Maximum H+/h{nu}PSI Stoichiometry of Proton Transport during Cyclic Electron Flow in Intact Chloroplasts Is at Least Two, but Probably Higher than Two

Effects of antimycin A on 9-aminoacridine (9AA) fluorescencequenching by intact chloroplasts during light-dependent electronflow to different electron acceptors indicated that considerablecyclic electron flow occurs concurrently with linear electrontransport already at low PFDs, when oxygen supported electronflow, but not, when nitrite or methylviologen (MV) were present.Quantum efficiencies of the use of 696 and 675 nm light werecalculated for oxygen-, nitrite- and MV-dependent linear electronflows. Since H⁺/e=3 during linear electron transport [Ivanov(1993) Photosynthesis, p. 111; Kobayashi et al. (1995) PlantCell Physiol. 36: 1613] and comparable 9AA fluorescence quenchingindicates comparable transthylakoid proton gradients, totalproton transport could be calculated and part of it could beassigned to linear and the remainder to cyclic electron transportwhen oxygen was electron acceptor. Quanta of 696 nm light notused to support linear electron flow to oxygen at h/e=2 wereassumed to be available for coupled proton transport duringcyclic electron flow. H⁺/h ratios for cyclic electron transportobtained on this basis were consistently higher than 1 and occasionallyapproached 3. No allowance was made in these calculations foroxidized P700 in the reaction center of PSI, which could notdonate electrons to the cyclic pathway, and for reduced Q_A inthe reaction center of PSII. It therefore appears likely thatmaximum H⁺/h ratios in cyclic electron transport are higherthan values calculated in this work. Our observations with intactchloroplasts agree in principle with those of [Heath (1972)Biochim. Biophys. Acta 256: 645] with thylakoids, who also reportedhigh H⁺/ e ratios in cyclic electron transport. These ratiosare briefly discussed in relation to the H⁺/ATP stoichiometryof ATP production during carbon assimilation of leaves and toprotection of chloroplasts against photoinactivation. ²Present address: Timiriasev Institute of Plant Physiology,Russian Academy of Sciences, Botanicheskaya, 35, Moscow, Russia ³Present address: Department of Forestry, Faculty of Agriculture,Kyushu University, Hakozaki, Higashi-ku, Fukuoka, 812 Japan     

Energy charge,phosphorylation potential and proton motive force in chloroplasts

Adenylate concentrations were measured in intact chloroplasts under a variety of conditions. Energy charge was significant in the dark and increased in the light, but remained far below values expected from observed phosphorylation potentials in broken chloroplasts, which were 80 000 M^?1 or more in the light. With nitrite as electron acceptor, phosphorylation potentials in intact chloroplasts were about 80 M^?1 in the dark and only 300 M^?1 in the light. Similar phosphorylation potentials were observed, when oxaloacetate, phosphoglycerate or bicarbonate were used as substrates. ΔG′_ATP was ?42 kJ/mol in darkened intact chloroplasts, ?46 kJ/mol in illuminated intact chloroplasts and ?60 kJ/mol in illuminated broken chloroplasts. Uncoupling by NH₄Cl, which stimulated electron transport to nitrite or oxaloacetate and decreased the proton gradient, failed to decrease the phosphorylation potential of intact chloroplasts. Also, it did not increase the quantum requirement of CO₂ reduction. It is concluded that the proton motive force as conventionally measured and phosphorylation potentials are far from equilibrium in intact chloroplasts. The insensitivity of CO₂ reduction and of the phosphorylation potential to a decrease in the proton motive force suggests that intact chloroplasts are over-energized even under low intensity illumination. However, such a conclusion is at variance with available data on the magnitude of the proton motive force.     

Light-dependent fluorescence variations in intact leaves

Transient variations in the fluorescence from intact Phytolaccaamericana leaves after the onset of illumination were measuredunder various light and dark conditions. Dark-adapted leaveswhen illuminated with strong light underwent an intensity variationwith a peak; the fluorescence intensity reaching its peak severalseconds after the onset of illumination then decreasing to asteady level. The peak height relative to the steady level increasedwith the increasing intensity of actinic light. Pre-illuminationof the dark-adapted leaves with strong light caused a markedlowering of the peak. About 20 min of dark incubation was requiredfor the light-adapted leaves to return to the dark-adapted state.All of the action spectra, for the peak, the steady level andthe effect of light in post-illumination to inhibit recoveryto the dark state, showed high bands due to chlorophyll b andcarotenoid absorption and low bands due to chlorophyll a absorption.We concluded that the light absorbed by photosystem 2 is responsiblefor these phenomena. (Received April 21, 1975; )     

