Characterization of a phototolerant mutant of Synechocystis sp. PCC 6803 created by random mutagenesis of psbAII gene

Photosensitivity and photosynthetic characteristics have been analyzed in wild type (KC) and its psbAII mutant (I6) of Synechocystis having three point amino acid substitutions, i.e., N322I, I326F and F328S, which are localized in the C-terminal extension of D1 protein of the photosystem II reaction center. Wild type and mutant cells show almost an identical growth pattern under normal/low light (30 mumol m-2s-1, 30 degrees C) liquid culture (BG-11) condition. However, upon shifting the cultures to high light (500 mumol m-2s-1, 30 degrees C), these two types of cells exhibit entirely different growth characteristics, i.e., the mutant cells continue to grow normally whereas, the control cells fail to adapt the light stress and eventually resulting in complete loss of the photosynthetic pigments. On the other hand, a quick loss in the Fv/Fm value with half--decay time of about 30 min is observed in the mutant, in contrast to 120-130 min in case of control, upon shifting to high light conditions. In spite of this, mutant cells are able to adapt and grow well under prolonged high light exposure even after losing a major part of the variable yield of chlorophyll fluorescence (Fv/Fm). The high light treatment also induced decrease in the level of D1 protein in the mutant. However, half-decay time for D1 is much longer (approximately 10 hr) than that of variable fluorescence. Thus, the mutant cells have shown an unique way for cell growth and maintenance under high light even after losing Fv/Fm and photosynthetic oxygen evolving capacity as well as D1 content to a great extent. Therefore, these results could extend an interesting insight to understand the coordination of physiological, biochemical and molecular mechanisms regulating phototolerance of the photosynthetic organisms.     

Characterization of cyclic AMP-resistant Chinese hamster ovary cell mutants lacking type I protein kinase

A group of three mutants of Chinese hamster ovary cells (10260, 10265, and 10223) which are resistant to cyclic AMP (Gottesman, M. M., LeCam, A., Bukowski, M., and Pastan I. (1980) Somatic Cell Genet. 6, 45-61) have been characterized in this work. By genetic analysis, these mutants are all recessive and fall into two complementation groups. Cycl AMP-stimulated protein kinase activity in crude extracts of these mutants using histone as a substrate is decreased to 10 and 7% (complementation group I), and 31% (complementation group II), respectively, of the activity found in wild type extracts. The binding of cyclic [3H]AMP by extracts of all of these mutants is decreased to 30 to 50% of the binding found in wild type extracts. We have used the photoaffinity label 8-azidoadenosine 3':5'-[32P]monophosphate to label the regulatory subunits of type I and type II protein kinase in wild type and mutant extracts analyzed by DEAE-cellulose and Sephadex chromatography. We find that all three mutants lack type I cyclic AMP-dependent protein kinase and have reduced amounts of type II kinase activity. The regulatory subunits of type I and type II kinase are present in both complementation groups. We conclude that type I protein kinase is not needed for normal growth of Chinese hamster ovary cells. The defect in both classes of mutants appears to be in the failure of the catalytic subunit to associate normally with its regulatory subunits.     

The menD and menE homologs code for 2-succinyl-6-hydroxyl-2,4-cyclohexadiene-1-carboxylate synthase and O-succinylbenzoic acid-CoA synthase in the phylloquinone biosynthetic pathway of Synechocystis sp. PCC 6803

