Absence of the Lhcb1 and Lhcb2 proteins of the light-harvesting complex of photosystem II - effects on photosynthesis,grana stacking and fitness

We have constructed Arabidopsis thaliana plants that are virtually devoid of the major light-harvesting complex, LHC II. This was accomplished by introducing the Lhcb2.1 coding region in the antisense orientation into the genome by Agrobacterium-mediated transformation. Lhcb1 and Lhcb2 were absent, while Lhcb3, a protein present in LHC II associated with photosystem (PS) II, was retained. Plants had a pale green appearance and showed reduced chlorophyll content and an elevated chlorophyll a/b ratio. The content of PS II reaction centres was unchanged on a leaf area basis, but there was evidence for increases in the relative levels of other light harvesting proteins, notably CP26, associated with PS II, and Lhca4, associated with PS I. Electron microscopy showed the presence of grana. Photosynthetic rates at saturating irradiance were the same in wild-type and antisense plants, but there was a 10-15% reduction in quantum yield that reflected the decrease in light absorption by the leaf. The antisense plants were not able to perform state transitions, and their capacity for non-photochemical quenching was reduced. There was no difference in growth between wild-type and antisense plants under controlled climate conditions, but the antisense plants performed worse compared to the wild type in the field, with decreases in seed production of up to 70%.     

State 1-State 2 transitions in the cyanobacterium Synechococcus 6301 are controlled by the redox state of electron carriers between Photosystems I and II

The mechanism by which state 1-state 2 transitions in the cyanobacterium Synechococcus 6301 are controlled was investigated by examining the effects of a variety of chemical and illumination treatments which modify the redox state of the plastoquinone pool. The extent to which these treatments modify excitation energy distribution was determined by 77K fluorescence emission spectroscopy. It was found that treatment which lead to the oxidation of the plastoquinone pool induce a shift towards state 1 whereas treatments which lead to the reduction of the plastoquinone pool induce a shift towards state 2. We therefore propose that state transitions in cyanobacteria are triggered by changes in the redox state of plastoquinone or a closely associated electron carrier. Alternative proposals have included control by the extent of cyclic electron transport around PS I and control by localised electrochemical gradients around PS I and PS II. Neither of these proposals is consistent with the results reported here.Abbreviations DBMIB 2,5-dibromo-3methyl-6-isopropyl-p-benzoquinone - Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DQH₂ duroquinol (tetramethyl-p-hydroquinone) - LHC II light-harvesting chlorophyll a/b-binding protein of PS II - Light 1 light predominantly exciting PS I - Light 2 light predominantly exciting PS II - M.V. methyl viologen - PS photosystem     

Consequences of state transitions on the structural and functional organization of Photosystem I in the green alga Chlamydomonas reinhardtii

State transitions represent a photoacclimation process that regulates the light‐driven photosynthetic reactions in response to changes in light quality/quantity. It balances the excitation between photosystem I (PSI) and II (PSII) by shuttling LHCII, the main light‐harvesting complex of green algae and plants, between them. This process is particularly important in Chlamydomonas reinhardtii in which it is suggested to induce a large reorganization in the thylakoid membrane. Phosphorylation has been shown to be necessary for state transitions and the LHCII kinase has been identified. However, the consequences of state transitions on the structural organization and the functionality of the photosystems have not yet been elucidated. This situation is mainly because the purification of the supercomplexes has proved to be particularly difficult, thus preventing structural and functional studies. Here, we have purified and analysed PSI and PSII supercomplexes of C. reinhardtii in states 1 and 2, and have studied them using biochemical, spectroscopic and structural methods. It is shown that PSI in state 2 is able to bind two LHCII trimers that contain all four LHCII types, and one monomer, most likely CP29, in addition to its nine Lhcas. This structure is the largest PSI complex ever observed, having an antenna size of 340 Chls/P700. Moreover, all PSI‐bound Lhcs are efficient in transferring energy to PSI. A projection map at 20 Å resolution reveals the structural organization of the complex. Surprisingly, only LHCII type I, II and IV are phosphorylated when associated with PSI, while LHCII type III and CP29 are not, but CP29 is phosphorylated when associated with PSII in state2.     

