Functional linkage between phycobilisome and reaction center in two phycobilisome oxygen-evolving photosystem II preparations isolated from the thermophilic cyanobacterium Synechococcus sp

Photosystem II oxygen-evolving preparations with attached phycobilisomes were isolated from the thermophilic cyanobacterium Synechococcus sp. with beta-octylglucoside or digitonin. Fluorescence emission spectra of the two preparations determined at 77 K largely lacked a far red band which originates from photosystem I. The spectrum of the digitonin preparation was otherwise similar to that of intact cells, whereas the beta-octylglucoside preparation showed a pronounced band at 687 nm, which is considered to be emitted from phycobilisomes. The relative yield of phycobilin fluorescence was similar between the digitonin preparations and the cells but was considerably larger in the beta-octylglucoside preparations at room temperature. The quantum yield of ferricyanide photoreduction determined with light which is absorbed mainly by phycobiliproteins was 0.85 for the digitonin preparation and 0.57 for the beta-octylglucoside preparation. The results indicate that excitation energy is transferred from phycobilisomes to photosystem II reaction centers in the digitonin preparation as efficiently as in intact cells, while a significant portion of light energy harvested by phycobilisomes is not utilized by the primary photochemistry in the beta-octylglucoside preparation. Digitonin and beta-octylglucoside preparations had 65 and 48 chlorophyll a molecules per photosystem II reaction center, respectively. The beta-octylglucoside preparation contained twice as much phycocyanin and allophycocyanin per photosystem II reaction center as the digitonin preparation, which has a phycobiliprotein-to-photosystem II reaction center ratio very similar to that of cells. It is concluded that whereas the beta-octylglucoside preparation contains a considerable amount of free phycobilisomes, all phycobilisomes present in the digitonin preparation are physically and functionally linked to photosystem II reaction center complexes.     

The heart-gill preparation of the eel and its perfusion for the study of a natural membrane in situ

Summary The isolated heart-gill preparation of the eel is presented. Essentially it is a double perfusion system in which both internal and external mediums are circulated separately. The applicability of this preparation to other species of fish besides the eel is indicated.The apparatus arrangement, operative technique, and the procedure for making the most carefully controlled studies of the permeability and chemical activity of the gills are described in detail.The composition and preparation of various concentrations of a perfusion medium developed for use in this preparation are given.It is shown that the preparation retains its vital properties for a period of hours and is physiologically acceptable.The utilization of the preparation as a potent research tool in the investigation of the physiology 6f the gills and heart, as well as its use from the aspect of the properties of membranes, are discussed.Fellow of the National Research Council of America.     

缩醛磷脂提取和制备的实验研究

通过将新鲜猪心切块、绞碎,制成组织匀浆后,再经一系列过程包括总脂的提取、从总脂提取物中制取磷脂酰乙醇胺、碱水解进一步纯化缩醛磷脂酰乙醇胺以及对获得的缩醛磷脂酰乙醇胺制剂鉴定其纯度。通过Ｉｏｄｉｎｅ ｄｉｓａｐｐｅａｒａｎｃｅ法测出缩醛磷脂酰乙醇胺制剂中的含量为１５．３５ｍｍｏｌ／Ｌ,通过Ｂａｒｔｅｌｌ法测出缩醛磷脂酰乙醇胺制剂中无机磷含量为１６．１０ｍｍｏｌ／Ｌ。因此,获得缩醛磷脂酰乙醇胺制剂的纯     

微生态制剂在水产养殖中的应用

随着健康养殖发展的需求,微生态制剂在水产动物中的应用逐渐成为当今的研究热点。有益菌在提高人和动物营养和防病方面的作用已得到证实。关于高等动植物的生物防治理论也应用于水产养殖中,微生态制剂的应用作为抗生素的替代品逐渐成为水产养殖动物病害防治的一种生物控制模式。本文简要介绍了微生态制剂概念的形成和发展过程,分别在营养特性、免疫特性和改善养殖生态环境三个方面阐述了其在水产养殖中的应用。虽然,微生态制剂在水产中的应用取得了一定的成效,但是仍然处于发展的初级阶段,有待进一步的研究。文章还阐明了微生态制剂的生产工艺和施用原则,最后就微生态制剂的应用问题提出了作者自己的观点和展望。     

The length-tension relationship of the dorsal longitudinal muscle of a leech.

