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The aba Mutant of Arabidopsis thaliana (L.) Heynh. Has Reduced Chlorophyll Fluorescence Yields and Reduced Thylakoid Stacking.

It has been shown that the aba mutant of Arabidopsis thaliana (L.) Heynh. is impaired in epoxy-carotenoid biosynthesis and accumulates the epoxy-carotenoid precursor, zeaxanthin (C.D. Rock, J.A.D. Zeevaart [1991] Proc Natl Acad Sci USA 88: 7496-7499). In addition to providing conclusive evidence for the indirect pathway of abscisic acid biosynthesis from epoxy-carotenoids, the aba mutation offers a powerful means to study the function of xanthophylls (oxygenated carotenoids) in photosynthesis. We measured in vivo the chlorophyll (Chl) fluorescence parameters F(o) (initial), F(m) (maximum), F(v) (variable = F(m) - F(o)), and t((1/2)) (half-rise time of fluorescence induction) of wild-type (WT) and three allelic aba mutants. The mutant genotypes had significantly lower F(o) and F(m) values relative to those of WT. The F(v)/F(m) ratio and t((1/2)), which are parameters affected by photochemical efficiency, photosystem II (PSII), and plastoquinone pool sizes, were similar in the aba alleles and WT. Because the aba genotypes accumulate high levels of zeaxanthin, which is involved in nonphotochemical quenching of Chl fluorescence, we propose that the reduced fluorescence yields in the aba genotypes are a consequence of the accumulated zeaxanthin. Measurement of PSII oxygen evolution rates in isolated thylakoid membranes of WT and aba-4 confirmed that quantum efficiency was not altered in aba-4 but indicated that the mutant had reduced PSII activity in vitro. Electron microscopy revealed an abnormal chloroplast ultrastructure in the aba plants: the mutants had significantly fewer thylakoid lamellae per granum stack but significantly more grana per chloroplast, as well as more chloroplasts per cell than WT. Immunoblot analysis established that aba-4 had normal levels of the Chl a/b-binding core polypeptide of PSII (CP29) and the PSII light-harvesting Chl a/b-binding complex. These results provide evidence for the role of zeaxanthin in nonphotochemical fluorescence quenching and suggest involvement of epoxy-carotenoids and/or zeaxanthin in thylakoid stacking and PSII activity.

Journal Article↗

Ascorbate-deficient mutants of Arabidopsis grow in high light despite chronic photooxidative stress.

Acclimation to changing environments, such as increases in light intensity, is necessary, especially for the survival of sedentary organisms like plants. To learn more about the importance of ascorbate in the acclimation of plants to high light (HL), vtc2, an ascorbate-deficient mutant of Arabidopsis, and the double mutants vtc2npq4 and vtc2npq1 were tested for growth in low light and HL and compared with the wild type. The vtc2 mutant has only 10% to 30% of wild-type levels of ascorbate, vtc2npq4 has lower ascorbate levels and lacks non-photochemical quenching of chlorophyll fluorescence (NPQ) because of the absence of the photosystem II protein PsbS, and vtc2npq1 is NPQ deficient and also lacks zeaxanthin in HL but has PsbS. All three genotypes were able to grow in HL and had wild-type levels of Lhcb1, cytochrome f, PsaF, and 2-cysteine peroxiredoxin. However, the mutants had lower electron transport and oxygen evolution rates and lower quantum efficiency of PSII compared with the wild type, implying that they experienced chronic photooxidative stress. The mutants lacking NPQ in addition to ascorbate were only slightly more affected than vtc2. All three mutants had higher glutathione levels than the wild type in HL, suggesting a possible compensation for the lower ascorbate content. These results demonstrate the importance of ascorbate for the long-term acclimation of plants to HL.

Arabidopsis↗

Induction of CO2 and Bicarbonate Transport in the Green Alga Chlorella ellipsoidea (II. Evidence for Induction in Response to External CO2 Concentration).

