Biomedical subjects
O Hansson
Publications and source records attributed to O Hansson.
Mitochondrial control of acute glutamate excitotoxicity in cultured cerebellar granule cells.
Mitochondria within cultured rat cerebellar granule cells have a complex influence on cytoplasmic free Ca2+ ([Ca2+]c) responses to glutamate. A decreased initial [Ca2+]c elevation in cells whose mitochondria are depolarized by inhibition of the ATP synthase and respiratory chain (conditions which avoid ATP depletion) was attributed to enhanced Ca2+ extrusion from the cell rather than inhibited Ca2+ entry via the NMDA receptor. Even in the presence of elevated extracellular Ca2+, when [Ca2+]c responses were restored to control values, such cells showed resistance to acute excitotoxicity, defined as a delayed cytoplasmic Ca2+ deregulation (DCD) during glutamate exposure. DCD was a function of the duration of mitochondrial polarization in the presence of glutamate rather than the total period of glutamate exposure. Once initiated, DCD could not be reversed by NMDA receptor inhibition. In the absence of ATP synthase inhibition, respiratory chain inhibitors produced an immediate Ca2+ deregulation (ICD), ascribed to an ATP deficit. In contrast to DCD, ICD could be reversed by subsequent ATP synthase inhibition with or without additional NMDA receptor blockade. DCD could not be ascribed to the failure of an ATP yielding metabolic pathway. It is concluded that mitochondria can control Ca2+ extrusion from glutamate-exposed granule cells by the plasma membrane in three ways: by competing with efflux pathways for Ca2+, by restricting ATP supply, and by inducing a delayed failure of Ca2+ extrusion. Inhibitors of the mitochondrial permeability transition only marginally delayed the onset of DCD.
Effects of single and double mutations in plastocyanin on the rate constant and activation parameters for the rearrangement gating the electron-transfer reaction between the triplet state of zinc cytochrome c and cupriplastocyanin.
The unimolecular rate constant for the photoinduced electron-transfer reaction 3Zncyt/pc(II) --> Zncyt+/pc(I) within the electrostatic complex of zinc cytochrome c and spinach cupriplastocyanin is kF. We report the effects on kF of the following factors, all at pH 7.0: 12 single mutations on the plastocyanin surface (Leu12Asn, Leu12Glu, Leu12Lys, Asp42Asn, Asp42Lys, Glu43Asn, Glu59Gln, Glu59Lys, Glu60Gln, Glu60Lys, Gln88Glu, and Gln88Lys), the double mutation Glu59Lys/Glu60Gln, temperature (in the range 273.3-302.9 K), and solution viscosity (in the range 1. 00-116.0 cP) at 283.2 and 293.2 K. We also report the effects of the plastocyanin mutations on the association constant (Ka) and the corresponding free energy of association (DeltaGa) with zinc cytochrome c at 298.2 K. Dependence of kF on temperature yielded the activation parameters DeltaH, DeltaS, and DeltaG. Dependence of kF on solution viscosity yielded the protein friction and confirmed the DeltaG values determined from the temperature dependence. The aforementioned intracomplex reaction is not a simple electron-transfer reaction because donor-acceptor electronic coupling (HAB) and reorganizational energy (lambda), obtained by fitting of the temperature dependence of kF to the Marcus equation, deviate from the expectations based on precedents and because kF greatly depends on viscosity. This last dependence and the fact that certain mutations affect Ka but not kF are two lines of evidence against the mechanism in which the electron-transfer step is coupled with the faster, but thermodynamically unfavorable, rearrangement step. The electron-transfer reaction is gated by the slower, and thus rate determining, structural rearrangement of the diprotein complex; the rate constant kF corresponds to this rearrangement. Isokinetic correlation of DeltaH and DeltaS parameters and Coulombic energies of the various configurations of the Zncyt/pc(II) complex consistently show that the rearrangement is a facile configurational fluctuation of the associated proteins, qualitatively the same process regardless of the mutations in plastocyanin. Correlation of kF with the orientation of the cupriplastocyanin dipole moment indicates that the reactive configuration of the diprotein complex involves the area near the residue 59, between the upper acidic cluster and the hydrophobic patch. Kinetic effects and noneffects of plastocyanin mutations show that the rearrangement from the initial (docking) configuration, which involves both acidic clusters, to the reactive configuration does not involve the lower acidic cluster and the hydrophobic patch but involves the upper acidic cluster and the area near the residue 88.
