Impact of age on cardiovascular function, inflammation, and oxidative stress in experimental asphyxial cardiac arrest.

BACKGROUND: Advanced age is an independent predictor of poor outcome after cardiac arrest (CA). From experimental studies of regional ischemia-reperfusion injury, advanced age is associated with larger infarct size, reduced organ function, and augmented oxidative stress. The objective of this study was to investigate the effect of age on cardiovascular function, oxidative stress, inflammation, and endothelial activation after CA representing global ischemia-reperfusion. METHODS: Aged (26 months) and young (5 months) rats were subjected to 8 min of asphyxia induced CA, resuscitated and observed for 360 min. Left ventricular pressure-derived cardiac function was measured at baseline and 360 min after CA. Blood samples obtained at baseline, 120 min, and 360 min after CA were analyzed for IL-1ß, IL-6, IL-10, TNF-α, elastase, sE-selectin, sL-selectin, sI-CAM1, hemeoxygenase-1 (HO-1) and protein carbonyl. Tissue samples of brain, heart, kidney, and lung were analyzed for HO-1. RESULTS: Cardiac function, evaluated by dP/dtmax and dP/dtmin , was decreased after CA in both young and aged rats, with no group differences. Mean arterial pressure increased after CA in young, but not old rats. Aged rats showed significantly higher plasma levels of elastase and sE-selectin after CA, and there was a significant different development over time between groups for IL-6 and IL-10. Young rats showed higher levels of HO-1 in plasma and renal tissue after CA. CONCLUSION: In a rat model of asphyxial CA, advanced age is associated with an attenuated hyperdynamic blood pressure response and increased endothelial activation.

Structure of porcine insulin cocrystallized with clupeine Z.

The crystal structure of NPH-insulin, pig insulin cocrystallized with zinc, m-cresol and protamine, has been solved by molecular replacement and refined using restrained least-squares refinement methods. The final crystallographic R factor for all reflections between 2 and 10 A is 19.4%. The insulin molecules are arranged as hexamers with two tetrahedrally coordinated Zn atoms in the central channel and one m-cresol bound to each monomer near His B5. One protamine binding site has been unequivocally identified near a dimer-dimer interface, although most of the polypeptide is crystallographically disordered. The conformation of the insulin moiety and the structural differences between the three unique monomers have been analysed. The zinc and m-cresol environments are described and the nature of the protamine binding site is outlined.

Proton nuclear magnetic resonance study of the B9(Asp) mutant of human insulin. Sequential assignment and secondary structure.

The sequence-specific 1H nuclear magnetic resonance (n.m.r.) assignment of 49 of the 51 amino acid residues of human B9(Asp) insulin in water at low pH is reported. Spin systems were identified using a series of two-dimensional n.m.r. techniques. For the majority of the amino acid residues with unique spin systems, particularly Ala, Thr, Val, Leu, Ile and Lys, the complete spin systems were identified. Sequence-specific assignments were obtained from sequential nuclear Overhauser enhancement (NOE) connectivities. The results indicate that the solution structure of the mutant closely resembles the crystal structure of native insulin. Thus, the NOE data reveal three helical domains all consistent with the secondary structure of the native human 2Zn insulin in the crystal phase. Numerous slowly exchanging amide protons support these structural elements, and indicate a relatively stable structure of the protein. A corresponding resemblance of the tertiary structures in the two phases is also suggested by slowly exchanging amide protons, and by the extreme chemical shift values observed for the beta-protons of B15(Leu) that agree with a close contact between this residue and the aromatic rings of B24(Phe) and B26(Tyr), as found in the crystal structure of the 2Zn insulin. Finally, there are clear indications that the B9(Asp) insulin mutant exists primarily as a dimer under the given conditions.

Magnetization-transfer n.m.r. investigation of the hydrogen exchange in H2O of the peptide fragment B23-B29 of insulin.

Detailed and precise information on the exchanges in water of the peptide hydrogens of the insulin fragment B23-B29 (Gly23-Phe24-Phe25-Tyr26-Thr27-Pro28 -Lys29) has been obtained from magnetization-transfer measurements, and nonlinear least-squares fits of the experimental spectra using the expression for the discrete Fourier transform of a sum of exponentially damped sinusoids. From a comparison of the differential exchange rates with those expected for completely solvent-exposed peptide hydrogens, specific conformational features of the heptapeptide are suggested.

Crystallization and X-ray data collection on human growth hormone.

Single crystals of natural sequence human growth hormone have been grown from media containing ethanol, acetone or paraldehyde. Recombinant growth hormone in its native and desamidated form and pituitary hormone have been crystallized. A full native set of diffraction data extending to 3.5 A resolution has been obtained with synchrotron radiation for crystals of recombinant human growth hormone grown from ethanol. The identity of the material in these crystals has been established by anion-exchange chromatography.

Plasma desorption mass spectrometry, an analytical tool in protein engineering: characterization of modified insulins.

