isolation of plastoquinone from spinach by hplc

We report a method for the purification of plastoquinone-9 (PQ), a prenylquinone cofactor involved in the photosynthetic electron transport chain. The described procedures relies on spinach-chloroplast isolation followed by PQ extraction and chromatographic fractionation. Extraction of PQ was achieved using partition of chloroplast suspension with methanol:petroleum ether. This procedure removed large amounts of green pigments from the extract and thus facilitates the subsequent chromatographic isolation of PQ. To obtain pure PQ, the developed extraction was combined with a two-step chromatographic approach using orthogonal stationary phases, i.e. alumina and octadecylsilane (C18). A small scale protocol for analytical reverse-phase high performance liquid chromatography (RP-HPLC), which may be implemented in most laboratories equipped with conventional systems, is described. The reported methodology represents a valuable tool for the fast production of small amounts of PQ, for which there are no commercial standards available

Effects of Chickpea in Substitution of Soybean Meal on Milk Production, Blood Profile and Reproductive Response of Primiparous Buffaloes in Early Lactation

This study aimed to evaluate the effects of the use of chickpea meal in substitution of soybean meal on plasma metabolites, reproductive response, milk yield and composition and milk coagulation traits of primiparous buffaloes in early lactation. Eighteen primiparous buffaloes were blocked by age, body weight and days in milk and equally allotted to two experimental groups from 10 to 100 days of lactation. The experimental diets consisted of the same forage integrated with two different isonitrogenous and isoenergetic concentrates containing either 210 g/kg of soybean meal or 371 g/kg chickpea. The use of chickpea meal had no negative effects on dry matter intake (p = 0.69), body condition score (p = 0.33) and milk yield (p = 0.15). Neither milk composition nor blood metabolites were influenced by dietary treatments (p > 0.05), but an increment of urea concentrations in milk (p 0.05) of the dietary treatment was highlighted on milk coagulation traits as well as buffalo reproductive responses. We concluded that soybean meal can be replaced by chickpea meal in the diet for primiparous dairy buffaloes in the early lactation period without impairing their productive and reproductive performance

Use of bacterial photosynthetic vesicles to evaluate the effect of ionic liquids on the permeability of biological membranes

: Ionic liquids (ILs) are salts composed of a combination of organic or inorganic cations and anions characterized by a low melting point, often below 100 °C. This property, together with an extremely low vapor pressure, low flammability and high thermal stability, makes them suitable for replacing canonical organic solvents, with a reduction of industrial activities impact on the environment. Although in the last decades the eco-compatibility of ILs has been extensively verified through toxicological tests performed on model organisms, a detailed understanding of the interaction of these compounds with biological membranes is far from being exhaustive. In this context, we have chosen to evaluate the effect of some ILs on native membranes by using chromatophores, photosynthetic vesicles that can be isolated from Rhodobacter capsulatus, a member of the purple non‐sulfur bacteria. Here, carotenoids associated with the light-harvesting complex II, act as endogenous spectral probes of the transmembrane electrical potential (ΔΨ). By measuring through time-resolved absorption spectroscopy the evolution of the carotenoid band shift induced by a single excitation of the photosynthetic reaction center, information on the ΔΨ dissipation due to ionic currents across the membrane can be obtained. We found that some ILs cause a rather fast dissipation of the transmembrane ΔΨ even at low concentrations, and that this behavior is dose-dependent. By using two different models to analyze the decay of the carotenoid signals, we attempted to interpret at a mechanistic level the marked increase of ionic permeability caused by specific ILs

Probing light-induced conformational transitions in bacterial photosynthetic reaction centers embedded in trehalose–water amorphous matrices

AbstractThe coupling between electron transfer and protein dynamics has been studied in photosynthetic reaction centers (RC) from Rhodobacter sphaeroides by embedding the protein into room temperature solid trehalose–water matrices. Electron transfer kinetics from the primary quinone acceptor (QA−) to the photoxidized donor (P+) were measured as a function of the duration of photoexcitation from 20 ns (laser flash) to more than 1 min. Decreasing the water content of the matrix down to ≈5×103 water molecules per RC causes a reversible four-times acceleration of P+QA− recombination after the laser pulse. By comparing the broadly distributed kinetics observed under these conditions with the ones measured in glycerol–water mixtures at cryogenic temperatures, we conclude that RC relaxation from the dark-adapted to the light-adapted state and thermal fluctuations among conformational substates are hindered in the room temperature matrix over the time scale of tens of milliseconds. When the duration of photoexcitation is increased from a few milliseconds to the second time scale, recombination kinetics of P+QA− slows down progressively and becomes less distributed, indicating that even in the driest matrices, during continuous illumination, the RC is gaining a limited conformational freedom that results in partial stabilization of P+QA−. This behavior is consistent with a tight structural and dynamical coupling between the protein surface and the trehalose–water matrix

