The pH-dependence of photochemical intermediates of O and P in bacteriorhodopsin by continuous light

The pH-dependence of the O and P intermediates in the photocycle of bacteriorhodopsin (bR) on the intensity and duration of the exciting flash was investigated for bR glycerol suspensions and bR gelatin films. Green and red laser flashes (532 and 670 nm) were utilized to generate a photoequilibrium state of bR and O at ambient temperature, and UV-vis spectroscopy was used to determine the photoconversion for the bR suspensions and films. The maximal concentration of the O intermediate was observed to be pH-dependent and the dependency was most pronounced at a slightly alkaline pH values. The photochemical conversion from the O to P intermediate was investigated for both bR suspensions and films. The P intermediate was only found in bR gelatin film. These results indicate that bR gelatin film may be an attractive candidate for the information storage based on P intermediate. It is possible, with red light, to create photoproducts which are thermally stable at ambient temperature and that can be photochemically erased.     

Two-photon absorption of bacteriorhodopsin: formation of a red-shifted thermally stable photoproduct F620

By means of high-intensity 532 nm laser pulses, a photochemical conversion of the initial B(570) state of bacteriorhodopsin (BR) to a stable photoproduct absorbing maximally at approximately 620 nm in BR suspensions and at approximately 610 nm in BR films is induced. This state, which we named F(620), is photochemically further converted to a group of three products with maximal absorptions in the wavelength range from 340 nm to 380 nm, which show identical spectral properties to the so-called P(360) state reported in the literature. The photoconversion from B(570) to F(620) is most likely a resonant two-photon absorption induced step. The formation of F(620) and P(360) leads to a distinguished photo-induced permanent optical anisotropy in BR films. The spectral dependence of the photo-induced anisotropy and the anisotropy orientations at the educt (B(570)) and product (F(620)) wavelengths are strong indicators that F(620) is formed in a direct photochemical step from B(570). The chemical nature of the P(360) products probably is that of a retro-retinal containing BR, but the structural characteristics of the F(620) state are still unclear. The photo-induced permanent anisotropy induced by short laser pulses in BR films helps to better understand the photochemical pathways related to this transition, and it is interesting in view of potential applications as this feature is the molecular basis for permanent optical data storage using BR films.     

电极介导的菌紫质干膜微分光电流响应的整流性质

在长方形光脉冲光照下,菌紫质(bacteriorhodopsin,BR)干膜组装成夹层光电池具有微分光电流响应．在氧化铟锡(ITO)导电玻璃/BR膜/封口膜/不锈钢形成的干膜电池下可观察到整流特性,而在不锈钢/BR膜/封口膜/ITO导电玻璃形成的干膜电池下则观察不到整流特性,这说明是电极介导的整流．平衡电压测定表明：工作电极/BR膜表面与对电极/BR膜表面有不同的性质,电极的界面效应控制了BR的取向．酸与碱产生的瞬间电流极性也证实了电极整流行为的存在．这些结果将有助于了解BR膜的微分光电响应．     

PCL film surfaces conjugated with P(DMAEMA)/gelatin complexes for improving cell immobilization and gene transfection

Successful gene transfection on a tissue scaffold is of crucial importance in facilitating tissue repair and regeneration by enabling the localized production of therapeutic drugs. Polycaprolactone (PCL) has been widely adopted as a scaffold biomaterial, but its unfavorable cell-adhesion property needs to be improved. In this work, the PCL film surface was conjugated with poly((2-dimethyl amino)ethyl methacrylate) (P(DMAEMA))/gelatin complexes via surface-initiated atom transfer radical polymerization (ATRP) for improving cell immobilization and subsequent gene transfection. A simple aminolysis-based method was first used for the covalent immobilization of ATRP initiators on the PCL film. Well-defined P(DMAEMA) brushes were subsequently prepared via surface-initiated ATRP from the initiator-functionalized PCL surfaces. The P(DMAEMA) chains with a pK(a) of 7.0-7.3 were used for conjugating gelatin with a pI of 4.7 via electrostatic interaction. The amount of complexed gelatin increased as that of the grafted P(DMAEMA) layer. The cell-adhesion property on the functionalized PCL surface could be controlled by adjusting the ratio of P(DMAEMA)/gelatin. It was found that the gene transfection property on the immobilized cells was dependent on the density of the immobilized cells on the functionalized PCL film. With the good cell-adhesive nature of gelatin and the efficient gene transfection on the dense immobilized cells, the incorporating the suitable of P(DMAEMA)/gelatin complexes onto PCL surfaces could endow the PCL substrates new and interesting properties for potential tissue engineering applications.     