Crystal structure of spinach plastocyanin at 1.7 A resolution.
The crystal structure of plastocyanin from spinach has been determined using molecular replacement, with the structure of plastocyanin from poplar as a search model. Successful crystallization was facilitated by site-directed mutagenesis in which residue Gly8 was substituted with Asp. The region around residue 8 was believed to be too mobile for the wild-type protein to form crystals despite extensive screening. The current structure represents the oxidized plastocyanin, copper (II), at low pH (approximately 4.4). In contrast to the similarity in the core region as compared to its poplar counterpart, the structure shows some significant differences in loop regions. The most notable is the large shift of the 59-61 loop where the largest shift is 3.0 A for the C(alpha) atom of Glu59. This results in different patterns of electrostatic potential around the acidic patches for the two proteins.
The involvement of the two acidic patches of spinach plastocyanin in the reaction with photosystem I.
Six different spinach plastocyanin mutants have been constructed by site-directed mutagenesis and expressed in Escherichia coli to probe the importance of the two acidic patches in the interaction with photosystem I. The mutants were: Asp42Lys, Glu43Asn, Glu43Lys, Glu43Gln/Asp44Asn, Glu59Lys/Glu60Gln and Glu43Asn/Glu59Lys/Glu60Gln and they have been characterised by optical absorption and EPR spectroscopy, redox titrations and isoelectric focusing. The electron transfer to photosystem I was investigated by flash-induced time-resolved absorption measurements at 830 nm. The kinetics were interpreted with a model that incorporates a rate-limiting conformational change from inactive to active forms of the plastocyanin-photosystem I complex. All mutations resulted in a displacement of the equilibrium towards the inactive conformation. The strongest impairment of the electron transfer was found for mutations in the larger acidic patch, in particular upon modification of residues 43 or 44. However, mutations of residues 59 and 60 in the smaller acidic patch also resulted in a lower reactivity.
Electron transfer between spinach plastocyanin mutants and photosystem 1.
Two distinct regions of plastocyanin, one hydrophobic and one acidic, are generally thought to be involved in the electron-transfer reactions with its physiological partners, cytochrome f and photosystem 1. To probe the importance of the hydrophobic patch in the reaction with photosystem 1, seven mutant plastocyanin proteins have been constructed with the following mutations: Gly7Ala, Gly8Asp, Ser11Asp, Ser11Gly, Pro36Gly, Ser85Thr and Gln88Asn. The electron-transfer reaction was investigated by transient flash-photolysis absorption spectroscopy. All proteins remained active in photosystem 1 reduction, showing a biphasic reaction. However, the substitution in position 36 resulted in a drastic decrease in efficiency, suggesting that this residue is involved in a specific contact with photosystem 1. Measurements over a wide range of plastocyanin concentration, ionic strength and pH, showed different properties for the two kinetic phases. A mechanism involving a rate-limiting conformational change accounts well for the observations. Electron transfer from plastocyanin to photosystem 1 would thus require a conversion from an inactive to an active conformation of the complex. Both hydrophobic and electrostatic interactions are important in the dynamics. The structural integrity of a few critical residues, including Pro36, is essential for efficient photosystem 1 reduction.
Effects of mutations in plastocyanin on the kinetics of the protein rearrangement gating the electron-transfer reaction with zinc cytochrome c. Analysis of the rearrangement pathway.
