Three dimensional structure of the light-harvesting chlorophyll a/b protein complex from plant chloroplasts

The light-harvesting chlorophyll a/b protein complex (LHC-II) is the major collector of solar energy in all plants and it binds about half of the chlorophyll in green plants. LHCII is a trimer in the photosynthetic membrane; each monomer consists of 232 amino acids, binds and orients a minimum of 12 chlorophyll molecules and three caroteinoids (two luteins and one neoxanthin) for light-harvesting and energy transfer. Although, the structure of LHC-II has been determined at 3.4 Å resolution by electron microscopy of two-dimensional crystals (Kühlbrandt et al., 1994), this is not sufficient to allow a complete understanding of the mechanism of energy transfer from LHC-II to the reaction centre, since the effective resolution in the z dimension is 4.9 Å. In fact, the chemical difference between Chl a and Chl b, which has a formyl group instead of the methyl group at the 7-position in the chlorin ring, is too small to be detected at this level of resolution. In addition, the orientation of the chlorophyll tetrapyrroles have not been determined unambiguously. This information is essential for a detailed understanding of the energy transfer within the complex and to the reaction centres of photosystem II and I (PSII and PSI). X-ray crystallography of three dimensional (3D) crystals may yield a more complete structure at high resolution. 3D crystals have been grown from LHC-II isolated from pea leaves using a standard purification procedure (Burke et al., 1978). The thylakoid membranes are solubilised in Triton X-100 and further purified by sucrose gradient ultra centrifugation. The LHC-II fraction is salt precipitated and pellets resuspended at the chlorophyll a/b ratio 2.8 mg/ml in 0.9 % Nonyl-glucoside. Crystals are currently obtained by vapour diffusion in hanging drops. These crystals are thin hexagonal plates, have a fairly large unit cell and diffract quite weakly. The high level of the background is due both to the detergent, necessary for protein solubilisation, and lipids, required for the trimer and crystals formation. However, three data sets, each from one single crystal have been collected up to 3.2 Å resolution over a rotation range of 135°. The crystals were exposed to a very highly collimated and brilliant beam (ID-14 EH1 at ESRF, Grenoble, France) and were kept under a stream of cold nitrogen to prevent radiation damage. Data were successfully integrated using the program XDS by Kabsch (1993). The crystals were found to belong to the space group P6 22 3 and have unit cell dimensions of a=128.45, b=128.45, c=135.32, a= ß=90º, ?=120. The solution of the phase problem was tackled by molecular replacement using, as a search model, the LHC-II structure solved by electron cryo-microscopy studies of twodimensional crystals (Kühlbrandt et al. 1994). Three different programs were tested: the most used AMoRe (Navaza et al., 1994) and the brute force based program Brute (FujinagaDer Chlorophyll a und b bindende Lichtsammelkomplex II (LHC-II) ist der Hauptantennenkomplex höherer Pflanzen. Er dient der Lichtabsorption und Energieweiterleitung und bindet fast die Hälfte der Chlorophylle in grünen Pflanzen. In der Thylakoidmembran liegt LHC-II als Trimer vor. Jedes Monomer besteht aus 232 Aminosäuren und bindet spezifisch mindestens 12 Chlorophyllmoleküle (Chl) und drei Carotinoide (zwei Luteine und ein Neoxanthin). Die Struktur von LHC-II wurde mittels Elektronenmikroskopie anhand von zweidimensionalen Kristallen mit einer Auflösung von 3.4 Å bestimmt. Diese Auflösung reicht jedoch nicht aus, um den Energieübertragungsmechanismus von LHC-II zu Photosystem II (PSII) zu verstehen, besonders da die tatsächliche Auflösung in der z- Dimension nur 4.9 Å beträgt. Der chemische Unterschied zwischen Chl a und Chl b besteht aus einer Formylgruppe anstelle einer Methylgruppe an der siebten Stelle des Chlorin-Rings und ist zu gering, um bei dieser Auflösung sichtbar zu werden. Zudem ist die Orientierung der Phorphyrinringe der Chlorophylle nicht eindeutig zu bestimmen. Diese Kenntnis ist jedoch wichtig für ein detailliertes Verständnis der Energieübertragung innerhalb des Komplexes und auf die Reaktionszentren von PSI und PSII. Um die Auflösung der Struktur zu verbessern und die EM Daten zu vervollständigen wurde Röntgenkristallographie an dreidimensionalen Kristallen (3D) durchgeführt. LHC-II wurde nach einem Standardprotokoll (Burge et al. 1978) aufgereinigt. Die in Triton X-100 solubilisierten Thylakoidmembranen wurden mittels eines linearen Saccharosegradienten aufgetrennt. Die fluoreszierende LHC-II Bande wurde mit KCl präzipitiert, gewaschen und in Nonyl-glucosid aufgenommen. Die Kristalle wurden mittels Dampfdiffusion in hängenden Tropfen erzeugt. Die Kristalle waren dünne hexagonale Platten, welche eine verhältnismäßig große Einheitszelle aufwiesen und schwach beugten. Die Kristalle waren zwischen 0.3 bis 0.4 mm groß und 10 bis 20 µm dick. Der hohe Hintergrund beruhte sowohl auf der Verwendung von Detergenz zur Solubilisierung des Proteins, als auch auf in der Präparation vorhandenen Lipiden. LHC- II bindet verschiedene Lipide: SQDG, MGDG, DGDG, PG und PC. Wie frühere Arbeiten gezeigt haben sind DGDG und PG für die 3D Kristallisation (Nußberger, 1994) notwendig, weshalb auf eine Entfernung der Lipide vor der Kristallisation verzichtet wurde. Die Datenaufnahme erfolgte an gefrorenen Kristallen um Strahlungsschäden zu verringern. Dies ermöglichte einen kompletten Datensatz über einen Rotationsbereich von 135º von einem einzelnen Kristall aufzunehmen. Insgesamt wurden 3 Datensätze mittels eines hoch kollimierten und brillianten Strahls (ID-14 EH1 am ESRF, Grenoble, Frankreich) mit einer Auflösung bis 3.2 Å aufgenommen. Die Datenanalyse erfolgte mittels des Programms XDS von Kabsch (1993). Dieses Programm verwendet einen R-faktor ( Rmrgd F . ) wie er in Diederich