The critical species and concentrations of dissolved inorganic carbon (DIC) required for the induction of DIC transport during adaptation to low CO2 were determined for the green alga Chlorella ellipsoidea. The concentration of dissolved CO2 needed for the induction of both CO2 and HCO3- transport was independent of pH during adaptation, whereas the total DIC concentration required increased at alkaline pH. At pH 7.5, the minimum equilibrium DIC concentration at which high CO2 characteristics were maintained, i.e. transport was repressed, was 2100 [mu]M, whereas the maximum equilibrium DIC concentration below which DIC transport was fully induced (DICIND) was 500 [mu]M. Intracellular DIC concentration during adaptation to DICIND decreased temporarily after 2 h to 60% of the maximum level but recovered after 3 h of adaptation. After 3 h of adaptation to DICIND, cells exhibited maximum O2 evolution rate at DICIND. When cells partially adapted to DICIND were returned to high CO2, there was an immediate halt to the induction of transport and a gradual decrease in transport capacity over 23 h. The capacity for the induction of transport was unaffected by the absence of light. These results indicate that changes in the internal DIC pool during adaptation to low CO2 do not trigger the induction of DIC transport and that the induction is not light dependent. Induction of DIC transport in C. ellipsoidea appears to occur in response to the continuous exposure of cells to a critical CO2 concentration in the external medium.

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Restriction of Chlorophyll Synthesis Due to Expression of Glutamate 1-Semialdehyde Aminotransferase Antisense RNA Does Not Reduce the Light-Harvesting Antenna Size in Tobacco.

The formation of 5-aminolevulinate is a key regulatory step in tetrapyrrole biosynthesis. In higher plants, glutamate 1-semialdehyde aminotransferase (GSA-AT) catalyzes the last step in the sequential conversion of glutamate to 5-aminolevulinate. Antisense RNA synthesis for GSA-AT leads to reduced GSA-AT protein levels in tobacco (Nicotiana tabacum L.) plants. We have used these transgenic plants for studying the significance of chlorophyll (Chl) availability for assembly of the light-harvesting apparatus. To avoid interfering photoinhibitory stress, plants were cultivated under a low photon flux density of 70 [mu]mol photons m-2 s-1. Decreased GSA-AT expression does not seem to suppress other enzymic steps in the Chl pathway, indicating that reduced Chl content in transgenic plants (down to 12% of the wild-type level) is a consequence of reduced GSA-AT activity. Chl deficiency correlated with a drastic reduction in the number of photosystem I and photosystem II reaction centers and their surrounding antenna on a leaf area basis. Different lines of evidence from the transgenic plants indicate that complete assembly of light-harvesting pigment-protein complexes is given preference over synthesis of new reaction center/core complexes, resulting in fully assembled photosynthetic units with no reduction in antenna size. Photosynthetic oxygen evolution rates and in vivo Chl fluorescence showed that GSA-AT antisense plants are photochemically competent. Thus, we suggest that under the growth conditions chosen during this study, plants tend to maintain their light-harvesting antenna size even under limited Chl supply.

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Transplantation Studies with Immature Fruit of Normal, and rin and nor Mutant Tomatoes.

The aim of the work reported herein was to determine whether the lack of normal ripening in fruits of rin and nor tomato mutants is due to the presence of ripening inhibitors or to the lack of ripening factors in the fruit. A fruit tissue transplantation technique was developed for this purpose.Disks of pericarp tissue were transplanted reciprocally between tomato fruits (Lycopersicon esculentum Mill.) of the rin and nor mutants and fruits of ;Rutgers,' a normally ripening cultivar. CO(2) and ethylene evolution rates were measured daily. To test whether materials are translocated between receptor fruits and transplanted disks, fruits were vacuum-infiltrated with (14)C-labeled amino acids and implanted with disks from unlabeled fruits.Normal ripening was not induced in disks of rin and nor tissues implanted in Rutgers fruit although development of yellow or yellow-orange colors associated with senescence of the mutant fruits was accelerated. Disks of Rutgers fruit tissue implanted in fruits of rin and nor ripened normally and concomitant with the intact Rutgers control fruits. The transplanted disks contained 28.8% as much label as found in disks of receptor fruits; thus, significant translocation into the implanted disks occurred. It is concluded that fruits of the mutants do not contain translocatable ripening inhibitors or lack translocatable ripening factors.

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Phycobilisome-thylakoid Topography on Photosynthetically Active Vesicles of Porphyridium cruentum.