We study, by flash kinetic spectrophotometry on the microsecond time scale, the effects of ionic strength and viscosity on the kinetics of oxidative quenching of the triplet state of zinc cytochrome c (3Zncyt) by the wild-type form and the following nine mutants of cupriplastocyanin: Leu12Glu, Leu12Asn, Phe35Tyr, Gln88Glu, Tyr83Phe, Tyr83His, Asp42Asn, Glu43Asn, and the double mutant Glu59Lys/Glu60Gln. The unimolecular rate constants for the quenching reactions within the persistent diprotein complex, which predominates at low ionic strengths, and within the transient diprotein complex, which is involved at higher ionic strengths, are equal irrespective of the mutation. Evidently, the two complexes are the same. In both reactions, the rate-limiting step is rearrangement of the diprotein complex from a configuration optimal for docking to the one optimal for the subsequent electron-transfer step, which is fast. We investigate the effects of plastocyanin mutations on this rearrangement, which gates the overall electron-transfer reaction. Conversion of the carboxylate anions into amide groups in the lower acidic cluster (residues 42 and 43), replacement of Tyr83 with other aromatic residues, and mutations in the hydrophobic patch in plastocyanin do not significantly affect the rearrangement. Conversion of a pair of carboxylate anions into a cationic and a neutral residue in the upper acidic cluster (residues 59 and 60) impedes the rearrangement. Creation of an anion at position 88, between the upper acidic cluster and the hydrophobic patch, facilitates the rearrangement. The rate constant for the rearrangement smoothly decreases as the solution viscosity increases, irrespective of the mutation. Fittings of this dependence to the modified Kramers's equation and to an empirical equation show that zinc cytochrome c follows the same trajectory on the surfaces of all the plastocyanin mutants but that the obstacles along the way vary as mutations alter the electrostatic potential. Mutations that affect protein association (i.e., change the binding constant) do not necessarily affect the reaction between the associated proteins (i.e., the rate constant) and vice versa. All of the kinetic and thermodynamic effects and noneffects of mutations consistently indicate that in the protein rearrangement the basic patch of zinc cytochrome c moves from a position between the two acidic clusters to a position at or near the upper acidic cluster.
Structural dynamics in the plastocyanin-photosystem 1 electron-transfer complex as revealed by mutant studies.
A series of plastocyanin mutants have been constructed by site-directed mutagenesis and expressed in Escherichia coli to elucidate the interaction between plastocyanin and photosystem 1 in the photosynthetic electron-transfer chain. Leu-12 has been replaced with alanine, asparagine, glutamate, and lysine, while Tyr-83 has been exchanged for histidine, phenylalanine, and leucine. Phe-35, Asp-42, and Gln-88 have been mutated to tyrosine, asparagine, and glutamate, respectively. The mutations that have been introduced do not seem to place any strain on the tertiary structure according to optical absorption and electron paramagnetic resonance (EPR) spectroscopic studies. However, there are changes in the reduction potential for the Leu-12 mutants that cannot be accounted for by electrostatic interactions alone. For some of the mutants, the pI shifts, in accordance with the changes in the number of titratable groups. Only the Leu-12 mutants show any major change in their photosystem 1 kinetics, while the mutants in the acidic patch show minor changes, suggesting that both the hydrophobic and acidic patches make contact with photosystem 1 but that the electron transfer occurs at the hydrophobic interface, most probably via the His-87 residue. The kinetics are best described with a model in which a rate-limiting conformational change occurs in the plastocyanin-photosystem 1 complex [Bottin, H., & Mathis, P. (1985) Biochemistry 24, 6453-6460; Sigfridsson, K., Hansson, O., Karlsson, B.G., Baltzer L., Nordling, M., & Lundberg, L. G. (1995) Biochim. Biophys. Acta 1228, 28-36], where the changes observed are attributed to changes in the dynamics within the electron-transfer complex.
A longitudinal study of resin-bonded prostheses.
Resin-bonded fixed partial dentures, without definitive tooth preparation and current resinous cements and perceptive treatment, have had an inordinate number of dislodgments in initial restorative efforts. A longitudinal examination of resin-bonded prostheses was performed after a mean observation time exceeding 6 years. To prevent stress fatigue fractures of the cement bondings, one proximal groove was prepared in dentin for each anterior abutment and the inner surfaces of the frameworks were silicoated. Five of 34 prostheses debonded, and the bond strength between resin and high gold alloys was too low to resist fatigue fractures during this study. The results of this study and other reports suggested a need for increased retention and resistance form of the frameworks to improve longevity of resin-bonded prostheses.
