Unbinding of oxidized cytochrome c from photosynthetic reaction center of Rhodobacter sphaeroides is the bottleneck of fast turnover

To understand the details of rate limitation of turnover of the photosynthetic reaction center, photooxidation of horse heart cytochrome c by reaction center from Rhodobacter spheroides in detergent dispersion has been examined by intense continuous illumination under a wide variety of conditions of cytochrome concentration, ionic strength, viscosity, temperature, light intensity, and pH. The observed steady-state turnover rate of the cytochrome was not light intensity limited. In accordance with recent findings [Larson, J. W., Wells, T. A., and Wraight, C. A. (1998) Biophys. J. 74 (2), A76], the turnover rate increased with increasing bulk ionic strength in the range of 0-40 mM NaCl from 1000 up to 2300 s(-)(1) and then decreased at high ionic strength under conditions of excess cytochrome and ubiquinone and a photochemical rate constant of 4500 s(-)(1). Furthermore, we found the following: (i) The contribution of donor (cytochrome c) and acceptor (ubiquinone) sides as well as the binding of reduced and the release of oxidized cytochrome c could be separated in the observed kinetics. At neutral and acidic pH (when the proton transfer is not rate limiting) and at low or moderate ionic strength, the turnover rate of the reaction center was limited primarily by the low release rate of the photooxidized cytochrome c (product inhibition). At high ionic strength, however, the binding rate of the reduced cytochrome c decreased dramatically and became the bottleneck. The observed activation energy of the steady-state turnover rate reflected the changes in limiting mechanisms: 1.5 kcal/mol at 4 mM and 5.7 kcal/mol at 100 mM ionic strength. A similar distinction was observed in the viscosity dependence of the turnover rate: the decrease was steep (eta(-)(1)) at 40 and 100 mM ionic strengths and moderate (eta(-)(0.2)) under low-salt (4 mM) conditions. (ii) The rate of quinone exchange at the acceptor side with excess ubiquinone-30 or ubiquinone-50 was higher than the cytochrome exchange at the donor side and did not limit the observed rate of cytochrome turnover. (iii) Multivalent cations exerted effects not only through ionic strength (screening) but also by direct interaction with surface charge groups (ion-pair production). Heavy metal ion Cd(2+) bound to the RC with apparent dissociation constant of 14 microM. (iv) A two-state model of collisional interaction between reaction center and cytochrome c together with simple electrostatic considerations in the calculation of rate constants was generally sufficient to describe the kinetics of photooxidation of dimer and cytochrome c. (v) The pH dependence of cytochrome turnover rate indicated that the steady-state turnover rate of the cytochrome under high light conditions was not determined by the isoelectric point of the reaction center (pI = 6. 1) but by the carboxyl residues near the docking site.     

Molecular rearrangements of thylakoids after heavy metal poisoning, as seen by Fourier transform infrared (FTIR) and electron spin resonance (ESR) spectroscopy

The specific effects exerted by different heavy metals on both the function and the structure of the photosynthetic apparatus were addressed. The functional analysis performed via the fluorescence induction kinetics revealed that the applied toxic heavy metals can be classified into two groups: Cd and Ni had no significant effect on the photosynthetic electron transport, while Cu, Pb and Zn strongly inhibited the Photosystem II (PS II) activity, as evidenced by the dramatic decreases in both the variable (F_v) and the maximal (F_m) fluorescence. The structural effects of the heavy metal ions on the thylakoid membranes were considered in three relations: (1) lipids, (2) proteins — studied by Fourier transform infrared (FTIR) spectroscopy, and (3) lipid—protein interactions — investigated by electron spin resonance (ESR) spectroscopy using spin-labeled probe molecules. The studied heavy metal ions had only a non-specific rigidifying effect on the thylakoid lipids. As regards proteins, Cd and Ni had no effect on the course of their heat denaturation. The heat denaturation of the proteins was accompanied by a decrease in the -helix content (1656 cm^-1), a parallel increase in the disordered segments (1651 cm^-1), a decrease in the intramolecular -sheet (1636 cm^-1) content and the concomitant appearance of an intermolecular -structure (1621 cm^-1). In contrast with Cd and Ni, Cu and Zn blocked the appearance of the intermolecular -structure. Pb represented an intermediate case. It seems that these heavy metals alter the native membrane structure in such a way that heat-induced aggregation becomes more limited. The ESR data revealed that certain heavy metals also affect the lipid—protein interactions. While Cd and Ni had hardly any effect on the solvation fraction of thylakoid lipids, Cu, Pb and Zn increased the fraction of lipids solvating the proteins. On the basis of the FTIR and ESR data, it seems that Cu, Pb, and Zn increase the surfaces available for lipid—protein interactions by dissociating membrane protein complexes, and that these lipidated proteins have a smaller chance to aggregate upon heat denaturation. The data presented here indicate that the damaging effects of poisonous heavy metals are element-specific, Cu, Pb and Zn interact directly with the thylakoid membranes of the photosynthetic apparatus, while Cd and Ni interfere rather with other metabolic processes of plants.     

Long-lasting antinociceptive effect of DAMGO chloromethyl ketone in rats

Previously, the opioid peptide Tyr-D-Ala-Gly-(NMe)Phe-CH2Cl (DAMCK) has been shown to bind irreversibly to mu opioid receptors in vitro. In the present work, the antinociceptive effect of DAMCK has been evaluated. Rats treated systemically with DAMCK (1-100 pg/kg) displayed a dose-dependent increase in tail-flick analgesia that peaked by 15 min, then stayed about the same until 60 min, followed by some decrease over time. Higher doses of DAMCK (10 ng/kg-100 microg/kg) produced a near-maximal antinociceptive effect that remained stable for 4 h. Significant antinociception was still detected 8 h, but not 24 h postinjection. In comparison, the parent peptide DAMGO (Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol) reached maximal effect by about 30 min, followed by a rapid cessation of its antinociceptive response. Naloxone administered before DAMCK antagonized the antinociceptive response of DAMCK, indicating that it was mediated via opioid receptors. Naloxone administered 45 min after DAMCK attenuated the tail-flick response to some extent, but a substantial part (40-60% depending on the peptide concentration and evaluation time) remained unaffected. Central administration of DAMCK also elicited time- and concentration-dependent, profound, opioid receptor mediated, apparently irreversible antinociception.     

