膜脂环境对紫膜蛋白的影响

紫膜蛋白菌紫质（bacteriorhodopsin,BR）处于不同的膜脂环境中,通过吸收光谱、荧光光谱和闪光动力学光谱的测定,比较了3种不同膜脂环境对菌紫质分子结构和功能的影响。实验结果表明：天然膜脂是BR最稳定的膜脂环境,可以形成以三聚体为单位的二维六角形品格结构。二豆蔻酰脂酰胆碱（dimyristoyl phosphatidylcholine,DMPC）也是BR分子的稳定脂环境,但以单体形式存在于膜脂环境中,功能受到一定影响。经TritonX-100增溶处理的紫膜膜脂环境中BR为单体,不稳定,容易发生结构和功能的变化。     

两种状态细菌视紫红质光循环中间产物与pH的关系

本文主要用微机控制的毫秒级闪光动力学光谱仪研究含三体细菌视紫红质(Bacteriorhodopsin,简称BR)的紫膜碎片和含单体BR的DMPC(dimyristoyl-Phosphatidyl-choline)脂质囊泡在不同pH条件下光循环中间产物M_(412)和O_(640)的变化,研究结果表明:BR单体与其三体状态相比,BR单体的光循环中间产物M_(412)的产量受介质pH变化的影响较大,其慢衰减成份的衰减比三体BR慢3—10倍.说明单体BR的结构状态较易受PH影响,单体BR光循环中间产物O_(640)随pH变化的趋势与三体BR的有很大区别,可能是由于不同状态的BR受pH的影响,但其具有不同的构型,导致光循环途径的变化.     

Bacteriorhodopsin/amphipol complexes: structural and functional properties

The membrane protein bacteriorhodopsin (BR) can be kept soluble in its native state for months in the absence of detergent by amphipol (APol) A8-35, an amphiphilic polymer. After an actinic flash, A8-35-complexed BR undergoes a complete photocycle, with kinetics intermediate between that in detergent solution and that in its native membrane. BR/APol complexes form well defined, globular particles comprising a monomer of BR, a complete set of purple membrane lipids, and, in a peripheral distribution, ∼2 g APol/g BR, arranged in a compact layer. In the absence of free APol, BR/APol particles can autoassociate into small or large ordered fibrils.     

Biodynamic response of human fingers in a power grip subjected to a random vibration

BACKGROUND: Knowledge of the biodynamic response (BR) of the human hand-arm system is an important part of the foundation for the measurement and assessment of hand-transmitted vibration exposure. This study investigated the BR of human fingers in a power grip subjected to a random vibration. METHOD: Ten male subjects were used in the experiment. Each subject applied three coupling actions to a simulated tool handle at three different finger grip force levels. RESULTS AND CONCLUSIONS: The BR is practically independent of the hand coupling actions for frequencies at or above 100 Hz. Above 50 Hz, the BR is correlated to finger and hand sizes. Increasing the finger coupling force significantly increases the BR. Therefore, hand forces should be measured and used when assessing hand-transmitted vibration exposure. The results also show that under a constant-velocity vibration, the finger vibration power absorption at frequencies above 200 Hz is approximately twice that at frequencies below 100 Hz. This suggests that the frequency weighting specified in the current ISO 5349-1 (2001) may underestimate the high frequency effect on vibration-induced finger disorders.     

The radical ions and photoionization of bile pigments

The semireduced, semioxidized, and OH(.)-adduct radicals of bilirubin (BR) and biliverdin (BV) have been characterized using pulse radiolysis techniques. Laser flash photolysis (265-nm) of these pigments led to monophotonic photoionization with quantum yields of 0.08 for BR and 0.03 for BV. No evidence for triplet formation or for photoisomerization was found after 265-nm laser excitation. However, 347-nm excitation of BR in chloroform led to simultaneous photoisomerization and radical formation, but the radicals are thought to have originated from a pathway other than photoionization. The relevance of these observations to BR photoreactivity is discussed. BR radical ions in alkaline solution did not react with tryptophan (TrpH), but the semioxidized TrpH radical oxidized BR with k = 4.3 X 10(8) dm3 mole-1 sec-1. When human serum albumin (HSA) was oxidized using radiolytically generated azide radicals, a radical transformation involving TrpH and TyrOH residues occurred with k = 3.8 X 10(3) sec-1. When BR was complexed with the protein the transformation rate was reduced to 1.6 X 10(3) sec-1. This was interpreted in terms of a conformational change in the protein. Identification of the probable residues involved provided information about the primary BR binding site which was consistent with an earlier report.     

Dimeric-like kinetic cooperativity of the bacteriorhodopsin molecules in purple membranes.