The genome of the cyanobacterium Synechocystis sp. PCC 6803 contains genes identified as menD and menE, homologs of Escherichia coli genes that code for 2-succinyl-6-hydroxyl-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase and O-succinylbenzoic acid-CoA ligase in the menaquinone biosynthetic pathway. In cyanobacteria, the product of this pathway is 2-methyl-3-phytyl-1,4-naphthoquinone (phylloquinone), a molecule used exclusively as an electron transfer cofactor in Photosystem (PS) I. The menD(-) and menE(-) strains were generated, and both were found to lack phylloquinone. Hence, no alternative pathways exist in cyanobacteria to produce O-succinylbenzoyl-CoA. Q-band EPR studies of photoaccumulated quinone anion radical and optical kinetic studies of the P700(+) [F(A)/F(B)](-) backreaction indicate that in the mutant strains, plastoquinone-9 functions as the electron transfer cofactor in the A(1) site of PS I. At a light intensity of 40 microE m(-2) s(-1), the menD(-) and menE(-) mutant strains grew photoautotrophically and photoheterotrophically, but with doubling times slower than the wild type. Both of which are sensitive to high light intensities. Low-temperature fluorescence studies show that in the menD(-) and menE(-) mutants, the ratio of PS I to PS II is reduced relative to the wild type. Whole-chain electron transfer rates in the menD(-) and menE(-) mutant cells are correspondingly higher on a chlorophyll basis. The slower growth rate and high-light sensitivity of the menD(-) and menE(-) mutants are therefore attributed to a lower content of PS I per cell.     

Changes in Thermostability of Photosystem Ⅱ and Leaf Lipid Composition of Rice Mutant with Deficiency of Light-harvesting Chlorophyll Protein Complexes

We studied the difference in thermostability of photosystem Ⅱ (PSⅡ) and leaf lipid composition between a T-DNA insertion mutant rice (Oryza sativa L.) VG28 and its wild type Zhonghua11. Native green gel and SDS-PAGE electrophoreses revealed that the mutant VG28 lacked all light-harvesting chlorophyll a/b protein complexes. Both the mutant and wild type were sensitive to high temperatures, and the maximal efficiency of PSⅡ photochemistry (Fv/Fm) and oxygen-evolving activity of PSⅡ in leaves significantly decreased with increasing temperature. However, the PSⅡ activity of the mutant was markedly more sensitive to high temperatures than that of the wild type. Lipid composition analysis showed that the mutant had less phosphatidylglycerol and sulfoquinovosyl diacylglycerol compared with the wild type. Fatty acid analysis revealed that the mutant had an obvious decrease in the content of unsaturation of membrane lipids on the thermostability of PSll are discussed.     

Recruitment of a foreign quinone into the A(1) site of photosystem I. I. Genetic and physiological characterization of phylloquinone biosynthetic pathway mutants in Synechocystis sp. pcc 6803

Genes encoding enzymes of the biosynthetic pathway leading to phylloquinone, the secondary electron acceptor of photosystem (PS) I, were identified in Synechocystis sp. PCC 6803 by comparison with genes encoding enzymes of the menaquinone biosynthetic pathway in Escherichia coli. Targeted inactivation of the menA and menB genes, which code for phytyl transferase and 1,4-dihydroxy-2-naphthoate synthase, respectively, prevented the synthesis of phylloquinone, thereby confirming the participation of these two gene products in the biosynthetic pathway. The menA and menB mutants grow photoautotrophically under low light conditions (20 microE m(-2) s(-1)), with doubling times twice that of the wild type, but they are unable to grow under high light conditions (120 microE m(-2) s(-1)). The menA and menB mutants grow photoheterotrophically on media supplemented with glucose under low light conditions, with doubling times similar to that of the wild type, but they are unable to grow under high light conditions unless atrazine is present to inhibit PS II activity. The level of active PS II per cell in the menA and menB mutant strains is identical to that of the wild type, but the level of active PS I is about 50-60% that of the wild type as assayed by low temperature fluorescence, P700 photoactivity, and electron transfer rates. PS I complexes isolated from the menA and menB mutant strains contain the full complement of polypeptides, show photoreduction of F(A) and F(B) at 15 K, and support 82-84% of the wild type rate of electron transfer from cytochrome c(6) to flavodoxin. HPLC analyses show high levels of plastoquinone-9 in PS I complexes from the menA and menB mutants but not from the wild type. We propose that in the absence of phylloquinone, PS I recruits plastoquinone-9 into the A(1) site, where it functions as an efficient cofactor in electron transfer from A(0) to the iron-sulfur clusters.     