Selective inhibition of neuronal nitric oxide synthase fails to alter the resting tension and the relaxant effect of bradykinin in isolated rat middle cerebral arteries

The role of the neuronal isoform of the nitric oxide (NO) synthase (nNOS) in the regulation of the cerebrovascular tone was studied in vitro. Selective inhibition of nNOS by 7-nitro indazole monosodium salt (7-NINA) failed to alter the resting tension and the relaxant effect of bradykinin in isolated rat middle cerebral arteries. These results indicate that 1./ 7-NINA is selective for nNOS and 2./ cerebrovascular nNOS is involved neither in the resting NO production nor in the mediation of the relaxant effect of bradykinin. Therefore, nNOS-derived NO that contributes to the maintenance of the resting cerebral blood flow in vivo appears to be released from neurons and/or glial cells.     

Cold pressor test and retinal capillary perfusion in vasospastic subjects with and without capsular glaucoma (a preliminary study)

PURPOSE: Using the cold pressor test the authors investigated the change in retinal and neuroretinal capillary perfusion in vasospastic patients suffering from capsular glaucoma (CG) and in vasospastic control subjects. METHODS: Changes in retinal and optic nerve head capillary perfusion induced by the cold pressor test (one hand immersed in 4 degrees C water for 30 seconds, then in 30 degrees C water for 2 minutes) was measured using the Heidelberg Retina Flowmeter in 4 patients with CG and in 5 healthy control subjects. Previously all subjects showed a reduction of cutaneous capillary flow higher than 70% in the cold pressor test (vasospastic reaction). One eye per subject was investigated. Two images were obtained for each phase (baseline, cold phase and warm phase), and the better quality image from each phase was selected for the measurements. One location on the temporal neuroretinal rim and one location on the temporal retina outside the peripapillary area were selected for the HRF measurements. RESULTS: In the CG group neuroretinal rim "Volume" decreased by 26.05%, "Flow" by 25.82% and "Velocity" by 23.91% (p<0.05), retinal "Volume" decreased by 12.30% (p=0.051), and retinal "Flow" by 22.36% (p=0.01) in the cold phase. All these parameters returned to the corresponding baseline values in the warm phase. In the control group a significant decrease was observed in retinal "Volume" (15.96%), "Flow" (17.81%), and "Velocity" (16.11%) in the cold phase (p<0.05), which diminished in the warm phase but remained still significant for "Flow" and "Velocity". CONCLUSION: Cutaneous cold provocation can induce an immediate decrease in retinal and optic nerve head capillary perfusion at least in a part of the vasospastic subjects with or without capsular glaucoma. This decrease diminishes or disappears quickly when the hand is immersed in warm water. To evaluate the potential role of cold-induced retinal and optic nerve head vasoconstriction in the pathogenesis of capsular glaucoma further investigations are necessary since this reaction was also present in the vasospastic control subjects.     

Application of an anti-HQIgY antibody for the measurement of IgY concentrations of hen's and quail's serum and yolk

The development of a sensitive ELISA for the measurement of quail IgY (QIgY) was the main purpose of our study. The suitable antibody (AB) was prepared in rabbits. Both quail IgY (QIgY) and hen IgY (HIgY) were precipitated by this developed AB. For this reason it was marked as anti-hen-quail-IgY (a-HQIgY). The purified AB was conjugated with horseradish peroxidase (aHQIgY-HRP) and a sensitive direct ELISA was developed, based on this labeled AB. The prepared aHQIgY AB which was used in this developed ELISA method was suitable for the measurement of total and specific IgY concentration in domestic hen (Gallus domesticus) and Japanese quail (Coturnix coturnix japonica) either. As a result of our experiments it is very likely that there are identical sequences of IgYs of both species. This part of IgY has good antigen character at the same time. Probably, this phenomenon has occurrence in other Galliform species, too. Further investigations will be carried out in this field.     

Hypoxia/reoxygenation and vitamin C intake influence NO synthesis and antioxidant defenses of neutrophils

Oxidative stress induced by hypoxia/reoxygenation mediates the pathophysiological consequence of ischemia/reperfusion and human diseases. Diving apnea could be a good model of oxidative stress induced by hypoxia/reoxygenation. We studied the influence of vitamin C diet supplementation on the response of neutrophil antioxidant defenses, NO production, and redox status to diving apnea. Seven professional apnea divers participated in a double-blind cross study. Divers were assigned to either vitamin C-supplemented (1 g/d for a week) or placebo groups. Blood samples were taken under basal conditions, immediately after diving apnea for 4 h and after 1 h of recovery. Plasma vitamin C increased only in the supplemented group after diving and was maintained high in recovery. Diving apnea decreased neutrophil GSH/GSSG ratio in both groups, but maintained protein carbonyl derivates. Neutrophil catalase activity and levels and glutathione peroxidase activity were lower in the supplemented group than in the placebo group after diving. iNOS and nitrite levels decreased only in the supplemented group after diving and recovery. Diving apnea induced oxidative stress and initiated neutrophil reactions that resemble the acute-phase immune response with increased myeloperoxidase activity in neutrophils. Diet supplementation with vitamin C reduced neutrophil iNOS levels and NO production.