Bromocriptine-induced inhibition of hydroxylase/lyase activity of adult rat Leydig cells

The present in vitro studies using a suspension of Leydig cells from adult rat testis demonstrated that bromocriptine (BR, 2 × 10⁻⁵M) inhibits hCG-stimulated testosterone production (in the presence of submaximal and maximal doses of hCG), while basal production was unaffected. When the cells were exposed to 8-bromo-cAMP either in the presence or absence of hCG, the inhibitory effect of BR was not reversed. In intact cells, BR inhibited conversion of progesterone and 17-hydroxy-progesterone to testosterone while conversion of androstenedione was not affected. Incubation of homogenates of Leydig cells in the presence of limiting NADPH concentrations ( 0.1 mM) resulted in significant BR-induced inhibition of conversion of progesterone (10 μM) to testosterone, while in the presence of “high” concentrations of NADPH ( 0.5 mM) BR was without effect. Present results suggest that BR inhibits androgen production at the level of the microsomal enzymes 17-hydroxylase and/or 17,20-lyase. The inhibitory effect of BR using homogenates of Leydig cells was evident only in the presence of limiting NADPH concentrations that suggests a competitive-like pattern of inhibition, but mechanisms by which BR decreases activity of microsomal enzymes remain to be determined.     

Transient behavior of a continuous stirred tank biological reactor utilizing phenol as an inhibitory substrate

Transient experiments were conducted on a Pseudomomas utilizing phenol in a continuous culture by disturbing the influent substrate concentration and dilution rate. Two stable steady states existed for some ranges of the parameters. Highly damped oscillations were observed in approaching a new high conversion steady state or in returning to a new high conversion steady state following a small disturbance. When a large disturbance was applied there was a smooth (overdamped) approach to a new low conversion steady state. The observed oscillatory behavior for small disturbances was predicted by a modified Powell-Ierusalemskii bottleneck model, but could not be predicted by a Monod-Haldane model; neither model was accurate for predicting the effect of large disturbances. A constant wall growth factor was used to account for microbial film activity, and the existence of two stable states was directly due to the presence of the film.     

Biomacromolecules electrostatic self-assembly on 3-dimensional tissue engineering scaffold

A poly(ethylenimine) (PEI) was employed to obtain a stable positively charged surface on a poly(D,L-lactide) (PDL-LA) tissue engineering scaffold. An extracellular matrix (ECM)-like biomacromolecule, gelatin, was selected as polyelectrolyte and deposit alternately with PEI on the activated PDL-LA scaffold via ESA technique. The zeta-potential result showed alternating charge of polyelectrolytes (PEI/gelatin) layering on PDL-LA microspheres. Quartz crystal microbalance (QCM) measurement further verified the gradual deposition of PEI/gelatin on the PDL-LA thin film. The combination of PEI aminolysis and the layer-by-layer technique was then explored to construct gelatin coating onto the 3-D porous PDL-LA scaffold. Scanning electronic microscopy showed that there is no notable difference between modified and unmodified PLA scaffolds, with regard to the porosity, pore diameter, and scaffold integration. The dual-tunnel confocal laser scanning microscopy indicated uniform gelatin distribution on the inner surface of the 3-D porous scaffold. The gradual build-up of protein layer on scaffold was investigated by radioiodination technique. Chondrocyte was chosen to test the cell behavior on modified and unmodified PDL-LA scaffolds. The results of the cell viability, total intracellular protein content, and cell morphology on the PEI/gelatin multilayers modified PDL-LA scaffold showed to promote chondrocyte growth. Comparing conventional coating methods, polyelectrolyte multilayers are easy and stable to prepare. It may be a promising choice for the surface modification of complex biomedical devices. These very flexible systems allow broad medical applications for drug delivery and tissue engineering.     

