Calphostin C induces tyrosine dephosphorylation/inactivation of protein kinase Fa/GSK-3α in a pathway independent of tumor promoter phorbol ester-mediated down-regulation of protein kinase C

The signal transduction mechanism of protein kinase FA /GSK-3α by tyrosine phosphorylation in A431 cells was investigated using calphostin C as an inhibitor for protein kinase C (PKC). Kinase Fa /GSK-3α could be tyrosine-dephosphorylated and inactivated to ∼ 10% of control in a concentration-dependent manner by 0.1–10 μM calphostin C (IC₅₀, ∼ 1 μM), as demonstrated by immunoprecipitation of kinase Fa /GSK-3α from cell extracts, followed by phosphoamino acid analysis and by immunodetection in an antikinase Fa /GSK-3α immunoprecipitate kinase assay. In sharp contrast, down-regulation of PKC by 0.05 μM calphostin C (IC₅₀, ∼ 0.05 μM for inhibiting PKC in cells) or by tumor promoter phorbol ester TPA was found to have stimulatory effect on the cellular activity of kinase Fa /GSK-3α, when processed under identical conditions. Furthermore, TPA-mediated down-regulation of PKC was found to have no effect on calphostin C-mediated tyrosine dephosphorylation/inactivation of kinase Fa /GSK-3α. Taken together, the results provide initial evidence that the PKC inhibitor calphostin C may induce tyrosine dephosphorylation/inactivation of kinase Fa /GSK-3α in a pathway independent of TPA-mediated down-regulation of PKC, representing a new mode of signal transduction for the regulation of this multisubstrate/multifunctional protein kinase by calphostin C in cells. Since kinase Fa /GSK-3α is a possible carcinoma dedifferentiation/progression-promoting factor, the results further suggest calphostin C as a potential anticancer drug involved in blocking carcinoma dedifferentiation/progression, possibly via inactivation of protein kinase FA /GSK-3α in tumor cells. © 1996 Wiley-Liss, Inc.     

珍珠芦荟失绿突变体‘MT-1’的农艺性状、解剖结构、光合特性和代谢组特征分析

为阐明珍珠芦荟叶色突变的变异机制, 以珍珠芦荟(Aristaloe aristata)叶色失绿突变体‘MT-1’及其叶色正常种质‘WT-18’为试材,对其表型特征、叶片超微结构、光合色素含量、叶绿素荧光参数、差异代谢产物等方面进行了对比研究。结果表明, 与‘WT-18’相比,突变体‘MT-1’从幼叶开始呈现出黄化条斑现象,且株型变小、生长缓慢;气孔数量少且小,形状由长方形变成正方形;叶绿体数量及内部片层数量变少、体积也明显变小;光合色素(叶绿素a、叶绿素b和类胡萝卜素)含量和叶绿素荧光动力参数(F_o、F_m、F_v/F_m、ФPSⅡ、qP、qN和ETR)均显著降低。‘MT-1’和‘WT-18’的代谢产物差异明显,KEGG代谢通路分析表明,大多数差异代谢物富集到氨基酸代谢、糖代谢、脂类代谢、核酸代谢、次级代谢产物生物合成等途径。因此,叶绿体相关基因的改变可能是发生叶色失绿突变的重要原因。     

The early formation of the photosynthetic apparatus in Rhodospirillum rubrum

The time dependent assembly of the photosynthetic apparatus was studied in Rhodospirillum rubrum after transfer of cells growing aerobically in the dark to low aeration. While bacteriochlorophyll (Bchl) cellular levels increase continuously levels of soluble cytochrome c ₂do not change significantly. Absorption spectra of membranes isolated at different times after transfer reveal that incorporation of carotenoids lags behind incorporation of Bchl. However, a carotenoid fraction exhibiting spectral properties of spirilloxanthin isomers was isolated apart from membranes. This carotenoid fraction even was present in homogenates from Bchl-free, aerobically grown cells. Incorporation of U-¹⁴C-proteinhydrolyzate into membrane proteins showed that proteins are mainly formed which are specific for photosynthetic membranes. Although the proportion of reaction center (RC) Bchl per light harvesting (LH) Bchl does not change the proportions of membrane proteins present in RC and LH preparations change initially. But later on the proportions of the different proteins also reach constant values. Concerning proteins characteristic for cytoplasmic membranes a differential incorporation of label can be observed. The data indicate that the photosynthetic apparatus in Rhodospirillum rubrum is assembled through a sequential mechanism.Abbreviations Bchl bacteriochlorophyll - LH light harvesting - RC reaction center - R. Rhodospirillum - R. Rhodopseudomonas     