Photosynthetic Properties of Photosystem II in Arabidopsis thaliana lpa1 Mutant

In a previous study, we characterized a high chlorophyll fluorescence lpa1 mutant of Arabidopsis thaliana, in which approximately 20% photosystem (PS) II protein is accumulated. In the present study, analysis of fluorescence decay kinetics and thermoluminescence profiles demonstrated that the electron transfer reaction on either the donor or acceptor side of PSII remained largely unaffected in the lpa1 mutant. In the mutant, maximal photochemical efficiency (Fv/Fm, where Fm is the maximum fluorescence yield and Fv is variable fluorescence) decreased with increasing light intensity and remained almost unchanged in wild-type plants under different light conditions. The Fv/Fm values also increased when mutant plants were transferred from standard growth light to low light conditions. Analysis of PSII protein accumulation further confirmed that the amount of PSII reaction center protein is correlated with changes in Fv/Fm in lpa1 plants. Thus, the assembled PSII in the mutant was functional and also showed increased photosensitivity compared with wild-type plants.(Author for correspondence. Tel: +86 (0)10 6283 6256; Fax: +86 (0)10 8259 9384; E-mail: zhanglixin@ibcas.ac.cn)     

Targeted mutagenesis of loop residues in the receptor-binding domain of the Bacillus thuringiensis Cry4Ba toxin affects larvicidal activity

Loop residues in domain II of Bacillus thuringiensis Cry delta-endotoxins have been demonstrated to be involved in insecticidal specificity. In this study, selected residues in loops beta6-beta7 (S(387)SPS(390)), beta8-beta9 (S(410), N(411), T(413), T(415), E(417) and G(418)) and beta10-beta11 (D(454)YNS(457)) in domain II of the Cry4Ba mosquito-larvicidal protein were changed individually to alanine by PCR-based directed mutagenesis. All mutant toxins were expressed in Escherichia coli JM109 cells as 130-kDa protoxins at levels comparable to the wild type. Only E. coli cells that express the P389A, S410A, E417A, Y455A or N456A mutants exhibited a loss in toxicity against Aedes aegypti mosquito larvae of approximately 30% when compared to the wild type. In addition, E. coli cells expressing double mutants, S410A/E417A or Y455A/N456A, at wild-type levels revealed a significantly higher loss in larvicidal activity of approximately 70%. Similar to the wild-type protoxin, both double mutant toxins were structurally stable upon solubilisation and trypsin activation in carbonate buffer, pH 9.0. These results indicate that S(410) and E(417) in the beta8-beta9 loop, and Y(455) and N(456) in the beta10-beta11 loop are involved in larvicidal activity of the Cry4Ba toxin.     

Polyphasic rise of chlorophyll a fluorescence in herbicide-resistant D1 mutants of Chlamydomonas reinardtii