In Vitro Application of Exogenous Fibrolytic Enzymes from Trichoderma spp. to Improve Feed Utilization by Ruminants

Treating ruminant feeds with exogenous fibrolytic enzymes may potentially increase forage cell wall degradability and thus feed efficiency. In nature, fungi biosynthesize lignocellulolytic enzymes that can break down lignocellulosic material into its sugar components, thereby providing ready fermentable substrates. This work showed the in vitro fibrolytic activity of three Trichoderma strains (T. atroviride strain P1, T. afroharzianum strain T22, T. reesei strain T67). Total protein concentration and enzymatic (e.g., glucanase, cellulase, and xylanase) activities were determined in fungal culture filtrates after 7 and 14 days of growth on different fiber-based media. The enzymatic mixtures produced by Trichoderma spp. showed the highest concentration of fibrolytic enzymes and were added to industrial feed to test their ability to hydrolyze insoluble fibers. The supplementation of industrial feeds containing medium-fiber or low-fiber concentrates with T22 enzymes produced in the presence of lyophilized mushrooms and durum wheat fiber reduced hemicellulose concentration up to 33% and 24%, respectively. These results may offer novel opportunities to develop livestock feeds with improved fiber digestibility

Changes in body composition and psychological profile when overcoming four Everesting bike challenges

Problem Statement: During ultra-endurance races, given the long duration of the competitions, athletes can experience variations in body composition and moods. These elements can greatly affect the athlete's performance. Purpose: To evaluate the effects of an ultra-endurance race (4 consecutive Everesting Bike Challenges) on the body composition and moods of an adult athlete. Material and Methods: A well-trained amateur cyclist (male; 46 years; 64 kg; 1.69 cm; BMI 22.4 kg/m2) was monitored during the 4 Everesting Bike Challenges. This test is an ultra-endurance challenge that involves overcoming 8848 meters by climbing a single peak several times. The changes in body composition and hydration, calculated by bio-impedentiometry, and the changes in moods, obtained by administering the Profile of Mood States (POMS), in addition to Rating of Perceived Exertion (RPE) and Visual Analogic Scale, were measured at the beginning, during or at the end of each Everesting passed. Results: The resting heart rate was 42 beats per minute. The estimated theoretical maximal heart rate was 174 bpm. The monitored athlete overcame the 4 Everesting Bike Challenges covering a total of 904.79 km. The time taken to complete the race was 113 hours and 18 minutes. The total height difference exceeded was 35395 m. During the race the athlete pedaled with an average heart rate of 97 bpm. Body mass dropped from 64.0 to 63.1 kg between the start and end of the test. Wide variations in the athlete's Vigor (T0=16:T5=6), fatigue (T0=0:T5=6) and Sleep quality (T0=100:T5= ≈0) were found during the competition. Regarding the Rating of Perceived Exertion scale, the results obtained indicate a medium-low value (RP=3). Conclusion: The results of this study showed negligible reduction in body mass in the athlete who performed an ultra-endurance challenge. During and at the end of the climbing challenge, a significant reduction in Vigor and an important increase in Fatigue levels was highlighted, as well as a very evident reduction in Sleep quality. From the analysis of the RPE scale, medium-low values emerge at the end of each EB

Structural role of PufX in the dimerization of the photosynthetic core complex of Rhodobacter sphaeroides.