Down-regulation of two non-homologous endogenous tomato genes with a single chimaeric sense gene construct

Tomatoes (Lycopersicon esculentum Mill cv. Ailsa Craig) were transformed with a gene construct having 244 bp of the 5 end of a polygalacturonase (PG) cDNA, coding for a 71 amino acid N-terminal extension to the mature protein, fused to 1320 bp of a pectinesterase (PE) cDNA encoding the full sequence of the mature PE protein. This chimaeric gene was inserted in a sense orientation between a CaMV 35S promoter and terminator for constitutive expression. In transformed tomato plants expression of the endogenous PG and PE genes in the fruit was inhibited; there was little or no observable PG and PE mRNA and a substantial reduction in the level of PG and PE enzyme activity. The transgene was expressed in the leaves of the transformed plants as demonstrated by the accumulation of mRNA, but no protein product could be identified. However, no transgene mRNA or protein were observed in the transgenic fruit.This paper represents the first report of the down-regulation of two non-homologous endogenous genes using a single gene construct. A sense gene construct was responsible for these effects. These findings are discussed in relation to possible mechanisms of action of co-suppression.     

No photosynthetic down-regulation in sweetgum trees (Liquidambar styraciflua L.) after three years of CO2 enrichment at the Duke Forest FACE experiment

Photosynthetic capacity and leaf properties of sun and shade leaves of overstorey sweetgum trees (Liquidambar styraciflua L.) were compared over the first 3 years of growth in ambient or ambient + 200 μL L^?1 CO₂ at the Duke Forest Free Air CO₂ Enrichment (FACE) experiment. We were interested in whether photosynthetic down‐regulation to CO₂ occurred in sweetgum trees growing in a forest ecosystem, whether shade leaves down‐regulated to a greater extent than sun leaves, and if there was a seasonal component to photosynthetic down‐regulation. During June and September of each year, we measured net photosynthesis (A) versus the calculated intercellular CO₂ concentration (C_i) in situ and analysed these response curves using a biochemical model that described the limitations imposed by the amount and activity of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Vc_max) and by the rate of ribulose‐1,5‐bisphosphate (RuBP) regeneration mediated by electron transport (J_max). There was no evidence of photosynthetic down‐regulation to CO₂ in either sun or shade leaves of sweetgum trees over the 3 years of measurements. Elevated CO₂ did not significantly affect Vc_max or J_max. The ratio of Vc_max to J_max was relatively constant, averaging 2·12, and was not affected by CO₂ treatment, position in the canopy, or measurement period. Furthermore, CO₂ enrichment did not affect leaf nitrogen per unit leaf area (N_a), chlorophyll or total non‐structural carbohydrates of sun or shade leaves. We did, however, find a strong relationship between N_a and the modelled components of photosynthetic capacity, Vc_max and J_max. Our data over the first 3 years of this experiment corroborate observations that trees rooted in the ground may not exhibit symptoms of photosynthetic down‐regulation as quickly as tree seedlings growing in pots. There was a strong sustained enhancement of photosynthesis by CO₂ enrichment whereby light‐saturated net photosynthesis of sun leaves was stimulated by 63% and light‐saturated net photosynthesis of shade leaves was stimulated by 48% when averaged over the 3 years. This study suggests that this CO₂ enhancement of photosynthesis will be sustained in the Duke Forest FACE experiment as long as soil N availability keeps pace with photosynthetic and growth processes.     

Stoichiometry of proton uptake in isolated pea chloroplasts under different light intensities

The number of protons released inside the chloroplast thylakoids per electron which is transferred through the electron transport chain (H⁺/e^– ratio) was measured in isolated pea chloroplasts at pH 6.0 under continuous illumination and with methyl viologen as an electron acceptor. At saturating light intensity (200 W · m^–2) (strong light) the H⁺/e^– ratio was 3. At low intensity (0.9 W · m^–2) (weak light) the H⁺/e^– ratio was 2 with dark-adapted chloroplasts, but it was close to 3 with chloroplasts that were preilluminated with strong light. It is shown that the presence of azide in the reaction mixture leads to errors in the determination of the H⁺/e^– ratio due to underestimation of the initial rate of H⁺ efflux on switching off the light. To explain the above data, we assume that transformation of the electron transport chain occurs during illumination with strong light, namely, the Q cycle becomes operative.     