Brassinosteroids, microtubules and cell elongation in Arabidopsis thaliana. II. Effects of brassinosteroids on microtubules and cell elongation in the bul1 mutant

In order to elucidate the involvement of brassinosteroids in the cell elongation process leading to normal plant morphology, indirect immunofluorescence and molecular techniques were use to study the expression of tubulin genes in the bul1-1 dwarf mutant of Arabidopsis thaliana (L.) Heynh., the characteristics of which are reported in this issue (M. Catterou et al., 2001). Microtubules were studied specifically in the regions of the mutant plant where the elongation zone is suppressed (hypocotyls and petioles), making the reduction in cell elongation evident. Indirect immunofluorescence of α-tubulin revealed that very few microtubules were present in mutant cells, resulting in the total lack of the parallel microtubule organization that is typical of elongating cells in the wild type. After brassinosteroid treatment, microtubules reorganized and became correctly oriented, suggesting the involvement of brassinosteroids in microtubule organization. Molecular analyses showed that the microtubule reorganization observed in brassinosteroid-treated bul1-1 plants did not result either from an activation of tubulin gene expression, or from an increase in tubulin content, suggesting that a brassinosteroid-responsive pathway exists which allows microtubule nucleation/organization and cell elongation without activation of tubulin gene expression. Received: 28 April 2000 / Accepted: 6 October 2000     

Light-adaptive state transitions in the Ross Sea haptophyte Phaeocystis antarctica and in dinoflagellate cells hosting kleptoplasts derived from it

Light state transitions (STs) is a reversible physiological process that oxygenic photosynthetic organisms use in order to minimize imbalances in the electronic excitation delivery to the reaction centers of Photosystems I and II, and thus to optimize photosynthesis. STs have been studied extensively in plants, green algae, red algae and cyanobacteria, but sparsely in algae with secondary red algal plastids, such as diatoms and haptophytes, despite their immense ecological significance. In the present work, we examine whether the haptophyte alga Phaeocystis antarctica, and dinoflagellate cells that host kleptoplasts derived from P. antarctica, both endemic in the Ross Sea, Antarctica, are capable of light adaptive STs. In these organisms, Chl a fluorescence can be excited either by direct light absorption, or indirectly by electronic excitation (EE) transfer from ultraviolet light absorbing mycosporine-like amino acids (MAAs) to Chl a (Stamatakis et al., Biochim. Biophys. Acta 1858 [2017] 189–195). Here we show that, on adaptation to PS II-selective light, dark-adapted P. antarctica cells shift from light state 1 (ST1; more EE ending up in PS II) to light state 2 (ST2; more EE ending up in PS I), as revealed by the spectral distribution of directly-excited Chl a fluorescence and by changes in the macro-organization of pigment-protein complexes evidenced by circular dichroism (CD) spectroscopy. In contrast, no STs are clearly detected in the case of the kleptoplast-hosting dinoflagellate cells, and in the case of indirectly excited Chls a, via MAAs, in P. antarctica cells.     

The High Efficiency of Photosystem I in the Green Alga Chlamydomonas reinhardtii Is Maintained after the Antenna Size Is Substantially Increased by the Association of Light-harvesting Complexes II

Photosystems (PS) I and II activities depend on their light-harvesting capacity and trapping efficiency, which vary in different environmental conditions. For optimal functioning, these activities need to be balanced. This is achieved by redistribution of excitation energy between the two photosystems via the association and disassociation of light-harvesting complexes (LHC) II, in a process known as state transitions. Here we study the effect of LHCII binding to PSI on its absorption properties and trapping efficiency by comparing time-resolved fluorescence kinetics of PSI-LHCI and PSI-LHCI-LHCII complexes of Chlamydomonas reinhardtii. PSI-LHCI-LHCII of C. reinhardtii is the largest PSI supercomplex isolated so far and contains seven Lhcbs, in addition to the PSI core and the nine Lhcas that compose PSI-LHCI, together binding ∼320 chlorophylls. The average decay time for PSI-LHCI-LHCII is ∼65 ps upon 400 nm excitation (15 ps slower than PSI-LHCI) and ∼78 ps upon 475 nm excitation (27 ps slower). The transfer of excitation energy from LHCII to PSI-LHCI occurs in ∼60 ps. This relatively slow transfer, as compared with that from LHCI to the PSI core, suggests loose connectivity between LHCII and PSI-LHCI. Despite the relatively slow transfer, the overall decay time of PSI-LHCI-LHCII remains fast enough to assure a 96% trapping efficiency, which is only 1.4% lower than that of PSI-LHCI, concomitant with an increase of the absorption cross section of 47%. This indicates that, at variance with PSII, the design of PSI allows for a large increase of its light-harvesting capacities.     

