Early clinical signs and imaging findings in Gerstmann-Sträussler-Scheinker syndrome (Pro102Leu).

OBJECTIVE: To determine the clinical and radiologic features of Gerstmann-Sträussler-Scheinker syndrome caused by Pro102Leu mutation in PRNP (GSS102). METHODS: The authors report 11 patients (nine families) with clinically and radiologically diagnosed GSS102. RESULTS: All patients showed mild gait disturbance, dysesthesia and hyporeflexia of the lower legs, and truncal ataxia, and 9 of 11 patients showed proximal leg muscle weakness during the early stage of the disease. Dementia was not a main symptom during the early stage. Brain MRI and EEG abnormalities were not prominent initially. SPECT (N-isopropyl-p-[(123)I]iodoamphetamine) analyzed by the three-dimensional stereotactic surface projection (SSP) method detected abnormalities in five patients early during the course of the illness. SPECT findings showed diffusely decreased cerebral blood flow, demonstrated by a mosaic pattern, with the lowest perfusion noted in the occipital lobes. In contrast, blood flow to the cerebellum was preserved. These studies suggested sites of pathology in GSS102, with the main lesions probably located in the cerebrum and the spinal cord (posterior horn and spinocerebellar tract) instead of the cerebellum. CONCLUSIONS: Key features for early diagnosis of Gerstmann-Sträussler-Scheinker syndrome caused by Pro102Leu mutation in PRNP (GSS102) are truncal ataxia, dysesthesia and hyporeflexia of the lower legs, and mild dysarthria. Normal cerebellar MRI and abnormal cerebral SPECT findings are characters of early GSS102.

Generation of free radicals and the damage done to the sarcoplasmic reticulum during reperfusion injury following brief ischemia in the canine heart.

Free radical generation was studied by the electron spin resonance (ESR) technique using alpha-phenyl N tert butyl nitrone (PBN) in a brief ischemia-reperfusion model of the canine heart, and correlated with biochemical changes of the sarcoplasmic reticulum (SR). ESR spectra (aH=0.3-0.4mT, aN=1.43-1.58mT) were observed as PBN spin adducts, which peaked at levels 5-fold above the control levels at 5 min after reperfusion. The simulated coupling constants of PBN spin adducts suggested that the sample should contain at least 2 carbon-centered radicals at 5 min after reperfusion (radical A: aH=0.350mT, aN=1.485mT; radical B: aH=0.370mT, aN=1.615 mT). At this time point, a significant reduction in Ca-ATPase activity of the SR was found without degradation of the major ATPase protein. Superoxide dismutase (SOD) significantly reduced the intensity of the PBN spin adduct signals and preserved the Ca-ATPase activity of the SR to 80% of the control level. Reperfusion injury after brief ischemia may be the result of inactivation of intracellular Ca-ATPase by free radicals generated during reperfusion, and SOD contributes to the protective effect by scavenging the radicals.

Intramolecular electron transport in quinoprotein alcohol dehydrogenase of Acetobacter methanolicus: a redox-titration study

Quinohemoprotein-cytochrome c complex alcohol dehydrogenase (ADH) of acetic acid bacteria consists of three subunits, of which subunit I contains pyrroloquinoline quinone (PQQ) and heme c, and subunit II contains three heme c components. The PQQ and heme c components are believed to be involved in the intramolecular electron transfer from ethanol to ubiquinone. To study the intramolecular electron transfer in ADH of Acetobacter methanolicus, the redox potentials of heme c components were determined with ADH complex and the isolated subunits I and II of A. methanolicus, as well as hybrid ADH consisting of the subunit I/III complex of Gluconobacter suboxydans ADH and subunit II of A. methanolicus ADH. The redox potentials of hemes c in ADH complex were -130, 49, 188, and 188 mV at pH 7.0 and 24, 187, 190, and 255 mV at pH 4.5. In hybrid ADH, one of these heme c components was largely changed in the redox potential. Reduced ADH was fully oxidized with potassium ferricyanide, while ubiquinone oxidized the enzyme partially. The results indicate that electrons extracted from ethanol at PQQ site are transferred to ubiquinone via heme c in subunit I and two of the three hemes c in subunit II. Copyright 1998 Elsevier Science B.V.

Protective effect of captopril on ischemic myocardium.