Chlorophyll (Chl) a fluorescence transient, a sensitive and non-invasive probe of the kinetics and heterogeneity of the filling up of the electron acceptor pool of Photosystem II (PS II), was used to characterize D1-mutants of Chlamydomonas reinhardtii. Using a shutter-less system (Plant Efficiency Analyzer, Hansatech, UK), which provides the first measured data point at 10 s and allows data accumulation over several orders of magnitude of time, we have characterized, for the first time, complete Chl a fluorescence transients of wild type (WT), cell wall less (CW-15) C. reinhardtii and several herbicide-resistant mutants of the D1 proteins: D1-V219I A251V, F255Y, S264A G256D and L275F. In all cases, the Chl a fluorescence induction transients follow a pattern of O-J-I-P where J and I appear as two steps between the minimum Fo (O) and the maximum Fmax (Fm, P) levels. The differences among the mutants are in the kinetics of the filling up of the electron acceptor pool of PS II (this paper) in addition to those in the re-oxidation kinetics of Q_A ^– to Q_A, published elsewhere (Govindjee et al. (1992) Biochim Biophys. Acta: 1101: 353–358; Strasser et al. (1992) Archs. Sci. Genève 42: 207–224) and not in the ratio of the maximal fluorescence Fm to the initial fluorescence Fo. The value of this experimental ratio is Fm/Fo = 4.4±0.21 independent of the mutation. At 600 W m^–2 of 650 nm excitation, distinct hierarchy in the fraction of variable Chl a fluorescence at the J level is observed: S264A > A251V G256A > L275F V219I > F255Y CW-15 WT. At 300 and 60 W m^–2 excitation, a somewhat similar hierarchy among the mutants was observed for the intermediate levels J and I. Addition of bicarbonate-reversible inhibitor formate did not change the O to J phases, slowed the I to P rise, and in many cases, slowed the decay of fluorescence beyond the P level. These observations are interpreted in terms of formate effect being on the acceptor rather than on the donor side (S-states) of PS II. The formate effect was different in different mutants, with L275F being the most insensitive mutant followed by others (V219I, F255Y, WT, A251V and S264A). Further, in the presence of high concentrations of DCMU, identical transients were observed for all the mutants and the WT.The quantum yield of photochemistry of PS II, calculated from 1-(Fo/Fm), is in the range of 0.73 to 0.82 for the WT as well as for the mutants examined. Thus, in contrast to differences in the kinetics of the electron acceptor side of PS II, there were no significant differences in the maximum quantum yield of PS II, among the mutants tested. We suggest that earlier photochemistry yield values were much lower (0.4–0.6) than those reported here due to either higher measured values of Fo by instruments using camera shutters, or due to the use of cells grown in less than-optimal conditions.

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Antioxidation of Anthocyanins in Photosynthesis Under High Temperature Stress

Chlorophyll fluorescence and antioxidative capability in detached leaves of the wild type Arabidopsis thaliana L. ecotype Landsberg erecta （Ler） and three mutants deficient in anthocyanins biosynthesis （tt3, tt4, and tt3tt4） were investigated during treatment with temperatures ranging 25-45 ℃. In comparison with the wild type, chlorophyll fluorescence parameters Fv/Fm, φps,, electron transport rate （ETR）, Fv/Fo and qP in three anthocyanin-deficient mutants showed a more rapidly decreasing rate when the temperature was over 35 ℃. Non-photochemical quenching （NPQ） in these mutants was almost completely lost at 44 ℃, whereas the content of heat stable protein dropped and the rate of the membrane leakage increased. Fo-temperature curves were obtained by monitoring Fo levels with gradually elevated temperatures from 22 ℃ to 72 ℃ at 0.5 ℃/min. The inflexion temperatures of Fo were 45.8 ℃ in Ler, 45.1℃ in tt3, 44.1℃ in tt4 and 42.3 ℃ in tt3tt4, respectively. The temperatures of maximal Fo in three mutants were 1.9-3.8℃ lower than the wild type plants. Meanwhile, three mutants had lower activities of superoxide dismutase （SOD） and ascorbate peroxidase （APX） and an inferior scavenging capability to DPPH （1.1-diphenyl-2-picrylhy.drazyl） radical under heat stress, and in particular tt3tt4 had the lowest antioxidative potential. The results of the diaminobenzidine-H2O2 histochemical staining showed that H2O2 was accumulated in the leaf vein and mesophyll cells of mutants under treatment at 40 ℃, and it was significantly presented in leaf cells of tt3tt4. The sensitivity of Arabidopsis anthocyanins-deficient mutants to high temperatures has revealed that anthocyanins in normal plants might provide protection from high temperature injury, by enhancing its antioxidative capability under high temperature stress.     