Electrogenic light reactions in photosystem I: resolution of electron-transfer rates between the iron-sulfur centers.
Flash-induced voltage changes (electrogenic events) in photosystem I particles from spinach, oriented in a phospholipid layer, have been studied at room temperature on a time scale ranging from 1 micros to several seconds. A phospholipid layer containing photosystem I particles was adsorbed to a Teflon film separating two aqueous compartments. Voltage changes were measured across electrodes immersed in the compartments. In the absence of added electron donors and acceptors, a multiphasic voltage increase, associated with charge separation, was followed by a decrease, associated with charge recombination. Several kinetic phases were resolved: a rapid (<1 micros) increase, ascribed to electron transfer from the primary electron donor P700 to the iron-sulfur electron acceptor FB, was followed by a slower, biphasic increase with time constants of 30 and 200 micros. The 30-micros phase is assigned to electron transfer from FB to the iron-sulfur center FA. The voltage decrease had a time constant of 90 ms, ascribed to charge recombination from FA to P700. Upon chemical prereduction of FA and FB the 30- and 200-micros phases disappeared and the decay time constant was accelerated to 330 micros, assigned to charge recombination from the phylloquinone electron acceptor (A1) or the iron-sulfur center FX to P700.
Clinical results with resin-bonded prostheses and an adhesive cement.
The longevity of resin-bonded prostheses made of a high-gold alloy and bonded with Super-Bond C&B (C&B Metabond) were tested clinically. Minimal preparation and large surface area of retainer coverage were used. The inner surfaces of the retainers were air abraded with aluminum oxide and heat treated in an oven at 400 degrees C to get an oxidized layer for bonding. The results of the adhesive technique were unsatisfactory compared with studies with resin-bonded prostheses using conventional mechanical retention techniques and nonadhesive resin cements. All fractures were at the resin-metal interface. Early debonds occurred; seven of 13 prostheses were detached after 11 months. The median survival time was only 9 months. The adhesion of resin-bonded prostheses must still be based on mechanical retention between cement and metal, because a way of attaining good durability of the bonding joints to water with Super-Bond is yet to be found.
Evaluation of three silicoating methods for resin-bonded prostheses.
Three different methods of increasing the bond strength of resin to metal were tested: the original Silicoater technique, the Silicoater MD technique, and the Rocatec system. Metals used for the resin-bonded prostheses were gold, a cobalt-chromium (Co-Cr) alloy, and titanium. Silicoating increased the bond strength to sandblasted specimens. The original Silicoater technique produced the highest bond strengths, especially when used with the Co-Cr alloy and titanium. The Silicoater MD technique showed the lowest bond strengths for all of the test metals. Storage for 7 days with protective film on the silicoated surface did not affect the bond strength, as compared with specimens bonded immediately. The retention of the resin to the gold specimens (Rocatec system) decreased after thermocycling, but retention to the Co-Cr alloy and titanium was not affected. Various resin cements were tested and showed the same retention with the exception of Microfill Pontic, which gave a weaker bond. Scanning electron microscope (SEM) and energy-dispersive X-ray analysis (EDAX) of the specimens before and after the measurements gave no clear explanation of the differences in bond strengths between the test metals. However, it was concluded that silicoating of the metal surfaces contributed to the retention of the resin by chemical action, as no differences in adaptation of the resin to the metal were seen between specimens pretreated by sandblasting and those which were also silicoated.
A membrane-bound monoheme cytochrome c551 of a novel type is the immediate electron donor to P840 of the Chlorobium vibrioforme photosynthetic reaction center complex.