Californium-252 plasma desorption mass spectrometry (PDMS) has been employed for the characterization of a series of human insulin derivatives in order to evaluate the performance of this technique as an analytical tool in protein engineering. Several of the characterized modifications result in a 1 a.m.u. mass change. The precision in mass determination obtainable by PDMS analysis is not sufficient for unambiguous verification of such modifications based on the molecular weight alone. It is, however, possible to carry out in situ enzymatic digestion of the sample. Subsequent PDMS analysis will in most cases reveal if the modification has been introduced as intended.

Phenol-promoted structural transformation of insulin in solution.

Phenolic additives widely used for the preservation of insulin preparations can have a profound effect on the hormone's conformation in solution. m-Cresol, for instance, increases the circular dichroism in the far ultraviolet by 10-20%, corresponding to an increase in helix, and around 255 nm. The CD-spectral changes are strikingly similar to those brought about by halide ions which have been identified to reflect the 2 Zn----4 Zn insulin transition. Its most prominent element is the helix formation at the B-chain N-terminus. In both cases the changes fail to occur with dimeric insulin in the absence of Zn2 and with monomeric des-(B26-B30)-insulin. In the presence of Ni2 which is unable to replace Zn2 in 4 Zn insulin for coordinative reasons, the effect of m-cresol is impeded. m-Cresol thus induces a transition identical with or closely similar to the 2 Zn----4 Zn transformation. 2 Zn insulin crystals, when soaked in m-cresol containing solvents, are destroyed. Crystals grown in the presence of m-cresol, however, are monoclinic and containing symmetrical hexamers of, notably, 4 Zn conformation. Phenol, o- and p-cresol, m-nitrophenol, Nipagin M and benzene were further additives tested, all of them inducing largely the same spectral effects except for benzene. The results presented corroborate the close correspondence of insulin's structure in solution and in the crystal as well as insulin's capacity for structural variation.

Klebsiella pneumoniae nifM gene product is required for stabilization and activation of nitrogenase iron protein in Escherichia coli.

A series of plasmids encoding various Klebsiella pneumoniae nif (nitrogen fixation) genes were constructed to determine which were required to produce active iron (Fe) protein in Escherichia coli, a species which does not normally fix nitrogen. The greatest success was achieved with binary plasmid systems that produced nifA regulatory protein under the control of a tac promoter on one plasmid, which then induced synthesis of nifH and nifM proteins from their native promoter sites on a second plasmid. nifH protein, the monomeric subunit of Fe protein, produced in the presence of nifM constituted nearly 10% of the whole cell protein and exhibited the corresponding amount of C2H2-reducing activity in nitrogenase assays conducted in vitro. nifH protein formed in the absence of nifM constituted 4.7% of the whole cell protein and exhibited no detectable activity in assays of whole cell extracts. The plasmid-encoded Fe protein was purified to homogeneity and was found to be indistinguishable from that isolated from derepressed wild type K. pneumoniae, having a similar specific activity, approximately 4 Fe/dimer of 68 kDa, and similar epr features. Although these experiments do not exclude the participation of other E. coli gene products in the maturation of nifH protein, they limit the nif-specific genes required for active Fe protein production to nifA, nifH, and nifM. Since nifA is thought to be the required activator protein involved in nif operon transcription, the simplest explanation for these observations is that nifH codes for the peptide of the Fe protein, while nifM acts to convert this nifH peptide to the functioning Fe protein of nitrogenase. In the absence of nifM, only an inactive nifH polypeptide is produced.

Solvent isotope effects and the pH dependence of laccase activity under steady-state conditions.

Solvent isotope effects and the pH dependence of laccase catalysis under steady-state conditions were examined with a rapid reductant to assess the potential roles of protein protic groups and the catalytic mechanism. The pH dependence of both reductant-dependent and reductant-independent steps showed bell-shaped profiles implicating at least two protic groups in each case. The apparent pKa values were: for the reductant-independent step(s), pK alpha 1 = 8.98 +/- 0.02 and pK alpha 2 = 5.91 +/- 0.03; for the reductant-dependent step(s), pK' alpha 1 = 7.55 +/- 0.12, pK' alpha 2 = 8.40 +/- 0.23. No solvent isotope effect on reductant-dependent steps was detected other than a standard shift effect. However, a significant solvent isotope effect on a reductant-independent step(s) was observed; kH/kD = 2.12 at the pH optimum of 7.5. The concentration dependence of the D2O effect indicated that a single proton was involved. Simulations of the p(H,D) data suggested that the solvent isotope effect was associated with the protein protic group required in its undissociated form (pK alpha 2). The pH effects on reductant-dependent steps are apparently associated with reductant-dependent steps that occur between O2 binding and water formation in the catalytic reaction sequence.

Effect of ionophores on carrier-mediated electron translocation in ferricyanide-containing liposomes.