The protective effect and mechanism of action of the angiotensin-converting enzyme inhibitor (ACE-I) captopril was investigated in organelles from ischemic myocardial cells in a canine coronary ligation model. Sarcoplasmic reticulum (SR) and mitochondrial fractions were extracted from ischemic and nonischemic myocardial cells from captopril- and saline-treated (control) hearts. Heart rate, cardiac output, and right ventricular systolic blood pressure were similar in the captopril-treated and control groups. Left ventricular systolic blood pressure (LVPs) decreased gradually to 89% of the baseline value after captopril administration, and to 78% of the baseline value after ligation. Ca-ATPase activity in the SR, the respiratory control ratio (RCR) in the mitochondria, and dinitrophenol (DNP)-stimulated ATPase activity were significantly higher in ischemic myocardium from the captopril-treated group than from the saline-treated (control) group. The SH group content of both organelles was higher in the captopril-treated group. Our results suggest that, in addition to their hemodynamic effects, ACE-I agents containing SH groups protect the myocardium from ischemic damage by preventing enzyme oxidation.

Some properties and occurrence of cytochrome c-552 in the aerobic photosynthetic bacterium Roseobacter denitrificans.

Characteristics and occurrence of cytochrome c-552 from an aerobic photosynthetic bacterium, Roseobacter denitrificans, were described. Relative molecular mass of the cytrochrome was 13.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 15,000 by gel filtration. This cytochrome was a acidic protein having a pI of 5.6 and Em was +215 mV at pH 7.0. Absorption peaks were at 278, 408 and 524 nm in the oxidized form and 416, 523 and 552 nm in the reduced form. Amino acid composition and N-terminal amino acid sequence of cytochrome c-552 determined for 24 residues had low similarities to those of cytochrome c-551 of this bacterium, which is homologous to cytochrome c2, although the physico-chemical properties of these two cytochromes were similar to each other. Cytochrome c-552 was maximally synthesized in the light under aerobic conditions but not in the dark. The synthesis also occurred in the presence of alternative acceptors such as trimethylamine N-oxide (TMAO) and nitrate under anaerobic conditions. Our results suggest that cytochrome c-552 is involved in TMAO respiration and denitrification in R. denitrificans, although the effect of light remains to be solved.

Purification and properties of trimethylamine N-oxide reductase from aerobic photosynthetic bacterium Roseobacter denitrificans.

Trimethylamine N-oxide (TMAO) reductase was purified from an aerobic photosynthetic bacterium Roseobacter denitrificans. The enzyme was purified from cell-free extract by ammonium sulfate fractionation, DEAE ion exchange chromatography, hydrophobic chromatography, and gel filtration. The purified enzyme was composed of two identical subunits with molecular weight of 90,000, as identified by SDS-polyacrylamide gel electrophoresis, containing heme c and a molybdenum cofactor. The molecular weight of the native enzyme determined by gel filtration was 172,000. The midpoint redox potential of heme c was +200 mV at pH 7.5. Absorption maxima appeared at 418,524, and 554 nm in the reduced state and 410 nm in the oxidized state. The enzyme reduced TMAO, nicotine acid N-oxide, picoline N-oxide, hydroxylamine, and bromate, but not dimethyl sulfoxide, methionine sulfoxide, chlorate, nitrate, or thiosulfate. Cytochrome c2 served as a direct electron donor. It probably catalyzes the electron transfer from cytochrome b-c1 complex to TMAO reductase. Cytochrome c552, another soluble low-molecular-weight cytochrome of this bacterium, also donated electrons directly to TMAO reductase.

Two Types of Channels Involved in the Malate Ion Transport across the Tonoplast of a Crassulacean Acid Metabolism Plant.

Ion channels in tonoplast of leaf cells of a Crassulacean acid metabolism plant, Graptopetalum paraguayense, using the patch clamp technique were investigated. Results showed the existence of two types of channels involved in the malate ion transport across the tonoplast. One type corresponded to the slow-activating vacuolar-type (R Hedrich, E Neher [1987] Nature 329: 833-836), probably taking part in the malate efflux from vacuoles. Another showed the membrane potential-dependent channel current of malate flux over a wide range of cytoplasmic free Ca(2+) concentration (10(-8)-10(-5) molar), a property favoring the malate uptake. This type seems to be different from the fast-activating vacuolar-type.

Trimethylamine N-oxide respiration by aerobic photosynthetic bacterium, Erythrobacter sp. OCh 114.