A photosynthetic reaction center complex has been isolated from the green sulfur bacterium Chlorobium vibrioforme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals polypeptides with apparent molecular masses of 80, 40, 18, 15, 9, and 6 kDa. Only the 18-kDa polypeptide is stained with 3,3',5,5'-tetramethylbenzidine, a heme-specific reagent. Oxidized minus reduced difference spectra show the presence of approximately one heme/P840 and the presence of a cytochrome c551. Flash photolysis of P840 was followed by rereduction of P840+ and oxidation of cytochrome c551, both with a biphasic kinetic with t1/2 values of 7 and 50 microseconds. Using oligonucleotide probes derived from an N-terminal amino acid sequence of the 18-kDa polypeptide, a genomic clone was isolated. The sequence of the gene, which we designate cycA, predicts a single heme binding site (Cys-Asn-Lys-Cys-His). The 621-base pair open reading frame encodes an apoprotein of 22,858 Da with three predicted membrane-spanning alpha-helices. No extensive sequence similarity is found to other cytochromes. Northern blotting indicates that the cycA gene is transcribed as a monocistronic mRNA. Southern blotting shows the presence of only one cycA gene in the C. vibrioforme and Chlorobium tepidum genomes. The unique membrane-bound monoheme cytochrome c551 of C. vibriforme is assigned to a new class of c-type cytochromes. The implications for the current view of evolution of photosynthetic reaction center complexes are discussed.
Reactivity of cytochromes c and f with mutant forms of spinach plastocyanin.
The reduction of plastocyanin by cytochromes c and f has been investigated with mutants of spinach plastocyanin in which individual, highly conserved surface residues have been modified. These include Leu-12 and Phe-35 in the 'northern' hydrophobic patch and Tyr-83 and Asp-42 in the 'eastern' acidic patch. The differences observed all involved binding rather than the intrinsic rates of electron transfer. The Glu-12 and Ala-12 mutants showed small but significant decreases in binding constant with cytochrome c, even though the cytochrome is not expected to make contact with the northern face of plastocyanin. These results, and small changes in the EPR parameters, suggested that these mutations cause small conformational changes in surface residues on the eastern face of plastocyanin, transmitted through the copper centre. In the case of cytochrome f, the Glu-12 and Ala-12 mutants also bound less strongly, but Leu12Asn showed a marked increase in binding constant, suggesting that cytochrome f can hydrogen bond directly to Asn-12 in the reaction complex. A surprising result was that the kinetics of reduction of Asp42Asn were not significantly different from wild type, despite the loss of a negative charge.
Flash-photolysis studies of the electron transfer from genetically modified spinach plastocyanin to photosystem I.
Plastocyanin (Pc) has been modified by site-directed mutagenesis at two separate electron-transfer (ET) sites: Leu-12-Glu at a hydrophobic patch, and Tyr-83-His at an acidic patch. The reduction potential at pH 7.5 is decreased by 26 mV in Pc(Leu-12-Glu) and increased by 35 mV in Pc(Tyr-83-His). The latter mutant shows a 2-fold slower intracomplex ET to photosystem I (PSI) as expected from the decreased driving force. The affinity for PSI is unaffected for this mutant but is drastically decreased for Pc(Leu-12-Glu). It is concluded that the hydrophobic patch is more important for the ET to PSI.
Strength of bond with Comspan Opaque to three silicoated alloys and titanium.
In Sweden high-gold alloys or cobalt-chromium alloys are used for resin-bonded prostheses. The bond strength between a resin cement and different sandblasted or silicoated metals were measured before and after thermocycling; in connection with this some rapid thermocycling methods were studied. The effect of different storage times and different protection coatings on bond strength were tested. Finally, the influence of rubbing and contamination with saliva on bond strength were investigated. Silicoating increased the bond strength significantly. The highest bond strengths were these of silicoated Wirobond and titanium, unsusceptible to thermal stress; the bond strengths of the sandblasted metals were the weakest, and sensitive to thermocycling as well. The influence on bond strength for silicoated gold alloys, protected with an unpolymerized composite resin coating, stored in sealed plastic bags up to 7 days, was negligible. Rubbing and contamination with saliva did not influence bond strength. Preferably, silicoated Wirobond and titanium should be used for resin-bonded prostheses, but gold alloys may still be adequate for clinical use. The experimental method described for storing, sealing, and cleaning the silicoated metal surfaces in this article can be recommended for laboratory and clinical use.
The Silicoater technique for resin-bonded prostheses: clinical and laboratory procedures.
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[Silicoater procedures for adhesive bridges (1). Clinical and laboratory procedures].
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