Conditions are described for isolating functional phycobilisome-thylakoid vesicles from the red alga Porphyridium cruentum. Phycobilisome-thylakoid vesicles were prepared by brief sonication and centrifugation in a medium containing 0.5 molar sucrose, 0.5 molar potassium phosphate, and 0.3 molar sodium citrate (pH 7.0). They required ferricyanide as an oxidant and had O(2) evolution rates (about 450 micromoles O(2) per hour per milligram chlorophyll) higher than whole cells (about 250 micromoles O(2) per hour per milligram chlorophyll). Energy transfer to photosystem II chlorophyll was evident from a high F695 nanometer (-196 C) emission peak. Preparations could be stored for over 24 hours and were considerably more stable than those from the cyanobacterium Anabaena variabilis (Katoh T, E Gantt 1979 Biochim Biophys Acta 546: 383-393). In electron micrographs of negatively stained material, the active thylakoid vesicles were found covered by closely spaced phycobilisomes on their external surface. The phycobilisome number in negatively stained vesicles was 450 per square micrometer, which was in the same range as the 400 per square micrometer observed in surface sections. A cell containing 1.5 x 10(-6) micrograms phycoerythrin and 1.3 x 10(-6) micrograms chlorophyll was found to contain 5 to 7 x 10(5) phycobilisomes on a thylakoid area of 1.1 to 1.6 x 10(3) square micrometers.

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Inorganic Carbon Accumulation and Photosynthesis in a Blue-green Alga as a Function of External pH.

The blue-green alga Coccochloris peniocystis photosynthesizes optimally over the pH range of 7.0 to 10.0, but the O(2)-evolution rate is inhibited below pH 7.0 and ceases below pH 5.25. Measurement of the inorganic carbon pool in this alga in the light, using the silicone-fluid filtration technique demonstrated that the rate of accumulation of dissolved inorganic carbon remained relatively constant over a wide pH range. At external dissolved inorganic carbon concentrations of 0.56 to 0.89 millimolar the internal concentration after 30 seconds illumination was greater than 3.5 millimolar over the entire pH range. Intracellular pH measured in the light using [(14)C]5,5-dimethyloxazolidine-2,4-dione and [(14)C]methylamine dropped from pH 7.6 at an external pH of 7.0 to pH 6.6 at an external pH of 5.25. Above an external pH of 7.0 the intracellular pH rose gradually to pH 7.9 at an external pH 10.0. Ribulose-1,5-bisphosphate carboxylase activity of cell-free algal extracts exhibited optimal activity at pH 7.5 to 7.8 but was inactive below pH 6.5. It is suggested that the inability of Coccochloris to maintain its intracellular pH when in an acidic environment restricts its photosynthetic capacity by a direct pH effect on the principal CO(2) fixing enzyme.

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Effect of salinity on tomato fruit ripening.

Tomato (Lycopersicon esculentum Mill) plants from various cultivars growing on half-strength Hoagland solution were exposed at anthesis to 3 or 6 grams per liter NaCl. Salinity shortened the time of fruit development by 4 to 15%. Fruits of salt-treated plants were smaller and tasted better than did fruits of control plants. This result was obtained both for ripe fruits tested on the day of picking and for those picked at 100% development and allowed to ripen at room temperature for 9 days. Percentage of dry weight, total soluble solids, and titratable acidity; content of reducing sugars, Cl(-), Na(+), and various pericarp pigments; and electrical conductivity of the juice were higher in fruits of saline-treated plants than they were in those of control plants, while the pH was lower. Ethylene and CO(2) evolution rates during ripening; as well as the activities of pectin methyl esterase, polymethylgalacturonase, and polygalacturonase; were also higher in fruits of the saline-treated plants. The treatment with 6 grams per liter NaCl shortened the fruit shelf life considerably.

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Anacystis nidulans Demonstrates a Photosystem II Cation Requirement Satisfied Only by Ca or Na.

Anacystis nidulans exhibits a total loss of photosystem II (PSII) activity upon incubation in a nutrient medium deficient in Ca(2+) and Na(+) and containing a divalent cation chelator. This loss of activity is light-dependent, which corresponds to an energy requirement. Likewise, Ca(2+) efflux takes place only in cells incubated in light. The loss of PSII activity is reversible by addition of submillimolar amounts of either Ca(2+) or Na(+) to the external medium but not by the addition of any other cation. Restoration of lost PSII activity also requires light. Light saturation curves for partially depleted cells demonstrate both lower maximum O(2) evolution rates and decreased relative quantum yields when compared to control cells. Partial electron transport reactions isolate the site of the Ca(2+)/Na(+) effect to the reaction center itself or immediately on its oxidizing side and exclude the water-splitting complex. O(2) flash yields decline during cation depletion, indicating a decrease in the number of functional PSII reaction centers, but the maximum turnover rate for still functional reaction centers does not decline. Thus, PSII of A. nidulans exhibits an all-or-none cation requirement, satisfied only by Ca(2+) or Na(+).

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Endogenous Auxin and Ethylene in the Lichen Ramalina duriaei.