Photochemical conversion of the O-intermediate to 9-cis-retinal-containing products in bacteriorhodopsin films.

The photochemical activity of the O-state was investigated in bacteriorhodopsin (BR) films containing wildtype BR at pH 6.5 in the presence of glycerol. The formation of a photoproduct of O with an absorption maximum at 490 nm and 9-cis-retinal configuration was found. This 490-nm product was named P and shows a slow thermal reaction into a compound with a maximal absorption at 380 nm which was named Q and contains free 9-cis-retinal in the proteins binding site. The photoproducts of O, i.e., P and Q, are very similar, or even identical, to those previously observed in blue membranes. Common to the O-state and blue membrane forms of bacteriorhodopsin is a protonated aspartic acid 85, and we suggest that it is the reduced negative charge around the Schiff base which is responsible for the 9-cis photoisomerization. The release of a proton from aspartic acid 85 is linked to the conversion of the O-state back to the initial state of BR. Therefore the conditions of low proton mobility in BR films containing glycerol favor the accumulation of the O-state. For optical and holographic applications such BR films are very attractive. It is possible to create photoproducts with red light which are thermally stable at room temperature and that can be photochemically erased. Dependent on the light composition both properties can be realized in the same sample material. This feature may bridge the gap between information processing and short-term and long-term storage of information with BR.     

A potent modifier of liver cancer risk on distal mouse chromosome 1: linkage analysis and characterization of congenic lines

The C3H/HeJ (C3H) and CBA/J (CBA) mouse strains are classical mouse models of cancer susceptibility, exhibiting high risks for both spontaneous and chemically induced liver cancer. By analysis of backcrosses and intercrosses between C3H or CBA and resistant B6 mice, we have mapped a potent modifier of hepatocellular carcinoma development to distal chromosome 1, linked to the marker D1Mit33 with combined LOD(W) scores of approximately 5.9 (C3H) and 6.5 (CBA). We previously identified this region as one of two that modify susceptibility in the more distantly related C57BR/cdJ (BR) strain. Congenic B6.C3H(D1Mit5-D1Mit17) and B6.BR(D1Mit5-D1Mit17) mice developed significantly more liver tumors than B6 mice did (6- to 13-fold, P < 10(-11), in males; 3- to 4-fold, P < 10(-3), in females). Thus, distal chromosome 1 carries one or more genes that are sufficient to confer susceptibility to liver cancer.     

Characteristics and mechanisms of the two types of photoelectric differential response of bacteriorhodopsin-based photocell

Bacteriorhodopsin (BR)-based photocells have been assigned possessing differential photoelectric response. But recently we found that the differential response described before, which occurred in milliseconds to seconds, outputting a positive pulse when light was on and a negative pulse when light was off, was not the intrinsic property of the BR molecule. It was partially caused by the measuring circuit. By measuring the photoelectric response signal of the BR film photocell to a short laser pulse, the impulse response function of the BR film photocell was obtained by data fitting with MATLAB software. A simulation system was accordingly developed. The output response signals of the BR film photocell under different stepping incident light were calculated. By simulation and analysis, it was concluded that the differential response caused by the intrinsic property of the BR molecule happened in microseconds time scale, and it produced a negative pulse when light was on and a positive pulse when light was off. It was much faster but much weaker than that described before.     

Bacteriorhodopsin can be refolded from two independently stable transmembrane helices and the complementary five-helix fragment.