The length-tension relationship of a preparation of the dorsal body wall of the leech Haemopis sanguisuga was determined. Passive tension is low except at very long lengths of the preparation, when it rises steeply. It is due mainly to the epidermis present in the preparation. The active tension curve is very flat, with tension being reduced only at very short and very long lengths. This shape is explained in the context of the myofilament arrangement of the muscle fibres. It may be that thin filaments can form cross-bridges with different thick filaments at different lengths of the preparation.     

Preparation of Escherichia coli elongation factor Tu-guanosine 5'-triphosphate analogs

A simple procedure for the bulk preparation of 20 mg of Escherichia coli elongation factor (EF)-Tu-GTP analogs is described. The protocol is based upon the preparation and stabilization of nucleotide-free EF-Tu using an EF-Ts affinity chromatographic resin. The procedure is a general one for the preparation of any GTP analog of EF-Tu.     

三种不同制剂来源的叶绿素蛋白质复合物CPa的光谱特性及其组分的比较研究

用 SDS-PAGE 方法分离了菠菜叶绿体制剂、放氧光系统Ⅱ制剂和放氧光系统Ⅱ反应中心核心复合物的色素蛋白质复合物,对它们的 CPa 带进行的光谱特性的对比研究表明,在前两种制剂中 CPa 带不仅含有 Chl a 的蛋白质复合物带,它还含有少量 Chl b。且叶绿体制剂的 CPa 带中的 Chl b 含量高于放氧光系统Ⅱ制剂中的含量。此外,根据光系统Ⅱ反应中心核心复合物只有一条叶绿素蛋白质复合物带(CPa)的实验结果,我们认为光系统Ⅱ反应中心叶绿素蛋白质复合物即在 CPa 带中。但在叶绿体制剂和放氧光系统Ⅱ制剂的情况下,CPa 带还含有其它组分。     

Physicochemical and biological properties of an allergen from Trichomonas vaginalis

The chemical and biological characterization of the preparation, found to be strictly specific, is presented. The allergen is nontoxic and has sensitizing properties. The active principle of the preparation is protein.     

Further characterization and chemical purity assessment of the human erythrocyte glucose transporter preparation

Chemical and functional purity of the human erythrocyte glucose transporter preparation obtained by DEAE column chromatography after octyl glucoside solubilization was assessed. The cytochalasin B binding capacity of the preparation indicates that the preparation is 60-85% functional glucose transporter. Gel filtration chromatography on TSK 250 column separates this preparation into at least three major peptide fractions, namely, P0, P1 and P2, with apparent Mr of approx. 80 000, 43 000 and 17 000, respectively. When the preparation is photolabelled with [3H]cytochalasin B prior to the separation only P0 and P1 are labelled. Exposure of the preparation to octyl glucoside or to ultraviolet light irradiation results in an increase in P0 in a time-dependent manner with a concomitant and proportional reduction in P1, without affecting P2 appreciably. For individual preparations, relative abundance of P0 and P1 vary widely in a reciprocal fashion, while that of P2 is practically fixed at approx. 10% of the total protein. The specific activity of cytochalasin B binding of each preparation correlates linearly with the relative abundance of P1 of the preparation, which gives a calculated specific binding activity of 22 nmol/mg protein for this fraction. These results indicate that P1 and P0 are native and denatured transporter, respectively, while P2 is contaminating protein impurities. These results demonstrate that the glucose transporter preparation contains approx. 10% of nontransporter protein impurities, with a varying amount (up to 30%) of denatured transporter, and that the transporter free of the chemical impurities and the denatured transporter can be obtained by a gel filtration chromatography of this preparation.     

Biochemical and structural characterization of actin from Dictyostelium discoideum.