Effects of Rubisco kinetics and Rubisco activation state on the temperature dependence of the photosynthetic rate in spinach leaves from contrasting growth temperatures

Recently, several studies reported that the optimum temperature for the initial slope [IS(Ci)] of the light-saturated photosynthetic rate (A) versus intercellular CO2 concentration (Ci) curve changed, depending on the growth temperature. However, few studies compare IS(Ci) with ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) properties. Here, we assessed Rubisco activation state and in vitro Rubisco kinetics, the main determinants of IS(Ci), in spinach leaves grown at 30/25 [high temperature (HT)] and 15/10 degrees C [low temperature (LT)]. We measured Rubisco activation state and A at a CO2 concentration of 360 microL L(-1) (A360) at various temperatures. In both HT and LT leaves, the Rubisco activation state decreased with increasing temperatures above the optimum temperatures for A360, while the activation state remained high at lower temperatures. To compare Rubisco characteristics, temperature dependences of the maximum rate of ribulose 1,5-bisphosphate (RuBP) carboxylation (Vcmax), specificity factor (Sc/o) and thermal stability were examined. We also examined Vcmax, and thermal stability in the leaves that were transferred from HT to LT conditions and were subsequently kept under LT conditions for 2 weeks (HL). Rubisco purified from HT, LT and HL leaves are called HT, LT and HL Rubisco, respectively. Thermal stabilities of LT and HL Rubisco were similar and lower than that of HT Rubisco. Both Vcmax and Sc/o in LT Rubisco were higher than those of HT Rubisco at low temperatures, while these were lower at high temperatures. Vcmax in HL Rubisco were similar to those of LT Rubisco at low temperatures, and to those of HT Rubisco at high temperatures. The predicted photosynthetic rates, taking account of the Rubisco kinetics and the Rubisco activation state, agreed well with A360 in both HT and LT leaves. This study suggests that photosynthetic performance is largely determined by the Rubisco kinetics at low temperature and by Rubisco Kinetics and the Rubisco activation state at high temperature.     

Time-resolved chlorophyll fluorescence studies on photosynthetic mutants of Chlamydomonas reinhardtii: origin of the kinetic decay components

The room temperature chlorophyll fluorescence decay kinetics of photosynthetic mutants of Chlamydomonas reinhardtii have been measured as a function of Photosystem 2 (PS2) trap closure, DNB-induced quenching at F_M, and time-resolved emission spectra. The overall decays have been analyzed in terms of three or four kinetic components where necessary. A comparison of the characteristics of the decay components exhibited by the mutants with the wild-type has been carried out to elucidate the precise origins of the different emissions in relation to the observed pigment-protein complexes. It is shown that a) charge recombination in PS2 is not necessary for the presence of long-lived decay components, b) there are two rapid PS1-associated emissions (=30 and 150–200 ps), c) a slow PS1 decay is observed (=1.73 ns) in the absence of PS1 reaction centres, d) the two variable components (=0.25–1.2 and 0.5–2.2 ns) observed in the wild-type arise from LHC2 and e) a rapid (=50–250 ps) decay is associated with the PS2 core antenna (CP3 and CP4). These results show that the intact thylakoid membrane system is too complex to distinguish all of the individual kinetic components.Abbreviations Aexp preexponential factor (Amplitude) - chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DNB m, dinitrobenzene - F_M maximum chl fluorescence level - F₀ initial chl fluorescence level - F_v variable chl fluorescence (F_M–F₀) - LHC light harvesting chl a/b protein complex - PS photosystem - Q_A primary stable electron acceptor of PS2     

Inhibition of extracellular signal-regulated kinase potentiates the apoptotic and antimetastatic effects of cyclin-dependent kinase inhibitors on metastatic DU145 and PC3 prostate cancer cells