Peptide Fractions Obtained from Rice By- Products by Means of an Environment- Friendly Process Show In Vitro Health-Related Bioactivities

Recently, the isolation of new health-related bioactive molecules derived from agro-food industrial by-products by means of environment-friendly extraction processes has become of particular interest. In the present study, a protein by-product from the rice starch industry was hydrolysed with five commercial proteolytic enzymes, avoiding the use of solvents or chemicals. The digestion processes were optimised, and the digestates were separated in fractions with four different molecular weight ranges by using a cross-flow membrane filtration technique. Total hydrolysates and fractions were tested in vitro for a wide range of biological activities. For the first time rice-derived peptides were assayed for anti-tyrosinase, anti-inflammatory, cytotoxicity and irritation capacities. Antioxidant and anti-hypertensive activities were also evaluated. Protamex, Alcalase and Neutrase treatments produced peptide fractions with valuable bioactivities without resulting cytotoxic or irritant. Highest levels of bioactivity were detected in Protamex-derived samples, followed by samples treated with Alcalase. Based on the present results, a future direct exploitation of isolated peptide fractions in the nutraceutical, functional food and cosmetic industrial fields may be foreseen

A new route of valorization of rice endosperm by-product: Production of polymeric biocomposites

Rice endosperm, an industrial by-product containing starch as the main component, has been used for the production of novel biocomposites, based on poly(butylene succinate) (PBS). The composites were prepared by melt-mixing, using original or enzymatically-treated rice endosperm. This enzymatic treatment enables the extraction of some high value molecules with antioxidant properties, such as polyphenols. It results that rice endosperm has a good compatibility with the matrix, does not modify the thermal properties of PBS, but notably improves its mechanical performances from a brittle towards a more ductile behaviour. Therefore, rice endosperm by-product, which actually is used for animal feeding, can potentially find a sustainable valorization into new biocomposites with reinforced properties

Mono-dimensional SDS-PAGE (16% w/v acrylamide) protein separation of digestates obtained with different E/S (U/g) ratios.