Actin has been purified from amoebae of Dictyostelium discoideum by a procedure which is notable in that proteolysis has been diminished to undetectable levels and "selective" purification steps have been avoided. The overall yield of this procedure is 5- to 10- fold greater than that of a previous report (Spudich, J. A. (1974) J. Biol. Chem. 249, 6013-6020). The detailed biochemical and structural properties of this new preparation (preparation B) have been compared to those of Dictyostelium actin prepared by the previous procedure (preparation A) as well as to rabbit skeletal muscle actin. Preparation B actin is similar to muscle actin in its molecular weight, ability to activate myosin, filament structure, and polymerization properties. Preparation B actin has the same molecular weight and isoelectric point as preparation A actin, which is more acidic than that of skeletal muscle actin. However, preparation B actin and muscle actin form longer filaments than preparation A actin, as judged by viscometry and electron microscopy.     

High-molecular antigenic complex of the pneumococcus: its properties

The study of an antigenic preparation isolated from pneumococci, serotype 3, by the method of alkaline hydrolysis has revealed that the preparation contains a high-molecular complex protein-polysaccharide composition of the cell wall of this microorganism. The preparation contains at least two immunologically active components: the type-specific polysaccharide of serotype 3 pneumococcus and a nonspecific protein component. This antigenic preparation is capable of the formation of immunological memory in mice and protecting them from experimental challenge with pneumococci of homologous and heterologous serotypes.     

Studies on the mechanism of inhibition of the mitochondrial electron transport by antimycin. IV. Effect of surface-active agents on the antimycin-inhibition curve and availability of sulfhydryl groups of the heart muscle preparation

1. Pretreatment of submitochondrial particles with anionic detergents, such as deoxycholate and dodecyl sulfate, results in a change in the curve describing inhibition by antimycin of the succinate-cytochrome c reductase from sigmoidal towards linear.

2. On treatment of the preparation with either nonionic (Triton X-100 or Tween 80) or cationic (Cetavlon) detergents, the sigmoidal inhibition curve is retained. However, the preparation preincubated with Tween 80 is one half as sensitive to antimycin as the untreated one despite the fact that the activity of the preparation is not affected by this detergent.

3. In the presence of the anionic detergents, much higher amounts of sulfhydryl groups of the preparation are titratable by 5,5′-dithiobis(2-nitrobenzoic acid) than those of the control preparation. Addition of antimycin is without effect.

4. Preincubation of the preparation with Cetavlon results in only a small increase in the amount of sulfhydryl groups, whereas the nonionic detergents are without effect on the sulfhydryl content of the preparation.

5. The results indicate that the anionic detergents at the concentration transforming the antimycin-inhibition curve from sigmoidal towards linear result in a rapid increase of the sulfhydryl content of the heart-muscle preparation.     

High-polymeric xenogenic RNA as an antitumor immunity stimulator

To immunize CC57BR mice a suspension of live cells of Krebs-2 ascites tumour was administered intradermally into the tail partially amputated afterwards. The growth of the tumour transplanted intraperitoneally was inhibited by 23% only after twofold immunization. Single immunization with tumour cell incubated with the cattle liver RNA preparation in conjunction with intraperitoneal administration of RNA following tumour transplantation inhibited its growth by 43--53%, while twofold administration by 84--88%. The high polymeric fraction of the preparation enhanced the immunization effect to the same measures the initial overall preparation. The treatment of the preparation with RNAase and partial depolymerization of RNA in the course of isolation resulted in the activity loss. It is concluded that the capacity of the RNA preparation for stimulating antitumour immunity is due to high polymeric fraction of RNA.     

Reach preparation enhances visual performance and appearance

We investigated the impact of the preparation of reach movements on visual perception by simultaneously quantifying both an objective measure of visual sensitivity and the subjective experience of apparent contrast. Using a two-by-two alternative forced choice task, observers compared the orientation (clockwise or counterclockwise) and the contrast (higher or lower) of a Standard Gabor and a Test Gabor, the latter of which was presented during reach preparation, at the reach target location or the opposite location. Discrimination performance was better overall at the reach target than at the opposite location. Perceived contrast increased continuously at the target relative to the opposite location during reach preparation, that is, after the onset of the cue indicating the reach target. The finding that performance and appearance do not evolve in parallel during reach preparation points to a distinction with saccade preparation, for which we have shown previously there is a parallel temporal evolution of performance and appearance. Yet akin to saccade preparation, this study reveals that overall reach preparation enhances both visual performance and appearance.     

Scalable transcriptome preparation for massive parallel sequencing

Background

The tremendous output of massive parallel sequencing technologies requires automated robust and scalable sample preparation methods to fully exploit the new sequence capacity.