Purvalanol and roscovitine are specific cyclin-dependent kinase (CDK) inhibitors, which have antiproliferative and apoptotic effects on various types of cancer. Although, the apoptotic accomplishment of purvalanol and roscovitine was elucidated at the molecular level, the underlying exact of drug-induced apoptosis through mitogen-activated protein kinase (MAPK) signaling still speculative. In addition, the role of CDK inhibitors in the downregulation of extracellular signal–regulated kinase 1/2 (ERK1/2)-mediated epithelial-mesenchymal transition (EMT) remains unclear. Here, we investigated the potential effect of each CDK inhibitors on cell proliferation, migration, and generation of reactive oxygen species due to the inhibition of MAPKs in metastatic DU145 and PC3 prostate cancer cells. We reported that purvalanol and roscovitine induced mitochondria membrane potential loss–dependent apoptotic cell death, which was also characterized by activation of several caspases, cleavage of poly (ADP-ribose) polymerase-1 in DU145 and PC3 cells. Cotreatment of either purvalanol or roscovitine with ERK1/2 inhibitor, U0126, synergistically suppressed cell proliferation, and induced apoptotic action. Also, ERK1/2 inhibition potentiated the effect of each CDK inhibitor on the downregulation of EMT processes via increasing the epithelial marker and decreasing mesenchymal markers through reduction of Wnt signaling regulators in DU145 cells. This study provides biological evidence about purvalanol and roscovitine have apoptotic and antimetastatic effects via MAPK signaling on prostate cancer cell by activation of GSK3β signaling and inhibition of phosphoinositide-3-kinase/AKT (PI3K/AKT) pathways involved in the EMT process.     

山奈酚与水的氢键作用对其抗氧化活性影响的理论研究

本文用密度泛函理论在(RO)B3LYP/6-31G(2d,2p)水平上对山奈酚及其与水分子之间形成的氢键复合物进行结构优化,通过热力学计算研究了不同位置的酚羟基发生抽氢反应的键离解能(BDE)、质子解离反应过程的质子解离能(DPE)受分子间氢键的影响。结果表明:与H2O形成的分子间氢键会影响化合物结构,改变化合物B环与AC环的二面角,A5位酚羟基更容易发生抽氢反应和质子解离反应,此位点的BDE和DPE均明显降低,同时也降低C3位质子解离的DPE。分子间氢键的形成促使酚羟基的抽氢和质子解离反应,提高化合物抗氧化活性。     

Systematic studies on some species of the genus Artemisia: biomolecular analysis

ABSTRACT

The internal transcribed spacers (ITS) of the ribosomal DNA gene of 11 taxa of the genus Artemisia were sequenced and compared with other 14 species taken from GenBank. The aims of this study are to clarify phylogenetic relationships for 25 taxa within the genus Artemisia, and to highlight the phylogenetic position of some species of geobotanical interest from the Alps or from other European areas. The results support the monophyly of the genus Artemisia, and the presence of the five main clades, corresponding to the morphologically based sections, Absinthium, Artemisia, Seriphidium, Dracunculus and Tridentatae. Only A. annua and A. genipi are not classified in the section in which they were traditionally included: A. annua is assigned to Seriphidium and not Artemisia, and A. genipi to Absinthium and not Artemisia. The basal structure of the tree differed in the 45 equally parsimonious MP trees, and thus appeared as a polytomy in the consensus tree. This does not allow us to completely solve the relationships among the clades. The molecular data are complementary with the morphological and biogeographical information and all are essential to draw valid conclusions on the relative closeness of the various taxa.     

The effect of fludrocortisone on the uterine receptivity partially mediated by ERK1/2-mTOR pathway