<p>(<b>A</b>) Alcalase and (<b>B</b>) Protamex hydrolysates after 2 hours of incubation. Loaded volume: 3 μL of total samples, 25 μL of supernatants. Loading scheme: lane 1) molecular weight markers; 2) ND, not digested total sample at room temperature; 3) ND 60°C, not digested total sample 2h at 60°C; 4) 0.5 U/g, total sample; 5) 1 U/g, total sample; 6) 2 U/g, total sample; 7) ND 60°C, not digested supernatant 2h at 60°C; 8) 0.5 U/g, supernatant; 9) 1 U/g, supernatant; 10) 2 U/g, supernatant.</p

Protein quantification and biological activities of peptide fractions.

<p>Protein concentration in g/L (A, B, C), antioxidant activity expressed as g of ascorbic acid (AA) per L (D, E, F), anti-hypertensive activity IC50 expressed as g/L (G, H, I, J), and anti-tyrosinase activity expressed as g of kojic acid (KA) per L (K, L, M) on fractions obtained from Neutrase, Alcalase and Protamex hydrolyses. Data represent the mean ± SD (at least n = 3).</p

Protein quantification on total and supernatant samples.

<p>Enzymatic hydrolyses (2 hours) with Neutrase (neu), papain (pap), Alcalase (alc), Protamex (pro) and Flavourzyme (flav), were performed. Numbers under the enzyme abbreviations indicate the E/S ratio in U/g (unit of enzyme per g of protein). Results are expressed as g of protein per litre of digestate (g/L) ± SD (at least n = 4).</p

<i>In vitro</i> biological activities of the supernatants collected after scale up of protease digestion in flasks.

<p><i>In vitro</i> biological activities of the supernatants collected after scale up of protease digestion in flasks.</p

Schematic representation of the proposed process.

<p>Rice protein by-product from starch industry was enzymatically hydrolysed by one of the three selected commercial proteases at the defined conditions. The resulting liquid supernatant was fractionated by a sequence of four cross-flow membrane filtrations at different molecular weight cut-offs, leading to the isolation of different peptide ingredients.</p

Characterisation of initial rice by-product.

<p>Characterisation of initial rice by-product.</p

Prosafe: a european endeavor to improve quality of critical care medicine in seven countries

BACKGROUND: long-lasting shared research databases are an important source of epidemiological information and can promote comparison between different healthcare services. Here we present ProsaFe, an advanced international research network in intensive care medicine, with the focus on assessing and improving the quality of care. the project involved 343 icUs in seven countries. all patients admitted to the icU were eligible for data collection. MetHoDs: the ProsaFe network collected data using the same electronic case report form translated into the corresponding languages. a complex, multidimensional validation system was implemented to ensure maximum data quality. individual and aggregate reports by country, region, and icU type were prepared annually. a web-based data-sharing system allowed participants to autonomously perform different analyses on both own data and the entire database. RESULTS: The final analysis was restricted to 262 general ICUs and 432,223 adult patients, mostly admitted to Italian units, where a research network had been active since 1991. organization of critical care medicine in the seven countries was relatively similar, in terms of staffing, case mix and procedures, suggesting a common understanding of the role of critical care medicine. conversely, icU equipment differed, and patient outcomes showed wide variations among countries. coNclUsioNs: ProsaFe is a permanent, stable, open access, multilingual database for clinical benchmarking, icU self-evaluation and research within and across countries, which offers a unique opportunity to improve the quality of critical care. its entry into routine clinical practice on a voluntary basis is testimony to the success and viability of the endeavor