Methodology

In this study, a method for automated library preparation of RNA prior to massively parallel sequencing is presented. The automated protocol uses precipitation onto carboxylic acid paramagnetic beads for purification and size selection of both RNA and DNA. The automated sample preparation was compared to the standard manual sample preparation.

Conclusion/Significance

The automated procedure was used to generate libraries for gene expression profiling on the Illumina HiSeq 2000 platform with the capacity of 12 samples per preparation with a significantly improved throughput compared to the standard manual preparation. The data analysis shows consistent gene expression profiles in terms of sensitivity and quantification of gene expression between the two library preparation methods.     

Miniaturized solid-phase extraction and sample preparation for MALDI MS using a microfabricated integrated selective enrichment target

A microfabricated proteomic sample preparation and sample presentation device, Integrated Selective Enrichment Target, (ISET), comprising an array of 96 perforated nanovials is described. Each perforated nanovial can be filled with solid-phase extraction media for purification and concentration of peptides prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). The validity of the ISET sample preparation is shown by analysis of low nM-pM standard samples, as well as biological samples. The ISET solid-phase extraction sample preparation was compared to ZipTip and MassPREP PROtarget sample preparation, demonstrating a superior performance with respect to number of detected peptides and signal intensity of detected peptides.     

The molecular composition of the volutin granule of yeast.

The effects of insulin on glucose uptake and lactate release in the perfused working rat heart have been investigated in three types of preparation: (i) a control low-workload preparation; (ii) an increased-pressure-workload preparation, simulating conditions of aortic pressure encountered in vivo; (iii) an increased-volume-workload preparation, where pumping work done is approximately the same as (ii) but coronary flow is restricted because of the decreased aortic pressure. Insulin stimulated glucose uptake and lactate release in preparations (i) and (ii), but failed to do so in preparation (iii). It was considered possible that preparation (iii) was hypoxic, thus necessitating a maximal stimulation of glucose uptake. This was confirmed by improving cardiac oxygenation by addition of stroma-free haemoglobin to the perfusate in preparation (iii). Under these conditions in the absence of insulin, glucose uptake and lactate release were decreased compared with perfusions in the absence of haemoglobin. Insulin stimulation of both processes was restored. We conclude that the failure of other workers to observe insulin effects on glucose uptake and lactate release under physiological workloads [preparation (ii)] may be a consequence of intracellular hypoxia in their preparations.     

Isolation of human erythrocyte membranes in glucose solution

A method is described for the preparation or removal of erythrocyte membranes from hemolysates by a glucose solution. The procedure is simple and rapid, requiring centrifugation at 8000g for 2 min. The preparation has microscopic shape and two-dimensional peptide patterns similar to those of the membrane isolated by conventional procedures (10,000g for 20 min). The present procedure is suitable for dealing with a bulky preparation or for removal of erythrocyte membranes from large volumes of hemolysates to purify enzymes and proteins of soluble or membrane fractions.     

Towards robust cell culture processes — Unraveling the impact of media preparation by spectroscopic online monitoring

Biopharmaceutical manufacturing processes can be affected by variability in cell culture media, e.g. caused by raw material impurities. Although efforts have been made in industry and academia to characterize cell culture media and raw materials with advanced analytics, the process of industrial cell culture media preparation itself has not been reported so far. Within this publication, we first compare mid‐infrared and two‐dimensional fluorescence spectroscopy with respect to their suitability as online monitoring tools during cell culture media preparation, followed by a thorough assessment of the impact of preparation parameters on media quality. Through the application of spectroscopic methods, we can show that media variability and its corresponding root cause can be detected online during the preparation process. This methodology is a powerful tool to avoid batch failure and is a valuable technology for media troubleshooting activities. Moreover, in a design of experiments approach, including additional liquid chromatography–mass spectrometry analytics, it is shown that variable preparation parameters such as temperature, power input and preparation time can have a strong impact on the physico‐chemical composition of the media. The effect on cell culture process performance and product quality in subsequent fed‐batch processes was also investigated. The presented results reveal the need for online spectroscopic methods during the preparation process and show that media variability can already be introduced by variation in media preparation parameters, with a potential impact on scale‐up to a commercial manufacturing process.