Evidences for interaction of PsbS with photosynthetic complexes in maize thylakoids

The PsbS subunit of Photosystem II (PSII) has received much attention in the past few years, given its crucial role in photoprotection of higher plants. The exact location of this small subunit in thylakoids is also debated. In this work possible interaction partners of PsbS have been identified by immunoaffinity and immunoprecipitation, performed with mildly solubilized whole thylakoid membrane. The interacting proteins, as identified by mass spectrometry analysis of the immunoaffinity eluate, include CP29, some LHCII components, but also components of Photosystem I, of the cytochrome b₆f complex as well as of ATP synthase. These proteins can be co-immunoprecipitated by using highly specific anti-PsbS antibodies and, vice-versa, PsbS is co-immunoprecipitated by antisera against components of the interacting complexes. We also find that PsbS co-migrates with bands containing PSII, ATP synthase and cytochrome b₆f as well as with LHCII-containing bands on non-denaturing Deriphat PAGE. These results suggest multiple location of PsbS in the thylakoid membrane and point to an unexpected lateral mobility of this PSII subunit. As revealed by immunogold labelling with antibody against PsbS, the protein is associated either with granal membranes or prevalently with stroma lamellae in low or high-intensity light-treated intact leaves, respectively. This finding is consistent with the capability of PsbS to interact with complexes located in stroma lamellae, even though the exact physiological condition(s) under which these interactions may take place remain to be clarified.     

8株光合细菌的鉴定及其系统进化关系分析

目的为8株光合细菌(photosynthetic bacteria,PSB)作为益生菌株提供系统资料。方法用常规方法对8株PSB菌株的形态、培养特性及生理生化特征进行鉴定,同时定性分析菌株产生的类胡萝卜素和CoQ,测定菌株16S DNA序列并分析其系统进化关系,在GenBank中获取了8个16S DNA序列号。结果菌株鉴定结果表明:菌株2C、2c和13ing为沼泽红假单胞菌,Ga、Il106、WS8N为类球红细菌,MT1131为荚膜红细菌,rub为深红红螺菌。基于菌株16S DNA序列的系统进化树显示,同一种菌并不总是聚为一簇,但相隔较近;种属完全不同的菌株,尽管序列相似性高达97%以上,在系统进化树上相隔较远。结论8株PSB菌株的鉴定和系统进化关系分析结果为后续研究提供了背景资料,同时菌株在GenBank中获得的16S DNA序列号为菌株作上了生物标记,也为菌株的产权保护提供了依据。     

烟草叶片发育过程中光合功能衰退与H_2O_2积累的关系

以烟草(NicotianatabacumL.cvNC89)为材料,研究了叶片发育过程中H2O2积累与叶绿体光合功能衰退、抗坏血酸-谷胱甘肽(AsA-GSH)循环的关联。结果表明,光合功能衰退过程中,各光合参数均表现为先缓慢后快速的下降趋势,核酮糖-1,5-二磷酸羧化酶(RuBPCase)活性下降较电子传递活性下降迅速,H2O2含量与叶绿素含量、光合速率、RuBPCase活性、抗坏血酸过氧化物酶(APX)、谷胱甘肽还原酶(GR)活性显著负相关。H2O2的定位染色也证实光合功能衰退与H2O2积累密切相关。APX和GR在光合功能可逆衰退阶段维持较高水平,不可逆衰退阶段下降稍快。烟草叶片光合功能衰退快于AsA-GSH循环运转的下调。     

Microalgae-based processes for the biodegradation of pretreated piggery wastewaters

The potential and limitations of photosynthetic oxygenation on carbon and nitrogen removal from swine slurry were investigated in batch experiments using Chlorella sorokiniana and an acclimated activated sludge as model microorganisms. While algal-bacterial systems exhibited similar performance than aerated activated sludge in tests supplied with four and eight times diluted slurry, a severe inhibition of the biodegradation process was recorded in undiluted and two times diluted wastewater. Daily pH adjustment to 7 in enclosed algal-bacterial tests at several swine slurry dilutions allowed the treatment of up to two times diluted slurries (containing up to 1,180 mg N-NH(4) (+) l(-1)). The combination of high pH levels and high NH(4) (+) concentrations was thus identified as the main inhibitory factor governing the efficiency of photosynthetically oxygenated processes treating swine slurry. Measurements of soluble total organic carbon (TOC) and volatile fatty acids (VFA) present in the slurry suggested that VFA degradation (mainly acetic and propionic acid) accounted for most of the soluble TOC removal, especially during the initial stages of the biodegradation process. On the other hand, assimilation into biomass and nitrification to NO(2) (-) constituted the main NH(4) (+) removal processes in enclosed algal-bacterial systems.