The kinetics of the absorption changes accompanying the photocycle of bacteriorhodopsin (BR) strongly depend on the intensity of the exciting short laser pulse. The decrease in the flash intensity dependence of the M kinetics after different extents of bleaching of the purple membranes by hydroxylamine proves the existence of a cooperative interaction between the photocycling BR molecules. The yield of the slow component of the M decay (M(s)) is a quadratic function of the extent of the fraction cycling. The slope of the relative weight of M(s) versus the fraction cycling is 0.5. This slope indicates a dimeric-like cooperative interaction, although the structural units of the purple membranes are the trimers of the BR molecules. For the most probable cooperative mechanism an asymmetric trimeric interaction is suggested, which accounts for the apparently dimeric features. A photocycling molecule may influence only one of its two neighbors in the trimer. From this asymmetric feature a deformative interaction is expected to be the cooperative mechanism, which would be an allosteric regulating mechanism in the purple membrane.     

Purification of bacteriorhodopsin and characterization of mature and partially processed forms

Bacteriorhodopsin (BR) essentially free of native lipids has been prepared in a highly stable state. Purple membrane was solubilized in Triton X-100 and BR was purified by size exclusion chromatography using 3-[cholamidopropyl)dimethylammonio]-2-hydroxyl-1-propanesulfonic acid (CHAPSO) detergent at pH 5. Molar ratios of phospholipid/BR ranged from 0.4 to 0.05 corresponding to 94-98% phospholipid removal. Purified BR has an absorbance ratio (A280nm/A548nm) of 1.5-1.6 in the dark-adapted state which is the highest purified BR/protein ratio reported to date. The purified BR in CHAPSO shows maximum stability in the pH range 5.0-5.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of native purple membrane and solubilized BR from most Halobacterium halobium JW-3 cultures show 3 higher molecular weight bands in addition to BR. Immunological staining and amino acid sequencing indicates that these additional proteins are partially processed forms of the BR precursor protein. The BR preprotein contains 13 additional amino acids on the NH2 terminus which are removed by post-translational processing in at least four steps. Isoelectric focusing separated most delipidated and non-delipidated BR samples into 8 bands. Incomplete BR post-translational processing BR is thought to be largely responsible for the multiplicity of isoelectric BR species. The principal components have pI values of 5.20 and 5.24 and both have absorption maxima at 550 nm, characteristic of detergent-solubilized BR. BR in Triton X-100 or nonylglucoside, delipidated BR in CHAPSO, and BR in intact purple membrane all have a dark-adapted ratio of 13-cis to all-trans-retinal of 1.9:1.     

The effect of charged lipids on bacteriorhodopsin membrane reconstitution and its photochemical activities

Bacteriorhodopsin (BR) was reconstituted into artificial lipid membrane containing various charged lipid compositions. The proton pumping activity of BR under flash and continuous illumination, proton permeability across membrane, as well as the decay kinetics of the photocycle intermediate M₄₁₂ were studied. The results showed that lipid charges would significantly affect the orientation of BR inserted into lipid membranes. In liposomes containing anionic lipids, BRs were more likely to take natural orientation as in living cells. In neutral or positively charged liposomes, most BRs were reversely assembled, assuming an inside out orientation. Moreover, the lipids charges also affect BR’s M intermediate kinetics, especially the slow component in M intermediate decay. The half-life M_412s increased significantly in BRs in liposomes containing cationic lipids, while decreased in those in anionic liposomes.     

Enhancement and phototransduction in the ventral eye of limulus

Limulus ventral photoreceptors were voltage clamped to the resting (dark) potential and stimulated by a 20-ms test flash and a 1-s conditioning flash. At a constant level of adaptation, we measured the response to the test flash given in the dark (control) and the incremental response produced when the test flash occurred within the duration of the conditioning flash. The incremental response is defined as the response to the conditioning and test flashes minus the response to the conditioning flash given alone. When the test flash was presented within 100 ms after the onset of the conditioning flash we observed that: (a) for dim conditioning flashes the incremental response equaled the control response; (b) for intermediate intensity conditioning flashes the incremental response was greater than the control response (we refer to this as enhancement); (c) for high intensity conditioning flashes the incremental response nearly equaled the control response. Using 10-μm diam spots of illumnination, we stimulated two spatially separate regions of one photoreceptor. When the test flash and the conditioning flash were presented to the same region, enhancement was present; but when the flashes were applied to separate regions, enhancement was nearly absent. This result indicates that enhancement is localized to the region of illumination. We discuss mechanisms that may account for enhancement.     