Indole-3-acetic acid (IAA) levels and ethylene evolution rates were measured in a fruticose lichen Ramalina duriaei collected from carob trees growing in northeast Israel. IAA levels were estimated by gas liquid chromatography with electron capture detection of the pentafluorobenzyl ester and also by enzyme-linked immunosorbent assay following methylation. The identity of the isolated IAA was confirmed by gas chromatography-mass spectrometry of both the methyl and the pentafluorobenzyl ester. IAA levels in lichens 1 year after transplanting to an air-polluted urban site were found to be lower than in the control thalli left at a nonpolluted, rural site. The material from the latter contained about 2.5 micrograms per gram fresh weight free IAA and 0.5 microgram per gram fresh weight conjugated IAA, while the urban material contained 0.3 microgram per gram each of free and conjugated IAA. Ethylene production rate was 1.0 nanoliter per gram fresh weight per hour in the material from the rural site and 1.5 nanoliters per gram fresh weight per hour in material from the urban site.

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Effects of Elevated CO(2) Concentrations on Glycolysis in Intact ;Bartlett' Pear Fruit.

Mature intact ;Bartlett' pear fruit (Pyrus communis L.) were stored under a continuous flow of air or air + 10% CO(2) for 4 days at 20 degrees C. Fruit kept under elevated CO(2) concentrations exhibited reduced respiration (O(2) consumption) and ethylene evolution rates, and remained firmer and greener than fruit stored in air. Protein content, fructose 1,6-bisphosphate levels, and ATP:phosphofructokinase and PPi:phosphofructokinase activities declined, while levels of fructose 6-phosphate and fructose 2,6-bisphosphate increased in fruit exposed to air + 10% CO(2). These results are discussed in light of a possible inhibitory effect of CO(2) at the site of action of both phosphofructokinases in the glycolytic pathway, which could account, at least in part, for the observed reduction in respiration.

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Functional Analysis of the Photosynthetic Apparatus of Prochlorothrix hollandica (Prochlorales), a Chlorophyll b Containing Procaryote.

Light-shade adaptation of the chlorophyll a/b containing procaryote Prochlorothrix hollandica was studied in semicontinuous cultures adapted to 8, 80 and 200 mumole quanta per square meter per second. Chlorophyll a contents based on dry weight differed by a factor of 6 and chlorophyll b by a factor of 2.5 between the two extreme light conditions. Light utilization efficiencies determined from photosynthesis response curves were found to decrease in low light grown cultures due to lower light harvesting efficiencies; quantum requirements were constant at limiting and saturating irradiances for growth. At saturating growth irradiances, changes in light saturated oxygen evolution rate originated from changes in chlorophyll a antenna relative to the number of reaction centers II. At light-limiting conditions both the number of reaction centers II and the antenna size changed. The amount of chlorophyll b relative to reaction center II remained constant. As in cyanobacteria, the ratio of reaction center I to reaction center II was modulated during light-shade adaptation. On the other hand, time constants for photosynthetic electron transport (4 milliseconds) were low as observed in green algae and diatoms. The occurrence of state one to two and state two to one transitions is reported here. Another feature linking photosynthetic electron transport in P. hollandica to that in the eucaryotic photosynthetic apparatus was blockage of the state one to two transition by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Although chlorophyll b was reported in association with photosystem I, the 630 nanometer light effect does not exclude that chlorophyll b is the photoreceptor for the state one to two transition.

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Dark Ammonium Assimilation Reduces the Plastoquinone Pool of Photosystem II in the Green Alga Selenastrum minutum.

The impact of dark NH(4) (+) and NO(3) (-) assimilation on photosynthetic light harvesting capability of the green alga Selenastrum minutum was monitored by chlorophyll a fluorescence analysis. When cells assimilated NH(4) (+), they exhibited a large decline in the variable fluorescence/maximum fluorescence ratio, the fluorescence yield of photosystem II relative to that of photosystem I at 77 kelvin, and O(2) evolution rate. NH(4) (+) assimilation therefore poised the cells in a less efficient state for photosystem II. The analysis of complementary area of fluorescence induction curve and the pattern of fluorescence decay upon microsecond saturating flash, indicators of redox state of plastoquinone (PQ) pool and dark reoxidation of primary quinone electron acceptor (Q(A)), respectively, revealed that the PQ pool became reduced during dark NH(4) (+) assimilation. NH(4) (+) assimilation also caused an increase in the NADPH/NADP(+) ratio due to the NH(4) (+) induced increase in respiratory carbon oxidation. The change in cellular reductant is suggested to be responsible for the reduction of the PQ pool and provide a mechanism by which the metabolic demands of NH(4) (+) assimilation may alter the efficiency of photosynthetic light harvesting. NO(3) (-) assimilation did not cause a reduction in PQ and did not affect the efficiency of light harvesting. These results illustrate the role of cellular metabolism in the modulating photosynthetic processes.