This paper describes experimental tests of the hypothesis that bacteriorhodopsin (BR) can fold by the association of independently stable transmembrane helices. Peptides containing the first and second helical segments of BR were chemically synthesized. These two peptides and the complementary five-helix fragment of BR were reconstituted in three separate populations of native-lipid vesicles which were then mixed and fused to allow the fragments to interact. After addition of retinal, absorption spectroscopy of the reconstituted BR and X-ray diffraction of two-dimensional crystals of this material showed that the native structure of BR was regenerated. The first two helices of BR can therefore be considered as independent folding domains, and covalent connections in the loops connecting the helices to each other and to the rest of the molecule are not essential for the appropriate association of the helices.     

Evidence that brassinosteroid stimulates auxin-induced ethylene synthesis in mung bean hypocotyls between S-adenosylmethionine and 1-aminocyclopropane-1-carboxylic acid

Brassinosteroid (BR) stimulation of auxin-induced ethylene production and the particular step at which BR acts to promote such synthesis were studied in mung bean ( Vigna radiata L. Rwilcz cv. Berken) hypocotyl segments. Increasing concentrations of methionine alone and in combination with 3 μ M BR and 10 μ M IAA had a minimal effect on ethylene production. With increasing concentrations of 1-aminocyclopro-pane-1-carboxylic acid (ACC), however, ethylene production increased. BR or IAA further enhanced ethylene production with maximum rates occurring when these compounds were added together with ACC. The addition of 10 μ M CoCl₂ in conjunction with BR and/or IAA resulted in 85–97% inhibition of ethylene production. When 20 μ M cycloheximide was used in conjunction with BR and/or IAA there was a complete inhibition of ethylene production. Total inhibition also resulted when 1.0 μ M aminoethoxy-vinylglycine (AVG) was used in combination with BR and/or IAA. AVG alone had no effect on ACC conversion to ethylene.     

Measuring net protease activities in biological samples using selective peptidic inhibitors

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is widely used for analysis of macromolecules like peptides and proteins. The analysis procedure is generally simple but must be adapted to the characteristics of the analytes. Therefore, specific matrices suitable for, e.g., hydrophobic proteins and peptides that are difficult to analyze would be preferable in order to optimize the outcome. In the present work, 2,6-dihydroxyacetophenone (DHAP) was shown to be beneficial in comparison to DHB for intact bacteriorhodopsin (BR) as well as for chemically digested BR.     

Arabidopsis CYP85A2 catalyzes lactonization reactions in the biosynthesis of 2-deoxy-7-oxalactone brassinosteroids

Brassinolide (BL), a plant 7-oxalactone-type steroid hormone, is one of the active brassinosteroids (BRs) that regulates plant growth and development. BL is biosynthesized from castasterone by the cytochrome P450 monooxygenase, CYP85A2. We showed that a Pichia pastoris transformant that synchronously expresses Arabidopsis P450 reductase gene ATR1 and P450 gene CYP85A2 converts teasterone and typhasterol to 7-oxateasterone and 7-oxatyphasterol, respectively. Thus, CYP85A2 catalyzes the lactonization reactions of not only castasterone but also teasterone and typhasterol. The two 2-deoxy-7-oxalactone-type BRs were identified in Arabidopsis plants. Although the reversible conversion between 7-oxateasterone and 7-oxatyphasterol was observed in vivo, no conversion of 7-oxatyphasterol to BL was observed. The biological activity of 7-oxatyphasterol toward Arabidopsis hypocotyl elongation was nearly the same as that of castasterone. These results suggest that a new BR biosynthetic pathway, a BR lactonization pathway, functions in Arabidopsis and plays an important role in regulating the concentration of active BRs, even though the metabolism of 7-oxatyphasterol to BL is still unknown.     

Refolding and proton pumping activity of a polyethylene glycol-bacteriorhodopsin water-soluble conjugate.