水在菌紫质光循环和质子泵中的作用

用圆二色仪和闪光动力学先谱仪分别测量了空气干燥紫膜薄层的圆二色谱及紫膜LB膜的M_(412)的衰减过程.在于燥紫膜的圆二色谱上出现412nm的正峰,它是光循环中间体M_(412)的特征峰.在无水介质中,紫膜LB膜中的BR仍能进行先化学循环而检测到中间体M_(412),但M_(412)的衰减速度减慢,产生M_(412)的堆积,质子化过程受阻.在有水的介质中,只要有足够的H~+存在,紫膜LB膜中的BR的中间体M_(412)的衰减速度明显加快.说明水介质的H~+是完成正常光化学循和质子化过程必不可少的.     

Brassinosteroids Positively Modulate Growth: Physiological,Biochemical and Anatomical Evidence Using Two Tomato Genotypes Contrasting to Dwarfism

Growth and development are vital processes in the life cycles of plants. Brassinosteroids (BRs) are steroids that when exogenously applied can regulate several biological responses. The aim of this research was to investigate the possible interferences caused by the exogenous application of BR on growth and metabolism using two genotypes of the DWARF gene, MT-d and MT-D, that are BR-deficient and BR-efficient, respectively. The experiment had four treatments with two genotypes (BR-efficient and BR-deficient) and two levels of brassinosteroids (0 and 100 nM BR, described as ??BR and +?BR, respectively). This study revealed that the exogenous application of BR promoted improvement in growth, inducing increases in all variables of both genotypes evaluated. In general, BR-deficient plants sprayed with BR had effects more intense, confirming the benefits of this steroid on photosynthetic apparatus and gas exchange. The changes in the anatomical characteristics of the leaf are related to the contribution of BR on the influx and consequent fixation of CO₂. In addition, modifications related to root anatomy occurred as a result of the BR action with the purpose of increasing the root protection and absorption of water and nutrients. Increases in photosynthetic pigments suggest that the role of BR is linked with chlorophyll biosynthesis and the maintenance of chloroplast integrity, resulting from associations with the increases found in the activities of antioxidant enzymes that modulate the accumulation of reactive oxygen species.     

酶切菌紫质(bR)C端对紫膜光循环和质子泵效率的影响

本文研究紫膜悬浮液经低剂量木瓜蛋酶处理去掉菌紫质(Bacteriorhodopsin简写bR)分子C-末端后。其光循环产物和质子泵效率的变化。实验发现经酶切后,M_(412)产物中慢衰减组份M_(412)降低了20％,O_(640)降低了50％,而质子泵效率降低了70％。双光脉冲实验表明酶解作用并不影响光循环周期。这些事实说明了去C-端所引起的质子泵效率降低,不是通过光循环的途径而产生的。介质中离子强度对正常紫膜和酶解紫膜的质子泵效率有明显不同的影响 说明了C端在不同盐浓度中的构象对质子泵行为有很重要的作用。     

表面活性剂TritonX－100对菌紫质蛋白紫外荧光性质的影响

用荧光光谱方法研究了ＴｒｉｔｏｎＸ－１００（以下缩写为ＴＸ－１００）对菌紫质蛋白（Ｂａｃｔｅｒｉｏｒｈｏｄｏｐｓｉｎ,ＢＲ）及视黄醛生色团漂白后的紫膜（Ｂａｔｅｒｉｏｐｓｉｎ,ＢＯ）紫外荧光性质的影响．结果表明：表面活性剂ＴＸ－１００使ＢＲ中色氨酸在３２６ｎｍ处的荧光发射强度增加。随着ＴＸ－１００对ＢＲ的增溶,改变了生色团的构象环境,破坏了ＢＲ中色氨酸与生色团之间的能量转移。增溶后的ＢＲ中,Ｔｒｐ趋向于更疏水性的环境。     

In vitro root induction by 24-epibrassinolide on hypocotyl segments of soybean [Glycine max (L.) Merr.]

Soybean hypocotyl segments were treated in the dark with 24-epibrassinolide (BR) at a range of concentrations for different durations. The maximum effect on adventitious root induction, both in terms of number and length was obtained at very low concentration (0.0001 ppm) of BR applied for 8 h. Higher concentrations were supraoptimal unless applied for a shorter period (4 h). BR was ineffective when applied at low concentration in continuous light.     

Improvement of bilirubin adsorption capacity of cellulose acetate/polyethyleneimine membrane using sodium deoxycholate

Bilirubin (BR) adsorbents have low removal efficiency because of the tight binding of BR with human albumin (HA) in a complicated blood system. Sodium deoxycholate (SDC) was selected as an adsorption promoter to improve the BR adsorption capacity of a cellulose acetate (CA)/polyethyleneimine (PEI) membrane. Static adsorption experiments show that the maximum BR adsorption capacity of the membrane with SDC in BR–HA mock solution is 100–200% at the molar ratio of SDC to HA ranging from 8 to 12, higher than that without SDC. It is also found that SDC is more efficiently adsorbed by the membrane than BR and HA. Absorption, circular dichroism, and zeta potential studies demonstrate that SDC can be bound with the BR–HA complex to form a ternary BR–HA-SDC_m complex. On the basis, the facilitated adsorption mechanism of BR with SDC was proposed that SDC aggregates or micelles form a quasi-multilayer adsorption on the membrane, increase approachable binding sites, and prolong the distance between the BR–HA complex and the membrane. Thus, SDC as a spacer reduces the influence of the steric hindrance of HA, resulting in an enhanced BR adsorption capacity. Dynamic adsorption results further evidence the facilitated adsorption mechanism.     