茄子光系统Ⅱ的热胁迫特性

以耐热性较弱的黑贝一号圆茄和耐热性较强的黑贝二号圆茄为试材,热胁迫处理后采用植物效率仪PEA进行快速叶绿素荧光诱导曲线及其参数测定.结果表明：当温度高于40 ℃,PSⅡ结构受热胁迫影响较为敏感,表现为初始荧光F_o缓慢上升;PSⅡ原初光化学效率F_v/F_m和ΔF/F_m′大幅度下降,且黑贝二号F_v/F_m的半衰时间T₅₀和ΔF/F_m′的半衰温度t₅₀分别大于黑贝一号.较高的热胁迫剂量（48℃处理5 min或44℃处理20～30min）下,快速荧光诱导动力学曲线呈现OKJIP型,在700μs处出现与放氧复合体失活有关的K相.黑贝一号在44 ℃下处理20 min才有K相出现,黑贝二号则晚10 min出现.与35℃相比,在48℃,特别是在52℃的较高剂量热胁迫下,Strasser能量流动模型参数中的DI_o/RC有大幅度地增加,体现了热耗散对PSⅡ的较强保护能力.随着热胁迫温度的升高和热胁迫时间的延长,两品种的无活性中心F_vi/Fv显著增加.     

Interaction of a non-peptide agonist with angiotensin II AT1 receptor mutants

To identify residues of the rat AT1A angiotensin II receptor involved with signal transduction and binding of the non-peptide agonist L-162,313 (5,7-dimethyl-2-ethyl-3-[[4-[2(n-butyloxycarbonylsulfonamido)-5-isobutyl-3-thienyl]phenyl]methyl]imidazol[4,5,6]-pyridine) we have performed ligand binding and inositol phosphate turnover assays in COS-7 cells transiently transfected with the wild-type and mutant forms of the receptor. Mutant receptors bore modifications in the extracellular region: T88H, Y92H, G1961, G196W, and D278E. Compound L-162,313 displaced [125I]-Sar1,Leu8-AngII from the mutants G196I and G196W with IC50 values similar to that of the wild-type. The affinity was, however, slightly affected by the D278E mutation and more significantly by the T88H and Y92H mutations. In inositol phosphate turnover assays, the ability of L-162,313 to trigger the activation cascade was compared with that of angiotensin II. These assays showed that the G196W mutant reached a relative maximum activation exceeding that of the wild-type receptor; the efficacy was slightly reduced in the G1961 mutant and further reduced in the T88H, Y92H, and D278E mutants. Our data suggest that residues of the extracellular domain of the AT1 receptor are involved in the binding of the non-peptide ligand, or in a general receptor activation phenomenon that involves conformational modifications for a preferential binding of agonists or antagonists.     

高、低温胁迫对牡丹叶片PSⅡ功能和生理特性的影响

以牡丹‘肉芙蓉’离体叶片为试材,以25 ℃为对照,研究了强光(1400 μmol·m-2·s-1)下高温(40℃)和低温(15℃)处理对牡丹叶片PSⅡ光化学活性和生理特性的影响.结果表明:随处理时间的延长,各处理叶片的PSⅡ最大光化学效率(Fv/Fm)、PSⅡ实际光量子效率(φPsⅡ)和光下开放的PSⅡ反应中心激发能捕获效率(Fv’/Fm’)均持续降低.暗恢复4h后,对照和15℃处理叶片的Fv/Fm基本上完全恢复,而40℃处理叶片仅恢复到处理前的75.5％,即使15 h后也不能完全恢复;强光下40℃处理使PSⅠ和PSⅡ间的激发能分配严重偏离平衡状态.强光下40 ℃处理抑制了超氧化物歧化酶活性,加剧了O2、H2O2、丙二醛的产生,导致叶绿素和可溶性蛋白含量不断下降.说明强光下40℃高温胁迫对牡丹叶片光合机构造成了不可逆的破坏,而15℃低温处理对其光合机构的影响相对较弱.     