Bacteriorhodopsin (BR), from the purple membrane (PM) of Halobacterium halobium, was chemically modified with methoxypolyethylene glycol (m-PEG; molecular weight = 5,000 Da) succinimidyl carbonate. The polyethylene glycol-bacteriorhodopsin (m-PEG-SC-BR33) conjugate, containing one polyethylene glycol chain, was water soluble. The secondary structure of the conjugate in water appeared partially denatured, but was shown to contain alpha-helical segments by circular dichroism spectroscopy. The isolated bacteriorhodopsin conjugate, with added retinal, was refolded in a mixed detergent-lipid micelle and had an absorption maximum at 555 nm. The refolded conjugate was transferred into vesicles that pumped protons, upon illumination, as efficiently as did native BR. Modification of the PM with m-PEG did not alter the native structure or inhibit proton pumping, and therefore it is suggested that the glycol polymer is present as a moiety covalently linked to residues unnecessary for proton pumping and proper folding. The site of attachment of m-PEG was determined to be at either Lys 129 or Lys 159, with position Lys 129 the most probable site of attachment. The m-PEG-SC-BR33 could be stepwise refolded to the native conformation by the addition of trifluoroethanol to lower the dielectric constant, simulating the insertion of the BR into the phospholipid bilayer.     

Dietary nitrate supplementation reduces the O2 cost of walking and running: a placebo-controlled study

Dietary supplementation with beetroot juice (BR) has been shown to reduce resting blood pressure and the O(2) cost of submaximal exercise and to increase tolerance to high-intensity cycling. We tested the hypothesis that the physiological effects of BR were consequent to its high NO(3)(-) content per se, and not the presence of other potentially bioactive compounds. We investigated changes in blood pressure, mitochondrial oxidative capacity (Q(max)), and physiological responses to walking and moderate- and severe-intensity running following dietary supplementation with BR and NO(3)(-)-depleted BR [placebo (PL)]. After control (nonsupplemented) tests, nine healthy, physically active male subjects were assigned in a randomized, double-blind, crossover design to receive BR (0.5 l/day, containing ～6.2 mmol of NO(3)(-)) and PL (0.5 l/day, containing ～0.003 mmol of NO(3)(-)) for 6 days. Subjects completed treadmill exercise tests on days 4 and 5 and knee-extension exercise tests for estimation of Q(max) (using (31)P-magnetic resonance spectroscopy) on day 6 of the supplementation periods. Relative to PL, BR elevated plasma NO(2)(-) concentration (183 ± 119 vs. 373 ± 211 nM, P < 0.05) and reduced systolic blood pressure (129 ± 9 vs. 124 ± 10 mmHg, P < 0.01). Q(max) was not different between PL and BR (0.93 ± 0.05 and 1.05 ± 0.22 mM/s, respectively). The O(2) cost of walking (0.87 ± 0.12 and 0.70 ± 0.10 l/min in PL and BR, respectively, P < 0.01), moderate-intensity running (2.26 ± 0.27 and 2.10 ± 0.28 l/min in PL and BR, respectively, P < 0.01), and severe-intensity running (end-exercise O(2) uptake = 3.77 ± 0.57 and 3.50 ± 0.62 l/min in PL and BL, respectively, P < 0.01) was reduced by BR, and time to exhaustion during severe-intensity running was increased by 15% (7.6 ± 1.5 and 8.7 ± 1.8 min in PL and BR, respectively, P < 0.01). In contrast, relative to control, PL supplementation did not alter plasma NO(2)(-) concentration, blood pressure, or the physiological responses to exercise. These results indicate that the positive effects of 6 days of BR supplementation on the physiological responses to exercise can be ascribed to the high NO(3)(-) content per se.     