Implantation of embryos needs endometrial receptivity. Mineralocorticoids is one of the causes influencing the implantation window. This study targeted to evaluation fludrocortisone different properties on endometrial receptivity. The objective of this study was to assess whether treatment with fludrocortisone could impact the expression of diverse genes and proteins that are involved in uterine receptivity in mice. In this study, 40 female adult BALB/c mice were used. The samples were allocated to four groups of ten. Control group (C) received: vehicle; fludrocortisone group (FCA): received 1.5 mg/kg fludrocortisone; PP242 group (PP242): received 30 mg/kg PP242; fludrocortisone+PP242 group (FCA+PP242): received fludrocortisone and PP242. Mice were killed on window implantation day after mating and confirmed pregnancy. The endometrial epithelium of mouse was collected to assess mRNA expression of leukemia inhibitory factor (LIF), mucin-1 (MUC1), heparin-binding epidermal growth factor (HB-EGF), (Msx.1), miRNA Let-7a, and miRNA 223-3p as well as protein expression of extracellular signal-regulated kinase 1/2 (ERK1/2), mammalian target of rapamycin (mTOR), and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) in the uterine using real-time PCR and western blot, respectively. In comparison with the control group, fludrocortisone administration upregulated the expression of LIF, HB-EGF, Msx.1, miRNA Let-7a, ERK1/2, and mTOR in the epithelial endometrium. The PP242-treated group demonstrated a significant rise in the expression of MUC1, miRNA 223-3p and a remarkable decline in ERK1/2 and p-4E-BP1 levels in comparison with the control group. Combination therapy of (FCA+PP242) resulted in a remarkable rise in LIF, Msx-1, HB-EGF, ERK1/2, and mTOR levels, in comparison with the PP242 group. Furthermore, combination therapy of (FCA+PP242) downregulated the expression of MUC1 in comparison with the PP242-treated group. According to the results, fludrocortisone affected uterine receptivity possibly by means of modulating the expression of genes involved in the uterine receptivity and activation of the ERK1/2-mTOR pathway.     

S-亚硝基-N-乙酰-DL-青霉胺对RAW264.7巨噬细胞亚型分化的影响

目的:探讨S-亚硝基-N-乙酰-DL-青霉胺(SNAP)对巨噬细胞亚型分化的影响及其机制。方法:以RAW264.7巨噬细胞为研究对象,分为空白对照组、SNAP组、SNAP+PBA(4-苯基丁酸)组,采用不同浓度(30、100、300、400、500μmol/L)的SNAP或300μmol/L SNAP+20 mmol/L PBA对巨噬细胞进行干预24 h,应用RT-PCR法检测RAW264.7巨噬细胞亚型分化标志物M1(iNOS,CD86)、M2(Arg-I,MR)及CHOP mRNA的表达,应用Western blot技术检测iNOS及ERS通路中相关蛋白CHOP、P-PERK的表达。结果:与空白对照组比较,SNAP组iNOS、CD86、CHOPmRNA的表达均明显降低(P0.05),Arg-ImRNA表达明显升高(P0.05),而MR mRNA表达升高,但差异无统计学意义(P0.05);与300μmol/L SNAP组比较,300μmol/L+PBA组iNOS、CHOP mRNA均无明显变化(P0.05),CD86 mRNA升高,Arg-I、MR mRNA均明显降低(P0.05)。SNAP组CHOP、iNOS、p-PERK蛋白表达均明显低于对照组(P0.05),300μmol/LSNAP+20 mmol/LPBA组与300μmol/LSNAP组比较iNOS蛋白、p-PERK、CHOP蛋白表达升高(P0.05)。结论:NO可能通过内质网应激机制抑制巨噬细胞向M1亚型分化。     

Effect of heat stress on the inhibition and recovery of the ribulose-1,5-bisphosphate carboxylase/oxygenase activation state