低温弱光胁迫对日光温室栽培杏树光系统功能的影响

以温室栽培的金太阳杏为材料,测定了金太阳杏叶片光合速率(P_n)、光系统Ⅱ(PSⅡ)光下实际光化学效率(Φ_PSⅡ)、光化学猝灭系数(q_P)和开放的PSⅡ反应中心的激发能捕获效率(F_v^′/F_m^′), 探讨了低温弱光(7 ℃、200 μmol·m^-2·s^-1 PFD)对叶片光系统Ⅰ(PSⅠ)和PSⅡ的抑制作用.结果表明：温室栽培的金太阳杏叶光合作用的最适温度在25 ℃左右.光下7 ℃的低温可使叶片净光合速率(P_n)大幅下降,造成激发压(1-q_P)增大,进而引起光抑制.低温弱光条件使PSⅠ和PSⅡ功能受到破坏,与单纯低温胁迫(7 ℃,黑暗)处理相比,经低温、弱光(7 ℃, 200 μmol·m^-2·s^-1PFD)胁迫2 h后,PSⅠ活性下降了28.26%,而PSⅡ最大光化学效率（F_v/F_m）没有发生显著变化,表明低温弱光条件下PSⅠ比PSⅡ 更易发生光抑制.     

Effects of Strong Light and Active Oxygen on Photosynthesis in Soybean

With the use of chlorophyll fluorescence technique, it was found that the net photosynthetic oxygen evolution rate decreased after strong light (2 000 μmol· m-2·2-1 ) treatment for two hours in soybean ( Glycine max L. ) leaves. The chlorophyll fluorescence parameters, Fm/Fo, Fv/Fm, ФPSII, qp and qN decreased along with the increase of light intensity. In strong light, exogenous active oxygen H202、·OH and 'O2 were harmful to soybean leaves. The destruction of 'O2 and·OH to leaves was most evident, as was shown that Fv/Fm and PS H decreased significantly. The antioxidants DABCO, mannitol, ascorbate and histidine protected the leaves, but weakly, from strong light. In darkness, the SOD inhibitor sodium diethyldithiocar- bamate (DDC) had no significant effect on Fm/Fo and Fv/Fm, but NAN,, the ascorbate peroxidase (APX)inhibitor, significantly decreased Fm/Fo, Fv/Fm and ФPS II. In strong light, however, beth DDC and NaN3 reduced the above-mentioned fluorescence parameters, but NaN3 was more effective than DDC. The results suggested that photoinhibition did take place in soybean leaves under strong light, and it was related to active oxygen in vivo.     

State 1-state 2 adaptation in the cyanobacteria Synechocystis PCC 6714 wild type and Synechocystis PCC 6803 wild type and phycocyanin-less mutant

The mechanism of excitation energy distribution between the two photosystems (state transitions) is studied in Synechocystis 6714 wild type and in wild type and a mutant lacking phycocyanin of Synechocystis 6803. (i) Measurements of fluorescence transients and spectra demonstrate that state transitions in these cyanobacteria are controlled by changes in the efficiency of energy transfer from PS II to PS I (spillover) rather than by changes in association of the phycobilisomes to PS II (mobile antenna model). (ii) Ultrastructural study (freeze-fracture) shows that in the mutant the alignment of the PS II associated EF particles is prevalent in state 1 while the conversion to state 2 results in randomization of the EF particle distribution, as already observed in the wild type (Olive et al. 1986). In the mutant, the distance between the EF particle rows is smaller than in the wild type, probably because of the reduced size of the phycobilisomes. Since a parallel increase of spillover is not observed we suggest that the probability of excitation transfer between PS II units and between PS II and PS I depends on the mutual orientation of the photosystems rather than on their distance. (iii) Measurements of the redox state of the plastoquinone pool in state 1 obtained by PS I illumination and in state 2 obtained by various treatments (darkness, anaerobiosis and starvation) show that the plastoquinone pool is oxidized in state 1 and reduced in state 2 except in starved cells where it is still oxidized. In the latter case, no important decrease of ATP was observed. Thus, we propose that in Synechocystis the primary control of the state transitions is the redox state of a component of the cytochrome b ₆/f complex rather than that of the plastoquinone pool.Abbreviations DCCD dicyclohexylcarbodiimide - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - EF exoplasmic face - PQ plasto-quinone - PS photosystem - PBS phycobilisome     