Crystallization of an R-Form Lipopolysaccharide from Klebsiella pneumoniae

An R-form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN-111 (O3^–:K1^–) formed crystals, whose shapes were elongated hexagonal plates, trapezoid plates, and rhomboid plates, and whose greatest dimensions were 3.1 × 0.8 μm, when it was suspended in 50 mM Tris buffer at pH 8.5 containing 5 mM MgCl₂ and kept at 4 C for as long as 870 days. K. pneumoniae LEN-111 synthesized LPS molecules possessing incomplete repeating units of the O-antigenic polysaccharide portion besides the R-form LPS because of a leaky characteristic, but crystals consisted exclusively of the R-form LPS. Although the size of crystals was not large enough for X-ray analysis and limited crystallographic information was available, it was suggested that the crystals consist of hexagonal lattices with an a axis of 4.62 Å and c axis of 79.8 ±2.6 Å. The present results showed that R-form LPS lacking the O-antigenic polysaccharide portion tends to form crystals during long-term incubation in Tris buffer at pH 8.5 containing MgCl₂ at 4 C.     

Protein-bound 4-hydroxy-2-hexenal as a marker of oxidized n-3 polyunsaturated fatty acids

In the present study, to investigate the contribution of n-3 PUFAs in the oxidative modification of protein in vivo, we characterize the covalent binding of 4-hydroxy-2-hexenal (HHE), a potent cytotoxic aldehyde originating from the peroxidation of n-3 PUFAs, to protein and describe the production of this aldehyde in oxidatively modified LDL and in human atherosclerotic lesions. Upon incubation with BSA, HHE was rapidly incorporated into the protein and generated the protein-linked carbonyl derivative, a potential marker of oxidatively modified proteins under oxidative stress. To detect the protein-bound HHE in vivo, we raised monoclonal antibody HHE53 (MAb HHE53) directed to the HHE-modified protein and identified the Michael addition-type HHE-histidine adduct as the major epitope. This antibody reacted with copper-oxidized LDL, suggesting that HHE was produced during the oxidative modification of LDL. In addition, we demonstrated that the materials immunoreactive to MAb HHE53 indeed constituted the atherosclerotic lesions, in which intense positivity was associated primarily with macrophage-derived foam cells. The results of this study suggest that the reaction between oxidized n-3 PUFAs and protein might represent a process common to the formation of degenerative proteins during aging and its related diseases.     

Genetic verification of Bordetella pertussis seed strains used for production of Japanese acellular pertussis vaccines

In Japan, the Bordetella pertussis strain Tohama provided by the National Institute of Health, Japan has been used for the production of acellular pertussis (aP) vaccines since 1981. In the present study, in order to verify the genetic consistency of B. pertussis vaccine seed strains, we analyzed the genetic properties of the working seeds obtained from five Japanese vaccine manufacturers, and compared them with those of B. pertussis Tohama reference strains (NIID L-7 and ATCC BAA-589). Genetic analyses with pulsed-field gel electrophoresis and allele typing showed 100% genetic identity among the five seed strains and the Tohama reference strains. In addition, Southern blot analyses revealed the absence of four orthologous genes (BB0537, BB0920, BB1149 and BB4885), which are specifically absent in the strain Tohama, and in the genome of all seed strains tested, suggesting that the regions of difference (RD11–RD14) are absent in their genomes. Consequently, no genetic difference was observed among the working seeds and Tohama reference strains. Our observations indicate that B. pertussis seed strains for Japanese aP vaccine production are genetically comparable with B. pertussis Tohama.     

Wave reduction in a mangrove forest dominated by Sonneratia sp.

Based on a field observation at the Vinh Quang coast in northern Vietnam, the characteristics of wave reduction due to the drag force of one mangrove species, Sonneratia sp., were quantitatively analyzed. The reduction rate of sea waves in this area changed substantially with the tidal phase, due to the unique vertical configuration of Sonneratia sp. At the shallow range of water depth, since the shape of pneumatophores of Sonneratia sp. tapers off upward, the effect of drag force by these roots on the wave reduction decreased with the increase in the water level, resulting in a decrease in the rate of wave reduction. On the other hand, when water levels rose above the height of thickly spread branches and leaves of these trees, the rate of wave reduction increased again with an increase in the water level. Further, at this high range of water level, the rate of wave reduction depended strongly on the incident wave height. These results indicate that the thickly grown mangrove leaves effectively dissipate huge wave energy which occurs during storms such as typhoons, and protect coastal areas. Referring to the past studies, our results suggest that the hydrodynamic knowledge in various mangrove conditions such as the vertical configuration of mangrove species, their vegetation conditions, water depth, incident wave condition and the relationships between these factors should be further accumulated and then quantitatively formulated in order to protect coastal areas from severe sea waves.