Experiments were conducted to determine the relative contributions of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) activation state vis-à-vis Rubisco activase and metabolite levels to the inhibition of cotton (Gossypium hirsutum L.) photosynthesis by heat stress. Exposure of leaf tissue in the light to temperatures of 40 or 45 °C decreased the activation state of Rubisco to levels that were 65 or 10%, respectively, of the 28 °C control. Ribulose-1,5-bisphosphate (RuBP) levels increased in heat-stressed leaves, whereas the 3-phosphoglyceric acid pool was depleted. Heat stress did not affect Rubisco per se, as full activity could be restored by incubation with CO₂ and Mg²⁺. Inhibition and recovery of Rubisco activation state and carbon dioxide exchange rate (CER) were closely related under moderate heat stress (up to 42.5 °C). Moderate heat stress had negligible effect on Fv/Fm, the maximal quantum yield of photosystem II. In contrast, severe heat stress (45 °C) caused significant and irreversible damage to Rubisco activation, CER, and Fv/Fm. The rate of Rubisco activation after alleviating moderate heat stress was comparable to that of controls, indicating rapid reversibility of the process. However, moderate heat stress decreased both the rate and final extent of CER activation during dark-to-light transition. Treatment of cotton leaves with methyl viologen or an oxygen-enriched atmosphere reduced the effect of heat stress on Rubisco inactivation. Both treatments also reduced tissue RuBP levels, indicating that the amount of RuBP present during heat stress may influence the degree of Rubisco inactivation. Under both photorespiratory and non-photorespiratory conditions, the inhibition of the CER during heat stress could be completely reversed by increasing the internal partial pressure of CO₂ (Ci). However, the inhibition of the CER by nigericin, a K⁺ ionophore, was not reversible when the Ci was increased at ambient or high temperature. Our results indicate that inhibition of photosynthesis by moderate heat stress is not caused by inhibition of the capacity for RuBP regeneration. We conclude that heat stress inhibits Rubisco activation via a rapid and direct effect on Rubisco activase, possibly by perturbing Rubisco activase subunit interactions with each other or with Rubisco. Received: 25 February 2000 / Accepted: 13 May 2000     

Proteomic‐based investigations on the mode of action of the marine anticancer compound rhizochalinin

Rhizochalinin (Rhiz) is a novel marine natural sphingolipid‐like compound, which shows promising in vitro and in vivo activity in human castration‐resistant prostate cancer. In the present study, a global proteome screening approach was applied to investigate molecular targets and biological processes affected by Rhiz in castration‐resistant prostate cancer. Bioinformatical analysis of the data predicted an antimigratory effect of Rhiz on cancer cells. Validation of proteins involved in the cancer‐associated processes, including cell migration and invasion, revealed downregulation of specific isoforms of stathmin and LASP1, as well as upregulation of Grp75, keratin 81, and precursor IL‐1β by Rhiz. Functional analyses confirmed an antimigratory effect of Rhiz in PC‐3 cells. Additionally, predicted ERK1/2 activation was confirmed by Western blotting analysis, and revealed prosurvival effects in Rhiz‐treated prostate cancer cells indicating a potential mechanism of resistance. A combination of Rhiz with MEK/ERK inhibitors PD98059 (non‐ATP competitive MEK1 inhibitor) and FR180204 (ATP‐competitive ERK1/2 inhibitor) resulted in synergistic effects. This work provides further insights into the molecular mechanisms underlying Rhiz bioactivity. Furthermore, our research is exemplary for the ability of proteomics to predict drug targets and mode of action of natural anticancer agents.     

Muscarinic receptors in pancreatic islets of the rat: Demonstration and dependence on long-term glucose environment

The presence of muscarinic receptors in islets of Langerhans was assessed by measurement of specific binding of [³H]methylscopolamine. Specific binding was defined as total binding minus binding obtained in the presence of 1000-fold or higher excess of unlabeled methylscopolamine. At 37°C specific binding was significant after 1 min and plateaued after 10 min of incubation. Displacement of label by increasing concentrations of unlabeled methylscopolamine indicated a dissociation constant of 1.5·10^?12 M. Effects of methylscopolamine on insulin release were evaluated from the inhibitions of cholinergic-induced insulin release. 4·10^?10 M methylscopolamine inhibited acetylcholine (20 μM)-induced insuliln release more than 60%. Binding was not influenced by the following variations during binding incubations: changing the glucose concentration from 0 to 83 mM, adding rotenon (1 μM) or omitting calcium from the incubation medium. Islets kept in tissue culture exhibited higher binding when cultured at 11.1 than at 3.3 mM glucose for 96 h. It is concluded that islets contain muscarinic receptors, the binding to which can be subject to alteration by the long-term glucose environment.     