Phycobilisome composition and possible relationship to reaction centers

The photosynthetic apparatus was studied in Anacystis nidulans wild type and in a spontaneous pigment mutant 85Y which had improved growth in far-red light (greater than 650 nm). Two phycobiliproteins, C-phycocyanin (lambda max 625) and allophycocyanin (lambda max 650), were present in a molar ratio of approximately 3:1 in the wild type and approximately 0.4:1 in the mutant. Phycobilisomes of wild type cells were larger (57 X 30 nm) than those of the mutant 85Y (28 X 15 nm). In the mutant they seemed to consist primarily of the allophycocyanin core. Fluorescence emission maxima of wild type and mutant 85Y phycobilisomes were at 680 nm (23 degrees C) and 685 nm (-196 degrees C). Excitation maxima of phycobilisomes were at 630 and 650 nm for the wild type and the mutant 85Y, respectively. The phycobilisomes of wild type cells whether grown in white or far-red light had the same size and pigment composition. A typical wild type cell in white light had a thylakoid area of 22.8 microns 2, but in far-red light the area was reduced to 13.5 microns 2, which was close to that of 85Y at 13.6 microns 2. Chlorophyll molecules per cell decreased in far-red light from 1.1 X 10(7) in wild type (white light) to 4.5 X 10(6) in mutant 85Y (far-red). The number of phycobilisomes per cell (approx 2 X 10(4)), calculated from the phycobiliprotein content and phycobilisome size, was about the same in wild type (white light) and mutant 85Y (far-red light), but the number of phycobilisomes per unit area of thylakoid was significantly greater in mutant 85Y than in wild type. The present results suggest that the phycobilisomes are linked with reaction centers and that the PSII complement (photo-system II and phycobilisome) was fully maintained in far-red light.     

The herbicide-resistant D1 mutant L275F of Chlamydomonas reinhardtii fails to show the bicarbonate-reversible formate effect on chlorophyll a fluorescence transients

Herbicide-resistant mutants of the eukaryotic green alga Chlamydomonas reinhardtii, that are altered in specific amino acids in their D-1 protein, show differential bicarbonate-reversible formate effects. These results suggest the involvement of D1 protein in the bicarbonate effect. A 25 mM formate treatment of mixotrophically or photoautotrophically grown wild type cells results in a slower rise of chlorophyll a fluorescence transient followed by a dramatically slowed decline during measurements in continuous light. These effects are fully reversed upon addition of 10 mM bicarbonate. The mutant BR-202 [L275F] is, however, highly insensitive to 25 mM formate suggesting that a significant change in formate (bicarbonate) binding has occurred in helix V of the D1 protein near histidine involved in Fe binding. With the exception of DCMU-4 [S264A], which is considerably more sensitive to formate than the wild type, five other different [V219I, A25IV, F255Y, G256D and cell-wall deficient CW-15] mutants display a relatively similar response to formate as wild type. Absence of formate effect on a PS II-lacking [FuD-7] mutant confirms the sole involvement of PS II in the bicarbonate effect.     

Wild type and mutant p53 differentially regulate the gene expression of human collagenase-3 (hMMP-13)

Matrix metalloproteinases (MMPs) are a family of secreted or transmembrane proteins that can degrade all the proteins of the extracellular matrix and have been implicated in many abnormal physiological conditions including arthritis and cancer metastasis. Recently we have shown for the first time that the human MMP-1 gene is a p53 target gene subject to repression by wild type p53 (Sun, Y., Sun, Y. I., Wenger, L., Rutter, J. L., Brinckerhoff, C. E., and Cheung, H. S. (1999) J. Biol. Chem. 274, 11535-11540). Here, we report that cotransfection of fibroblast-like synoviocytes with p53 expression and hMMP13CAT reporter plasmids revealed that (i) hMMP13, another member of the human MMP family, was down-regulated by wild type p53, whereas all six of the p53 mutants tested lost the wild type p53 repressor activity in fibroblast-like synoviocytes; (ii) this repression of hMMP-13 gene expression by wild type p53 could be reversed by overexpression of p53 mutants p53-143A, p53-248W, p53-273H, and p53-281G; (iii) the dominant effect of p53 mutants over wild type p53 appears to be a promoter- and mutant-specific effect. An intriguing finding was that p53 mutant p53-281G could conversely stimulate the promoter activity of hMMP13 up to 2-4-fold and that it was dominant over wild type p53. Northern analysis confirmed these findings. Although the significance of these findings is currently unknown, they suggest that in addition to the effect of cytokines activation, the gene expression of hMMP13 could be dysregulated during the disease progression of rheumatoid arthritis (or cancer) associated with p53 inactivation. Since hMMP13 is 5-10 times as active as hMMP1 in its ability to digest type II collagen, the dysregulation or up-modulation of MMP13 gene expression due to the inactivation of p53 may contribute to the joint degeneration in rheumatoid arthritis.     