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Ethylene Evolution from Maize (Zea mays L.) Seedling Roots and Shoots in Response to Mechanical Impedance.

The effect of mechanical impedance on ethylene evolution and growth of preemergent maize (Zea mays L.) seedlings was investigated by pressurizing the growth medium in triaxial cells in a controlled environment. Pressure increased the bulk density of the medium and thus the resistance to growth. The elongation of maize primary roots and preemergent shoots was severely hindered by applied pressures as low as 10 kilopascals. Following a steep decline in elongation at low pressures, both shoots and roots responded to additional pressure in a linear manner, but shoots were more severely affected than roots at higher pressures. Radial expansion was promoted in both organs by mechanical impedance. Primary roots typically became thinner during the experimental period when grown unimpeded. In contrast, pressures as low as 25 kilopascals caused a 25% increase in root tip diameter. Shoots showed a slight enhancement of radial expansion; however, in contrast to roots, the shoots increased in diameter even when growing unimpeded. Such morphological changes were not evident until at least 3 hours after initiation of treatment. All levels of applied pressure promoted ethylene evolution as early as 1 hour after application of pressure. After 1 hour, ethylene evolution rates had increased 10, 32, 70, and 255% at 25, 50, 75, and 100 kilopascals respectively, and continued to increase linearly for at least 10 hours. When intact corn seedlings were subjected to a series of hourly cycles of pressure, followed by relaxation, ethylene production rates increased or decreased rapidly, illustrating tight coupling between mechanical impedance and tissue response. Seedlings exposed to 1 microliter of ethylene per liter showed symptoms similar to those shown by plants grown under mechanical impedance. Root diameter increased 5 times as much as the shoot diameter. Pretreatment with 10 micromolar aminoethoxyvinyl glycine plus 1 micromolar silver thiosulfate maintained ethylene production rates of impeded seedlings at basal levels and restored shoot and root extension to 84 and 90% of unimpeded values, respectively. Our results support the hypothesis that ethylene plays a pivotal role in the regulation of plant tissue response to mechanical impedance.

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Survivin expression, apoptosis and proliferation in chronic myelomonocytic leukemia.

We analyzed the expression of the inhibitor of apoptosis survivin by immunocytochemistry in bone marrow cells from patients with chronic myelomonocytic leukemia (CMML) to evaluate possible abnormalities in comparison with other myelodysplastic (MDS) and myeloproliferative syndromes, and to investigate a possible correlation between survivin expression and altered apoptosis or proliferation, or relevant laboratory and clinical findings. Thirty-four patients with CMML [18 MDS-CMML and 16 myeloproliferative disorder (MPD)-CMML], 90 with MDS, 41 with acute myeloid leukemia (AML), 19 with chronic MPD and 25 control subjects were studied. In normal samples survivin was never detectable. In CMML survivin levels higher than in MDS and AML (P < 0.0001), but similar to those found in MPD were observed. In CMML and MDS apoptosis was significantly higher compared to normal controls and all other subtypes of leukemias (P < 0.0001). Proliferation did not differ significantly in normal controls, MDS and CMML; the lowest levels were observed in AML and MPD (P < 0.0001). In CMML there was no correlation between survivin expression and blast cell percentage, apoptosis or proliferation, FAB or WHO subgroup. Proliferation was higher in MDS-CMML and tended to correlate with overall survival. CMML-2 cases with higher survivin expression showed higher evolution rate and shorter survival. In conclusion, CMML is characterized by high proliferation and apoptosis. Survivin overexpression, by disrupting the balance between cell proliferation/differentiation and apoptosis, may play an important role in its pathophysiology. The detection of survivin-deregulated expression may provide a useful tool for diagnosis, prognosis and a possible target for experimental treatments.

Acute Disease↗

The primary structure of muskrat pancreatic ribonuclease.