Erythrobacter sp. OCh 114, an aerobic photosynthetic bacterium, had trimethylamine N-oxide (TMAO) reductase activity, which increased when the culture medium contained TMAO. The reductase was located in the periplasm. The bacteria grew anaerobically in the presence of TMAO. These results suggested that Erythrobacter OCh 114 has the ability to reduce TMAO through the respiratory chain. The TMAO respiration system of this organism was different from those of facultative purple photosynthetic bacteria in two respects: (a) TMAO reductase did not have activity to reduce dimethyl sulfoxide and (b) soluble c-type cytochrome, cytochrome c551, and cytochrome b-c1 complex appeared to be involved. The photochemical activity, which is usually inoperative in the anaerobic cell suspension, was restored by TMAO, suggesting that the photosynthesis and the TMAO respiration share a common electron transfer chain.

Flash-induced proton release in Rhodopseudomonas sphaeroides spheroplasts.

Proton release by flash excitations was measured with right-side-out vesicles prepared from Rhodopseudomonas sphaeroides by lysozyme-EDTA treatment followed by hypotonic treatment. Absorbance change at 586 nm in the presence of bromcresol purple was measured to monitor the pH change. In the presence of horse heart cytochrome c, which catalyzes the electron transfer from the cytochrome b-c1 complex to the primary electron donor, the single-turnover flash elicited release of about two protons per primary electron donor, which was rereduced rapidly by the added cytochrome c. The halftime of the proton release was about 70 ms at pH 6.3 and at a redox potential of about 150 mV. The rate was considerably lower than that of the electron transfer from the cytochrome b-c1 complex to cytochrome c. However, multiple flashes with intervals of 60 ms caused release of the same amount of protons as that by flashes with longer intervals. This indicated that the proton release itself was rapid, but delocalization was slower. Antimycin A inhibited the proton release, and myxothiazol almost completely abolished it.

Transmembrane orientation of reaction centers in proteoliposomes from Rhodopseudomonas sphaeroides.

The photochemical reaction centers from Rhodopseudomonas sphaeroides were reconstituted with soybean phospholipids into liposomes by the cholate-dialysis method. The transmembrane orientation of the reaction centers in the proteoliposomes and the morphology of the vesicles were investigated. The orientation was determined by the reduction of externally added cytochrome c after its photooxidation by a flash. The structure of the vesicles was examined by electron microscope. Discontinuous sucrose density gradient centrifugation yielded several proteoliposome fractions with different vesicular sizes and reaction-center orientations. The proportion of the reaction centers that exposed their cytochrome c reacting sites to the outside of the vesicles increased from 45 to 85% with an increase of the vesicular size. The proportion also depended on the ionic composition of the dialysis buffer. The optimal ionic environment during the dialysis (100 mM NaCl or 2.5 mM MgSO4) gave a liposome yield of 25-30% with a highly asymmetric orientation (greater than 60%). Entrapping of cytochrome c molecules into the phospholipid vesicles had little effect on the orientation of the reaction centers.

Enthalpy and volume changes accompanying electron transfer from P-870 to quinones in Rhodopseudomonas sphaeroides reaction centers.

A capacitor microphone was used to measure the enthalpy and volume changes that accompany the electron transfer reactions, PQAhv leads to P+Q-A and PQAQBhv leads to P+QAQ-B, following flash excitation of photosynthetic reaction centers isolated from Rhodopseudomonas sphaeroides. P is a bacteriochlorophyll dimer (P-870), and QA and QB are ubiquinones. In reaction centers containing only QA, the enthalpy of P+Q-A is very close to that of the PQA ground state (delta Hr = 0.05 +/- 0.03 eV). The free energy of about 0.65 eV that is captured in the photochemical reaction evidently takes the form of a substantial entropy decrease. In contrast, the formation of P+QAQ-B in reaction centers containing both quinones has a delta Hr of 0.32 +/- 0.02 eV. The entropy change must be near zero in this case. In the presence of o-phenanthroline, which blocks electron transfer between Q-A and QB, delta Hr for forming P+Q-AQB is 0.13 +/- 0.03 eV. The influence of flash-induced proton uptake on the results was investigated, and the delta Hr values given above were measured under conditions that minimized this influence. Although the reductions of QA and QB involve very different changes in enthalpy and entropy, both reactions are accompanied by a similar volume decrease of about 20 ml/mol. The contraction probably reflects electrostriction caused by the charges on P+ and Q-A or Q-B.

Thermodynamics of electron transfer and its coupling to vectorial processes in biological membranes.