Monomeric and dimeric PufX-containing core complexes have been purified from membranes of wild-type Rhodobacter sphaeroides. Reconstitution of both samples by detergent removal in the presence of lipids leads to the formation of two-dimensional crystals constituted of dimeric core complexes. Two-dimensional crystals were further analyzed by cryoelectron microscopy and atomic force microscopy. A projection map at 26-A resolution reveals that core complexes assemble in an "S"-shaped dimeric complex. Each core complex is composed of one reaction center, 12 light-harvesting 1 alpha/beta-heterodimers, and one PufX protein. The light-harvesting 1 assemblies are open with a gap of density of approximately 30-A width and surround oriented reaction centers. A maximum density is found at the dimer junction. Based on the projection map, a model is proposed, in which the two PufX proteins are located at the dimer junction, consistent with the finding of dimerization of monomeric core complexes upon reconstitution. This localization of PufX in the core complex implies that PufX is the structural key for the dimer complex formation rather than a channel-forming protein for the exchange of ubiquinone/ubiquinol between the reaction center and the cytochrome bc1 complex

Role of brain perfusion SPECT with 99mTc HMPAO in the assessment of response to drug therapy in patients with autoimmune vasculitis: a prospective study

Abstract BACKGROUND: The diagnosis of vasculitis in the brain remains a quite difficult achievement. To the best of our knowledge, there is no imaging method reported in literature which is capable of reaching to a diagnosis of vasculitis with very high sensitivity. AIM: The aim of this study was to determine whether perfusion brain single photon emission computed tomography (SPECT) can be usefully employed in monitoring the treatment of vasculitis, allowing treating only potentially responder patients and avoiding the side effects on patients who do not respond. MATERIALS AND METHODS: Twenty patients (two males and 18 females) suffering from systemic lupus erythematosus (SLE; n = 5), Behcet's disease (BD; n = 5), undifferentiated vasculitis (UV; n = 5), and Sjogren's syndrome (SS; n = 5) were included in the study. All patients underwent a wide neurological anamnestic investigation, a complete objective neurological examination and SPECT of the brain with 99mTc-hexamethyl-propylene-aminoxime (HMPAO). The brain SPECT was then repeated after appropriate medical treatment. The neurological and neuropsychiatric follow-up was performed at 6 months after the start of the treatment. RESULTS: Overall, the differences between the scintigraphic results obtained after and before the medical treatment indicated a statistically significant increase of the cerebral perfusion (CP). In 19 out of 200 regions of interest (ROI) studied, the difference between pre- and post treatment percentages had negative sign, indicating a worsening of CP. This latter event has occurred six times (five in the same patients) in the UV, 10 times (eight in the same patients) in the SLE, never in BD, and three times (two in the same patient) in the SS. CONCLUSION: The reported results seem to indicate the possibility of identifying, by the means of a brain SPECT, responder and nonresponder (unchanged or worsened CP) patients, affected by autoimmune vasculitis, to the therapy

High fiber cakes from mediterranean multipurpose oilseeds as protein sources for ruminants

Fifteen oilseed cakes from sunflower, pomegranate, cardoon, tobacco and hemp were characterized with regard to chemical composition, Cornell Net Carbohydrate and Protein System (CNCPS) fractionation, in vitro digestibility of dry matter, neutral detergent fiber, and crude protein. All the cakes presented low moisture, rather variable ether extract contents and medium to high levels of crude protein and neutral detergent fiber. The cakes significantly differed in terms of CNCPS partitioning and in vitro digestibility. Tobacco and hemp cakes presented high contents of slow degradable fractions of crude protein and carbohydrate joined to good post-ruminal protein digestibility. Cardoon cakes presented the highest rumen protein degradability. Based on crude protein content and intestinal digestibility of rumen undegraded protein, cakes of tobacco and hemp showed the better potential as alternative protein supplements for ruminants, while pomegranate appears to be the least suitable for ruminant feeding

Oxidative stress and cardiovascular disease

The endothelium is one of the most important, and certainly the most extensive, organs involved in cardio- vascular homeostasis. The endothelium-derived vasoactive factors inhibiting smooth muscular cells contraction and proliferation, and platelet function, include nitric oxide (NO), prostacyclin and endothelial-derived hyperpolarizing factor. However, endothelial cells can also produce vasoconstrictive, proaggregant, promitogen mediators, such as thromboxane A2, prostaglandin H2, endothelin 1, and angiotensin II. Therefore, any impair- ment of endothelial function may trigger the typical chain of events of atherogenesis, characterised by vasocon- striction, cellular proliferation and thrombosis. In this regard, the biological link between endothelial dysfunction and atherosclerosis is a reduced bioavailability of NO. However, the precise mechanisms by which the endothelial dysfunction occurs remain still unclear. A decreased bioavailability of NO can be caused by its enhanced reactive oxygen species (ROS) breakdown. Oxidative stress may represent a common mechanism by which different cardiovascular risk factors cause endothelial dysfunction and trigger atherothrombotic process