Brassinolide alleviated the adverse effect of water deficits on photosynthesis and the antioxidant of soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L.)

Brassinolide (BR) is a relatively new plant growth regulator. To test whether BR could be used to increase tolerance to water deficits in soybean, the effects of BR application on photosynthesis, assimilate distribution, antioxidant enzymes and seed yield were studied. BR at 0.1 mg l⁻¹ was foliar applied at the beginning of bloom. Two levels of soil moisture (80% field capacity for well-watered control and 35% for drought-stressed treatment) were applied at pod initiation. BR treatment increased biomass accumulation and seed yield for both treatments. Drought stress inhibited translocation of assimilated ¹⁴C from the labeled leaf, but BR increased the translocation for both treatments. Drought stress depressed chlorophyll content and assimilation rate (A), while chlorophyll content and A of BR-treated plants were greater than that of drought-stressed plants. BR treatment increased maximum quantum yield of PS II, the activity of ribulose-1,5-bisphosphate carboxylase, and the leaf water potential of drought-stressed plants. Treatment with BR also increased the concentration of soluble sugars and proline, and the activities of peroxidase and superoxide dismutase of soybean leaves when drought-stressed. However, it decreased the malondialdehyde concentration and electrical conductivity of leaves under drought stress. This study show that BR can be used as a plant growth regulator to enhance drought tolerance and minimize the yield loss of soybean caused by water deficits.     

Retardation of abscission of citrus leaf and fruitlet explants by brassinolide

Brassinolide (BR), a novel plant growth-regulating steroidal lactone, markedly retarded the abscission of leaf explants of Calamondin (Citrus madurensis Lour.), when dissolved in water and fed through the petiole. BR was effective at concentrations as low as 0.021 M, and showed a stronger effect than IAA which also retarded abscission. Trifluoperazine (TFP), an inhibitor of the calmodulin-calcium complex, accelerated abscission, and this acceleration could be counteracted by a simultaneous addition of IAA or BR, the effect of IAA being stronger. BR in lanolin applied to the cut surface of the leaf blade of the explant showed a weaker abscission-retarding effect than that applied in water via the petiole. BR and IAA also markedly retarded the abscission of fruitlet explants of Calamondin.     

Genetic transfer of the pigment bacteriorhodopsin into the eukaryote Schizosaccharomyces pombe

The gene encoding for bacterio-opsin (bop gene) from Halobacterium halobium has been introduced in a yeast expression vector. After transformation in Schizosaccharomyces pombe, bacterio-opsin (BO) is expressed and was detected by antisera. The precursor protein of BO (pre-BO) is processed by cleavage of amino acids at the N-terminal end as in H. halobium. Addition of the chromophore, retinal, to the culture medium results in a slight purple colour of the yeast cells indicating the in vivo regeneration of BO to bacteriorhodopsin (BR) and its incorporation into membranes. Therefore, in contrast to the expression in E. coli, isolation of the membrane protein and reconstitution in lipid vesicles is not necessary for functional analysis. The kinetics of the ground state signal of the photocycle BR in protoplasts is demonstrated by flash spectroscopy and is comparable to that of the natural system. The present investigation shows for the first time the transfer of an energy converting protein from archaebacteria to eukaryotes by genetic techniques. This is a basis for further studies on membrane biogenesis, genetics, and bioenergetics by analysis of in vivo active mutants.     

Dependence of Balbiani ring puffing on protein synthesis

The possible dependence of puffing of the Balbiani rings (BRs) on the protein synthesis has been investigated by studying the response of these structures to protein synthesis inhibition induced by cycloheximide and anisomycin. When larvae of Chironomus thummi belonging to middle IV instar (BR1 repressed, BR2 expanded) are subjected to short treatment (3–6 h) with these drugs, BR1 and BR2 puffing states remain essentially unaffected. But when the same treatments are applied to galactose-pretreated larvae (BR1 expanded, BR2 repressed), selective reactivation of the collapsed BR2 occurs. These observations suggest that maintenance of a given puffing state can be dependent, to a variable extent, on the supply of newly synthesized proteins. In particular, selective re-expansion of galactose-repressed BR2 induced by the drugs seem to indicate the existence of repressor-like factor whose activity would be triggered by the galactose treatment.