Determination of alpha-helix and beta-sheet stability in the solid state: a solid-state NMR investigation of poly(L-alanine)

The relative stability of alpha-helix and beta-sheet secondary structure in the solid state was investigated using poly(L-alanine) (PLA) as a model system. Protein folding and stability has been well studied in solution, but little is known about solid-state environments, such as the core of a folded protein, where peptide packing interactions are the dominant factor in determining structural stability. (13)C cross-polarization with magic angle spinning (CPMAS) NMR spectroscopy was used to determine the backbone conformation of solid powder samples of 15-kDa and 21.4-kDa PLA before and after various sample treatments. Reprecipitation from helix-inducing solvents traps the alpha-helical conformation of PLA, although the method of reprecipitation also affects the conformational distribution. Grinding converts the secondary structure of PLA to a final steady-state mixture of 55% beta-sheet and 45% alpha-helix at room temperature regardless of the initial secondary structure. Grinding PLA at liquid nitrogen temperatures leads to a similar steady-state mixture with 60% beta-sheet and 40% alpha-helix, indicating that mechanical shear force is sufficient to induce secondary structure interconversion. Cooling the sample in liquid nitrogen or subjecting it to high pressure has no effect on secondary structure. Heating the sample without grinding results in equilibration of secondary structure to 50% alpha-helix/50% beta-sheet at 100 degrees C when starting from a mostly alpha-helical state. No change was observed upon heating a beta-sheet sample, perhaps due to kinetic effects and the different heating rate used in the experiments. These results are consistent with beta-sheet approximately 260 J/mol more stable than alpha-helix in solid-state PLA.     

表皮生长因子对游离细胞核特异基因体外转录的影响

ＥＧＦ作用于ＮＣ３Ｈ１０和ＴＣ３Ｈ１０细胞核,对ＲＮＡ聚合酶Ⅱ有促进作用,但对ＲＮＡ聚合酶Ⅰ和酶Ⅲ没有影响,此外还发现转化细胞核内的ＲＮＡ聚合酶Ⅰ和酶Ⅱ的活性比正常细胞高１倍多。但两种细胞的ＲＮＡ聚合酶Ⅱ差别不大。同时,以非放射性标记的ｃ－ｆｏｓ、ＣＬＮ１、ＣＬＮ３探针进行点杂交,结果发现,ＥＧＦ直接作用于细胞核可使ｃ－ｆｏｓ、ＣＬＮ１基因的转录水平提高;但是,对ＣＬＮ３无影响。     

Phospholipid dependence of the anion transport system of the human erythrocyte membrane. Studies on reconstituted band 3/lipid vesicles

Band 3 protein extracted from human erythrocyte membranes by Triton X-100 was recombined with the major classes of phospholipid occurring in the erythrocyte membrane. The resulting vesicle systems were characterized with respect to recoveries, phospholipid composition, protein content and vesicle size as well as capacity and activation energy of sulfate transport. Transport was classified into band-3-specific fluxes and unspecific permeability by inhibitors. Transport numbers (sulfate ions per band 3 per minute) served as a measure of functional recovery after reconstitution. The transport properties of band 3 proved to be insensitive to replacement of phosphatidylcholine by phosphatidylethanolamine, while sphingomyelin and phosphatidylserine gradually inactivated band-3-specific anion transport when present at mole fractions exceeding 30 mol%. The activation energy of transport remained unaltered in spite of the decrease in transport numbers. The results, which are discussed in terms of requirements of band 3 protein function with respect to the fluidity and surface charge of its lipid environment, provide a new piece of evidence that the transport function of band 3 protein depends on the properties of its lipid environment just as the catalytic properties of some other membrane enzymes. The well-established species differences in anion transport (Gruber, W. and Deuticke, B. (1973) J. Membrane Biol. 13, 19–36) may to some extent reflect this lipid dependence.     

Further studies on the spreading of biomembranes at the air/water interface Structure,composition, enzymatic activities of human erythrocyte and sarcoplasmic reticulum membrane films

Air/water interface films were obtained from human erythrocytes and rabbit sarcoplasmic reticulum membranes at 'zero surface pressure, according to Verger, R. and Pattus, F. (Chem. Phys. Lipids (1976) 16, 285–291). The lipid and protein distribution of these membrane films suggest that the film composition is determined by the composition of the membrane and the mode of integration of its components. When kept at low surface pressure, slow film expansion occured due to unfolding of proteins at the interface. This process can be stopped by compressing the films at a higher surface pressure than 15 dyn/cm. Acetylcholinesterase activity from human erythrocyte films is highly dependent on the condensation state of the film. Ca²⁺-ATPase from sarcoplasmic reticulum films was still activable by Ca²⁺. Freeze-fracture studies on erythrocyte membrane films suggest that such films are monolayers in which proteins are randomly distributed.