Ferricyanide-containing liposomes were used as a system to compare the electron- and proton-translocating properties of six redox reagents commonly used as electron donors for biochemical systems. The effects of different ionophore combinations on the ferricyanide-reduction rate were generally consistent with the expected proton- and electron-translocating properties of the mediators. The transmembrane pH gradient produced by hydrogen carriers was demonstrated. Nigericin or valinomycin plus carbonyl cyanide p-trifluoromethoxyphenylhydrazone are capable of collapsing this gradient and of stimulating ferricyanide reduction mediated by this type of carrier. No pH gradient is produced with the electron carrier 1,1'-dibutylferrocene. In the presence of tetraphenylboron anion, which is needed for this carrier to act as an efficient mediator, addition of valinomycin alone is sufficient to obtain full stimulation of ferricyanide reduction. NNN'N'-Tetramethyl-p-phenylenediamine does not behave as a simple electron carrier. During NNN'N'-tetramethyl-p-phenylenediamine-mediated ferricyanide reduction protons are translocated across the membrane and accumulated in the vesicles. This is not due to the presence of demethylated impurities in the NNN'N'-tetramethyl-p-phenylenediamine sample, but may be the result of an accumulation of oxidation products other than the Wurster's Blue radical. These results suggest a reconsideration of studies on protonmotive forces across membranes where NNN'N'-tetramethyl-p-phenylenediamine is used as a mediator.

Control of respiration in proteoliposomes containing cytochrome aa3. II. Inhibition by carbon monoxide and azide.

1. Carbon monoxide (CO) acts competitively towards oxygen when the latter is taken up in respiration by cytochrome aa3-containing proteoliposomes, both in the presence of p-trifluoromethoxy carbonyl cyanide phenylhydrazone and valinomycin (deenergized state) and in their absence (energized state). At high levels of CO, the double reciprocal plots (1/v vs. 1/[O2]) in the energized and deenergized states are parallel, i.e. energization acts "anti-competitively" towards oxygen, and the "respiratory control ratio" decreases as the oxygen concentration decreases. 2. Azide acts non-competitively towards cytochrome c when the latter is oxidized by cytochrome aa3-containing proteoliposomes both in the energized and deenergized (plus p-trifluoromethoxy carbonyl cyanide phenylhydrazone and valinomycin) conditions. At low azide concentrations the apparent Ki for azide is unaffected by energization, but at high azide levels the Ki increases in energized liposomes, i.e. the "respiratory control ratio" decreases as the azide concentration increases. 3. It is concluded that the inhibitor experiments are consistent with but do not prove the concept that the oxidase molecules in a single vesicle are responding to a single "energization state" or set of electrochemical gradients. This and other models are discussed.

Control of respiration in proteoliposomes containing cytochrome aa3. I. Stimulation by valinomycin and uncoupler.

1. Both valinomycin and p-trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP) are required for full release of respiration by cytochrome c oxidase-containing proteoliposomes (prepared by sonicating beef heart cytochrome aa3 in salt solution with 4 parts phosphatidylcholine, 4 parts phosphatidylethanolamine and 2 parts cardiolipin) in the presence of external ascorbate and cytochrome c. In the absence of valinomycin the response to FCCP is rather sluggish, as reported by Wrigglesworth et al. (1976) (Abstracts, 10th Int. Congr. Biochem., No. 06-6-230). 2. The Km for cytochrome c in 67 mM, pH 7.4, phosphate buffer with ascorbate as substrate, was 9 micrometer in both absence and presence of valinomycin and FCCP. Energization thus acts non-competitively towards cytochrome c oxidation. 3. The apparent Km for oxygen is greater in the energized than in the deenergized state; double reciprocal plots of respiration rate versus oxygen concentration are concave downward in the absence of uncouplers, as found with intact mitochondria. Energization thus acts "competitively" towards oxygen. 4. Despite the lack of a functional ATPase system, all the kinetic features of energization found in intact mitochondria can be mimicked in the reconstituted liposomes. This supports the chemiosmotic idea that electrical and perhaps H+ gradients modify the oxidase activity in reconstituted vesicles.

Ligand-induced spectral changes in cytochrome c oxidase and their possible significance.

1. The spectral shifts induced on the binding of H2S to ferric cytochrome aa3 are similar to those induced by cyanide, reflecting a possible high- to low-spin state change in the a3 haem. Opposite shifts are seen with either formate or low azide concentrations, while high azide concentrations reverse the change induced at lower concentrations. The unusually high Soret band in the half-reduced sulphide-inhibited species (a2+a33+H2S) results from the superposition of cytochrome a2+ and cytochrome a33+H2S peaks. 2. The difference spectra in the visible region for cytochrome a2+ minus cytochrome a3+ obtained with four inhibitors (cytochrome a2+ a3+I minus minus a3+a33+I)are similar, except that azide and sulphide induce blue shifts of the alpha-peak. The trough in the Soret region for the azide complex is much deeper than that for the other complexes, suggesting changes in the cytochrome a33+HN3 centre on reduction of cytochrome a. 3. The "oxygenated" and "high-energy" forms of cytochrome aa3 both involve spectral changes at the a3 haem similar to the changes induced by cyanide and sulphide. The spectrum of partially reduced cytochrome aa3 in the presence of reductant and oxygen indicates the steady-state occurrence of appreciable levels of low-spin (oxygenated) cytochrome aa3. These may be important for energy conservation during the action of cytochrome aa3 in the intact mitochondrial membrane.