A method is developed to express the flux of an electron transfer reaction as a function of the conjugate force, the redox potential difference, throughout the nonlinear region. The flux can be expressed by a product of the hyperbolic sine of the force, a factor ("redox-poising parameter") determined by the redox potentials of subsystem (in certain cases by local pH's and pK's of subsystems), and some constants. This is analogous to the expression of the flux of a diffusion process by the product of its force and the concentration of the diffusing species. The redox-poising parameter corresponds to the concentration term. The expression is applied to redox chains in which electron transfers are coupled to vectorial processes such as proton translocation or electric current.

Energetic coupling in the primary processes of photosynthesis in Chromatium. pH dependence of delayed fluorescence, electron transfer and degree of coupling.

The effects of pH on the thermodynamic properties of the proton-translocating cyclic electron transfer system in a purple photosynthetic bacterium Chromatium vinosum were studied. Two thermodynamic parameters, the flux (Je) and force (deltamue) of the electron transfer process, were analyzed. The rate of electron transfer in the re-reduction of photooxidized reaction-center bacteriochlorophyll was used as Je. deltamue was determined from the intensity of the delayed fluorescence from bacteriochlorophyll. deltamue is composed of the redox potential difference and the electrical potential difference between two electron transfer components. In the steady state under illumination, the flux-to-force ratio is determined by the following relationship: Je = (1--q2)Lee deltamue where q is the "degree of coupling" of electron transfer to proton translocation and Lee is the value of Je/delta-approximately similar e when there is no back pressure by formation of delta approximately muH+ (electrochemical potential difference of H+). The value of (1--q2) Lee increased with increasing pH in the neutral pH range. Uncouplers and ionophores that dissipate delta-approximately muH+ increased Je and decreased deltamue. The effects were more prominent in the lower pH range. Therefore, q must be smaller at higher pH. The coupling is probably tight when redox components are saturated with protons. The experimental results agreed with the theoretical predictions for a system where a hydrogen-translocating component functions as an electron-proton symport carrier.

Delayed fluorescence from bacteriochlorophyll in Chromatium vinosum chromatophores: characteristics in the presence of o-phenanthroline.

Delayed fluorescence from bacteriochlorophyll in the chromatophores of Chromatium vinosum, a photosynthetic purple sulfur bacterium, was studied in the presence of o-phenanthroline (o-phen) under intermittent illumination. Re-reduction of the photooxidized reaction center bacteriochlorophyll (P+) in the dark interval was accelerated by o-phen. This effect was attributed to the return of electrons trapped in the primary electron acceptor (A) to P+. In the presence of o-phen, the time course of the decay of delayed fluorescence was not coincident with that of the re-reduction of P+. The delayed fluorescence was somewhat intensified at the early stage (within 30 ms) of relaxation in the dark period. Prolonged illumination (longer than 20 ms) or uncouplers such as carbonylcyanide m-chlorophenylhydrazone (CCCP) or valinomycin plus nigericin decreased the intensity of delayed fluorescence and suppressed the stimulation of delayed fluorescence at the early stage. Delayed fluorescence from reaction center-rich subchromatophore particles decayed with a time course identical to that of the reduction of P+ and was not affected by CCCP, in the presence of o-phen. The intensification at the early stage in the chromatophores can be interpreted in terms of charge separation between pairs of P and A, primary electron donor and acceptor molecules, oriented perpendicular to the intact chromatophore membrane, the effect decreasing in parallel with the recombination of P+ and A-.

Delayed fluorescence from bacteriochlorophyll in Chromatium vinosum chromatophores.

Delayed fluorescence from bacteriochlorophyll in Chromatium vinosum chromatophores was studied at room temperature and under intermittent illuminations. The decay of delayed fluorescence was constituted of two components; a fast component decayed with a half time of about 8 ms, a slow one decayed in parallel with the reduction of photooxidized bacteriochlorophyll (P+) with a half time of 100-200 ms. The biphasic decay of delayed fluorescence indicated that a rapid equilibrium was established between the primary electron acceptor and the secondary acceptor. In the presence of o-phenanthroline, the time course of the decay of delayed fluorescence was identical with that of the reduction of P+ in reaction center-rich subchromatophore particles, although they did not necessarily coincide with each other in "intact" chromatophores. The intensity of the slow component was increased and the decay was accelerated at basic pH values. Reagents that dissipate the proton gradient across the chromatophore membranes such as carbonylcyanide m-chlorophenylhydrazone (CCCP) and nigericin accelerated the decay of the slow component. These effects are probably resulting from changes in internal pH of chromatophore vesicles. Reagents that dissipate the membrane potential such as CCCP and valinomycin decreased the intensity.