in the apparent activation energy for crack growth. In the 0.06 wt pct copper alloy, overaging did not increase the activation energy for crack growth but did lower the pre-exponential factor (v ₀), resulting in a modest (∼2.5 times at 25 °C) decrease in the crack growth rate. These results indicate that alloyed copper and thermal aging affect the kinetic factors that govern stage II HEE crack growth rates. The OA, copper-bearing alloys are not intrinsically immune to hydrogen-environment-assisted cracking, but are more resistant due to an increased apparent activation energy for stage II crack growth.     

Calculation of the reduction rate of copper,nickel, and cobalt from oxide melts by carbon(II) oxide

The kinetic equation for the calculation of the reduction rate, which takes into account the physicochemical properties of contacting phases, is derived under the assumption of the electrochemical nature of interaction between the reducing gas (CO) and the oxide melt. Satisfactory agreement of calculated and experimental data is shown by the example of the interaction of the oxide melt of the CaO-SiO₂-Al₂O₃ system containing up to 6.0 wt % Me _n O _m (NiO, CoO, Cu₂O) blown by the gas phase with partial pressure P _CO = (0.4–5.0) × 10² MPa at T = 1623 K.     

Effect of microstructure,strength, and oxygen content on fatigue crack growth rate of Ti-4.5AI-5.0Mo-1.5Cr (CORONA 5)

Fatigue crack growth rate behavior in CORONA 5, an alloy developed for applications requiring high fracture toughness, has been examined for eight material conditions. These conditions were designed to give differences in microstructure, strength level (825 to 1100 MPa [120 to 160 ksi]), and oxygen content (0.100 to 0.174 wt pct), in such a manner that the separate effects of these variables could be defined. For all eight conditions, fatigue crack growth rates (da/dN) are virtually indistinguishable over the full spectrum of stress-intensity range (ΔK) examined,viz., 8 to 40 MPa√m (7 to 36 ksi√in). Concomitantly, it is noted that over the sizable solution annealing range studied (830° to 915 °C [1525° to 1675 °F]), the primary α-phase morphology was substantially invariant. Eachda/dN curve exhibits a bilinear form with a transition point (ΔKT) between 16 and 19 MPa√m (15 and 17 ksi√in). A change in microfractographic appearance occurs at ΔKT, as extensive secondary cracking along α/β interfaces is observed at all hypertransitional levels ofAK, but not for AK < ΔKT. For each material condition, the mean length of primary α platelets is approximately the same as the cyclic plastic zone size at ΔKT. Accordingly, locations ofAKT (and their similarity for the different material conditions) are rationalized in conformance with a cyclic plastic zone model of fatigue crack growth. Finally, the difference in behavior of CORONA 5, as compared to conventional α/β alloys such as Ti-6A1-4V, is rationalized in terms of crack path behavior.     

Rapid epitaxial growth of conducting and insulating III-V compounds on (001), (110), (111)A, (311)A, and (311)B surfaces by hydride vapor phase epitaxy

Rapid growth of conducting or insulating InP, GaAs, Ga_0.47In_0.53As, and GaAs_0.6P_0.4 on one or more of substrates of orientations (001), (110), (111)A, (311)A, and (311)B by hydride vapor phase epitaxy (HVPE) is demonstrated. The maximum growth rate of the binaries lies between 12 and 300 μm/h and that of the ternaries between 7 and 170 μm/h. A simple model is developed to describe the influence of crystallographic orientation on temperature-dependent growth rates. The model is compared with the experimental data. Room-temperature resistivity of insulating InP:Fe can be as high as 5 × 10⁹ ohm cm. Temperature-dependent differential resistivity is also analyzed. This feasibility of growing conducting and insulating layers rapidly on substrates of different orientations is very useful for electronic and optoelectronic devices.     

Kinase suppressor of Ras inhibits the activation of extracellular ligand-regulated (ERK) mitogen-activated protein (MAP) kinase by growth factors, activated Ras, and Ras effectors

Kinase suppressor of Ras (KSR) is a loss-of-function allele that suppresses the rough eye phenotype of activated Ras in Drosophila and the multivulval phenotype of activated Ras in Caenorhabditis elegans. Genetic and biochemical studies suggest that KSR is a positive regulator of Ras signaling that functions between Ras and Raf or in a pathway parallel to Raf. We examined the effect of mammalian KSR expression on the activation of extracellular ligand-regulated (ERK) mitogen-activated protein (MAP) kinase in fibroblasts. Ectopic expression of KSR inhibited the activation of ERK MAP kinase by insulin, phorbol ester, or activated alleles of Ras, Raf, and mitogen and extracellular-regulated kinase. Expression of deletion mutants of KSR demonstrated that the KSR kinase domain was necessary and sufficient for the inhibitory effect of KSR on ERK MAP kinase activity. KSR inhibited cell transformation by activated RasVal-12 but had no effect on the ability of RasVal-12 to induce membrane ruffling. These data indicate that KSR is a potent modulator of a signaling pathway essential to normal and oncogenic cell growth and development.     

Mouse serum amyloid A (SAA) proteins isolated by two-dimensional electrophoresis: characterization of isotypes and the effect of separate and combined administrations of cytokines, dexamethasone and lipopolysaccharide (LPS) on serum levels and isotype distribution

Hydrophobic interaction chromatography and two-dimensional electrophoresis were used to isolate and characterize mouse SAA, and to study the in vivo effect of separate or combined administrations of cytokines, dexamethasone (DEX) and LPS on mouse SAA. Four SAA spots containing partial amino acid sequence in accordance with mouse apoSAA and apoSAA2/SAA(SJL/J) pI 5.9 were demonstrated in serum. One of these proteins represents a previously undescribed, acidic acute-phase mouse SAA protein. Both DEX and interferon-gamma (IFN-gamma) proved to be capable of increasing SAA serum levels. In contrast to what has been shown in previous in vivo studies, administration of IL-6 did increase the SAA levels to nearly the same magnitude as IL-1, and the effect of IL-6 and LPS on SAA production was not significantly altered by the addition of DEX. Irrespective of the inflammatory stimuli that was administered, a non-selective production of SAA1 and SAA2 was observed in most groups, including the group that received IL-6. The results illustrate that data obtained about mouse SAA are highly dependent on which models, isolation and identification methods are used.