西南旱地油菜田土壤水分和作物光合特征对覆盖材料和垄沟比的响应

为了应对西南地区频发的季节性干旱,提高旱作农田水分利用效率和作物光能利用效率,设置大田试验,研究不同覆盖材料(普通白膜、普通黑膜、生物降解膜和不覆膜)和不同垄沟比(40 cm∶40 cm和40 cm∶80 cm)对土壤贮水量和油菜光合特性、荧光参数、叶绿素相对含量(SPAD)的影响。结果表明: 油菜生育期不同覆盖材料下的土壤贮水量为:普通黑膜垄作(BR)≈普通白膜垄作(WR)≈生物降解膜垄作(BDR)＞不覆膜垄作(NR)＞平作(FP);在同一覆盖材料下,垄沟比对土壤贮水量无显著影响。与平作相比,垄沟集雨处理的净光合速率、气孔导度、最大荧光、可变荧光、PSⅡ最大光化学量子产量、PSⅡ潜在活性、光化学猝灭系数和非光化学猝灭系数均有所提高;与平作相比,WR、BR、BDR和NR处理的SPAD值分别提高6.1%、8.6%、8.5%和3.6%,瞬时水分利用效率分别提高18.3%、11.4%、16.3%和10.4%。相比于平作,BR、WR和BDR处理可显著增加油菜产量,NR处理增产不显著,普通黑膜垄作+垄沟比40 cm∶40 cm处理的经济效益最高。可见,垄沟集雨种植可以改善西南旱地油菜田土壤水分,增强油菜叶片光合能力,提高油菜产量,增加农民收入。     

Adsorptive membranes for bilirubin removal

In this study, we employed ethylene vinyl alcohol (EVAL) adsorptive membranes with bovine serum albumin (BSA) as bioligand for affinity supports for bilirubin (BR) retention. Microfiltration membranes were prepared from ternary or quaternary water/(1-octanol)/DMSO/EVAL systems. To obtain active binding sites for BSA, the EVAL membranes were either chemically functionalized in aqueous and organic medium and by plasma dischargement or physically activated by entrapping of active particles. Static BR removal was determined for all EVAL-BSA membranes. BR retentions relevant for human plasma were gained for the mixed adsorber membranes and additionally investigated in the dynamic mode.     

Enzymatic hydrolysis of gelatin layers on used lith film using thermostable alkaline protease for recovery of silver and PET film

To develop a new efficient and potential industrial enzymatic process for the recovery of silver and poly(ethylene terephthalate) (PET) from used lith film for printing, which has not been recycled at all, enzymatic hydrolysis of gelatin layers on lith film was investigated using the thermostabilized mutant enzyme of the alkaline protease from alkaliphilic Bacillus sp. B21-2. The rate of gelatin hydrolysis of lith film in a stirred-tank reactor increased with the temperature and enzyme concentration. The time required to complete the hydrolysis of gelatin on lith film was longer than that on X-ray film because of the tightly cross-linked structure of the gelatin layers of lith film. The time required to complete the hydrolysis by using the mutant enzyme was less than that using the wild-type enzyme. The gelatin hydrolysis of lith film was well explained by a model that took into consideration a number of physical processes in addition to the chemical process.     

Photoinduced transformation of 14-F-bacteriorhodopsin gelatin films based on both wild type and D96N mutant

Spectral and kinetic transformations were studied in gelatin films made with 14-F wild type (WT) bacteriorhodopsin (BR) and 14-F D96N mutant BR. Unlike the recent study of water suspensions of the same pigments, where a red shifted species at 660 nm was shown to form under the light in 14-F WT only, there are no drastic differences in photoinduced behavior between gelatin films based on 14-F WT and 14-F D96N. It is not observed any photoinduced formation of red shifted species at 660 nm for both types of films as it is observed for corresponding pigments in water suspension. The observed results are explained in a terms of relationship between the rates of two photoinduced processes that occur in suspensions and films of corresponding pigments. Kinetic characteristics of the photoinduced processes for the films with chemical additives suggest that there are no advantages in using 14-F D96N films when compared to films based on 14-F WT.