重金属对盐生草光合生理生长特性的影响

以盐生草幼苗为试验材料,分别设置0(CK)、50、100、200、400μg?g-1的Ni2+、Cu2+处理,研究重金属Ni2+和Cu2+对盐生草光合生理特性的影响.结果表明:盐生草叶片光合色素含量、净光合速率(Pn)、气孔导度Gs、蒸腾速率Tr、PSⅡ最大光化学效率Fv/Fm、非光化学猝灭系数qN及生长指标(株高、地上部干重和鲜重)在50μg?g-1的Ni2+处理时均达到最大值,后随Ni2+浓度继续增加,其叶片叶绿素a、叶绿素b、Pn、Gs、Tr、Fv/Fm、PSⅡ电子传递量子产率ΦPSⅡ、光化学猝灭系数qP、qN及各项生长指标逐步下降并低于对照水平,而细胞间隙CO2浓度(Ci)较对照呈增加趋势.在50μg?g-1的Cu2+处理时,盐生草叶片光合色素含量、Pn、Gs、Tr、Ci、Fv/Fm、ΦPSⅡ、qP、qN及各项生长指标均达峰值;在100μg?g-1Cu2+处理时,光合色素含量、Pn、Gs、Tr、Fv/Fm、ΦPSⅡ、qN及各项生长指标较对照仍有增加,而后随Cu2+浓度继续增加,其叶绿素a、叶绿素b、各光合参数、叶绿素荧光参数及生长指标均逐步降低并低于对照.可见,盐生草Pn在Ni2+胁迫下的下降主要是由非气孔限制所致,而Cu2+胁迫下的下降主要是由气孔限制所致;低浓度Ni2+和Cu2+对盐生草生长具有一定促进作用,过高浓度Ni2+和Cu2+则会通过抑制盐生草叶片叶绿素合成,影响其光合作用,从而抑制植株生长.     

Isoleucine 10 is essential for DNA gyrase B function in Escherichia coli

DNA gyrase is an essential enzyme that regulates the DNA topology in bacteria. It belongs to the type II DNA topoisomerase family and is responsible for the introduction of negative supercoils into DNA at the expense of hydrolysis of ATP molecules. The aim of the present work was to study the contribution of I10, one of the most important residues responsible for the stabilization of GyrB dimer and involved in the ATP-binding step, in the ATP-hydrolysis reaction and in the DNA supercoiling mechanism. We constructed MBP-tagged GyrB mutants I10G and Delta4-14. Our results demonstrate that both mutations severely affect the DNA-dependent ATPase activity and DNA supercoiling. Mutation of Y5 residue involved in the formation of ATPase catalytic site (Y5G mutant) had only little effect on the DNA-dependent ATPase activity and DNA supercoiling. Interestingly, the DNA-relaxation activity of MBP-GyrB mutants and wild type was completely inhibited by ATP. Binding of ADPNP to MBP-tagged mutants was significantly decreased. ADPNP had no effect on DNA-relaxation activity of MBP-tagged mutants but was able to inhibit MBP-tagged wild type enzyme. Our results demonstrate that GyrB N-terminal arm, and specially I10 residue is essential for ATP binding/hydrolysis efficiency and DNA transfer through DNA gyrase.