Pancreatic ribonuclease from muskrat (Ondatra zibethica) was isolated and its amino acid sequence was determined from tryptic digests of the performic acid-oxidized and the reduced and aminoethylated enzyme. The peptides have been positioned in the sequence by homology with other ribonucleases. This could be done unambiguously for all peptides except Arg-Arg (tentative position 32-33) and Ser-Arg (tentative position 75-76). The amino acid sequences of the peptides were determined by the dansyl-Edman method, with the exception of residues 23-25 and 99-102, which were positioned by homology. The enzyme differs in 38 positions from the enzyme from rat and in 31-42 positions from other mammalian pancreatic ribonucleases, while rat ribonuclease differs at 44-52 positions from the other enzymes. These data point to a common ancestry of the enzymes from muskrat and rat and an increased evolution rate of rat ribonuclease after divergence of the ancestors of both species. Muskrat ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn-Val-Thr (62-64).

Amino Acid Sequence↗

Levocetirizine in children: evidenced efficacy and safety in a 6-week randomized seasonal allergic rhinitis trial.

Studies evaluating newer antihistamines in children are few. Levocetirizine is a potent and highly selective H1-antihistamine with a proven efficacy in adults. Primary objective was to assess the efficacy of levocetirizine 5 mg once-daily in reducing seasonal allergic rhinitis (SAR) symptoms, as measured by Total Four Symptom Score (T4SS = sum of sneezing, rhinorrhea, nasal and ocular pruritus), over the first 2 wk of treatment. Efficacy over 4 and 6 wk of treatment, effect on nasal congestion and on health-related quality of life as measured by PRQLQ (Paediatric Rhinoconjunctivitis Quality of Life Questionnaire) were among the major secondary objectives. A double-blind, randomized, placebo-controlled study including 177 children with a documented SAR (to grass and/or weed) for at least a year and having a mean baseline T4SS > or = 6 (out of 12). Children evaluated daily the severity of T4SS and nasal congestion on a scale from 0 (absent) to 3 (severe). PRQLQ responses were assessed on a scale from 0 (not bothered) to 6 (extremely bothered) and analysed descriptively. Global evaluation of disease evolution judged by investigators, parents and children was made on a scale from 1 (marked worsening) to 7 (marked improvement). For the primary objective, levocetirizine was statistically highly superior to placebo with a difference in adjusted means of 1.29 (95% CI: 0.66-1.92) in favour of levocetirizine (p < 0.001). The effect of levocetirizine was almost twice that of placebo (94.1% relative improvement over placebo). Nasal congestion was improved with levocetirizine reaching maximum difference to placebo of 0.31 (p < 0.05), a relative improvement over placebo of 77.5%. PRQLQ scores at week 2 improved with levocetirizine more than with placebo (0.85 vs. 0.51, respectively) remaining larger after 4 and 6 wk of treatment. In the study, 84.3%, 80.9%, 80.9% of children had their disease evolution rated as slightly-to-markedly improved by, respectively, the investigators, the parents and children themselves. Incidence of treatment-emergent adverse events was similar in both groups (33.7% with levocetirizine; 30.7% with placebo). No child in the levocetirizine group discontinued treatment because of adverse events. The 6-wk duration of this study was longer than the usual 2-4-wk duration for similar studies and shows that levocetirizine controls SAR symptoms in children over the entire pollen season.

Adolescent↗

Functional and structural analysis of photosystem II core complexes from spinach with high oxygen evolution capacity.

Oxygen-evolving photosystem II core complexes were prepared from spinach by solubilizing photosystem II membrane fragments with dodecyl-beta-D-maltoside. The core complexes consist of the intrinsic 47-kDa, 43-kDa, D1 and D2 polypeptides, the two subunits of cytochrome b559 and the extrinsic 33-kDa protein. In the presence of 50 mM CaCl2 they exhibit a high oxygen evolution rate of 1.3 +/- 0.2 mmol O2.mg chlorophyll-1.h-1 with either 2,6-dichloro-benzoquinone or K3[Fe(CN)6] as acceptor. Electron micrographs of these complexes reveal an obtuse triangular structure in when viewed from the top measuring 15.3 nm on one side and 10.6 nm on the other two sides. An average height of 7.3 nm was determined from the side view position. These data are in good agreement with previously reported dimensions for photosystem II core complexes [Irrgang, K.-D., Boekema, E. J., Vater, J. and Renger, G. (1988) Eur. J. Biochem. 178, 209-217]. In contrast to previous reports the extrinsic 33-kDa subunit could be resolved for the first time. It appears as a small protrusion when the complex is viewed from the side and seems to cover the lumenal side of the core complex appearing as a disk with a thickness of 1.5-3.3 nm.

Centrifugation, Density Gradient↗