Antioxidant activity of <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis> cells grown in continuous culture as a function of their carotenoid and fatty acid content

The influence of culture conditions on the quality of Haematococcus pluvialis biomass is assessed. Continuously grown cells have been characterised with respect to their astaxanthin, fatty acid content, and antioxidant activity and compared with those of non-growing haematocysts. Moderate limitation of nitrate availability (1.7 mM) under continuous growth conditions favoured the production of reddish palmelloid cells whose extracts possessed antioxidant activity equivalent to that of haematocyst extracts, despite the lower astaxanthin content (0.6%d.wt.), which is compensated by a higher fatty acid level (7.6%d.wt.). Green cells produced under nitrate saturation conditions (>4.7 mM) exhibit only 40% antioxidant activity than palmelloid. In addition, the major fatty acid present in palmelloid cells was oleic acid (40%f.a.), whereas, in both green cells and haematocysts, the main fatty acids were myristic, palmitic, and oleic acid (20–30%f.a. each). Biomass extracts were fractionated and analysed. The antioxidant capacity was a function of both the carotenoid and the fatty acid profiles, the antioxidant capacity of astaxanthin diesters fraction being 60% higher than astaxanthin monoesters fraction and twice than free astaxanthin. In such a way, the evaluation of the quality of H. pluvialis biomass must take into account both variables. When considering the production of H. pluvialis biomass for human consumption, special attention should be paid to the one-step continuous system approach for the generation of cells rich in both astaxanthin and fatty acids, as they have high antioxidant activity but without thick hard cell wall.     

Changes in photosynthetic electron transfer and state transitions in an herbicide-resistant D1 mutant from soybean cell cultures

Anomalies in photosynthetic activity of the soybean cell line STR7, carrying a single mutation (S268P) in the chloroplastic gene psbA that codes for the D1 protein of the photosystem II, have been examined using different spectroscopic techniques. Thermoluminescence emission experiments have shown important differences between STR7 mutant and wild type cells. The afterglow band induced by both white light flashes and far-red continuous illumination was downshifted by about 4 °C and the Q band was upshifted by 5 °C. High temperature thermoluminescence measurements suggested a higher level of lipid peroxidation in mutant thylakoid membranes. In addition, the reduction rate of P700⁺ was significantly accelerated in STR7 suggesting that the mutation led to an activation of the photosystem I cyclic electron flow. Modulated fluorescence measurements performed at room temperature as well as fluorescence emission spectra at 77 K revealed that the STR7 mutant is defective in state transitions. Here, we discuss the hypothesis that activation of the cyclic electron flow in STR7 cells may be a mechanism to compensate the reduced activity of photosystem II caused by the mutation. We also propose that the impaired state transitions in the STR7 cells may be due to alterations in thylakoid membrane properties induced by a low content of unsaturated lipids.     

Differential protein expression in ovaries of uninfected and Babesia-infected southern cattle ticks, Rhipicephalus (Boophilus) microplus

We used gel electrophoresis and mass spectrometry to investigate differences in protein expression in ovarian tissues from Babesia bovis-infected and uninfected southern cattle tick, Rhipicephalus (Boophilus) microplus. Soluble and membrane proteins were extracted from ovaries of adult female ticks, and analyzed by isoelectric focusing (IEF) and one-dimensional or two-dimensional (2-D) gel electrophoresis. Protein patterns were analyzed for differences in expression between infected and uninfected ticks. 2-D separation of proteins revealed a number of proteins that appeared to be up- or down-regulated in response to infection with Babesia, in particular membrane/membrane-associated proteins and proteins in a low molecular mass range between 6 and 36 kDa. A selection of differentially expressed proteins was subjected to analysis by capillary-HPLC-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS). Among the ovarian proteins that were up-regulated in infected ticks were calreticulin, two myosin subunits, an endoplasmic reticulum protein, a peptidyl-prolyl cis–trans isomerase (PPIase), a cytochrome c oxidase subunit, a glutamine synthetase, and a family of Kunitz-type serine protease inhibitors. Among the down-regulated ovarian proteins were another PPIase, a hemoglobin subunit, and a lysozyme. This study is part of an ongoing effort to establish a proteome database that can be utilized to investigate specific proteins involved in successful pathogen transmission.     

Microbial biomass and activity of an agricultural soil amended with the solid phase of pig slurries

Information about the mineralisation rates and effects on soil microorganisms must be obtained prior to the rational use of organic wastes in agriculture or forestry. The objective of this work was to study the mineralisation of two manures derived from the solid phase of pig slurries and the effects on the soil microbial biomass of an agricultural soil. Samples of this soil were mixed at two different rates with two manures derived from the solid phase of pig slurry (composted, CSP, and non-composted, NSP), and then were incubated during 163 days. Carbon mineralised from manures was fitted to first-order kinetic model, and small differences were found between manures despite the composting of one of them. Approximately 45% of the C added was mineralised in the experimental period. The soil microbial biomass C (C(mic)) was increased by the amendments according to the application rate. The sudden increases of the qCO(2) in the treated samples were ephemeral. The most appreciable differences between these manures were those related with net N mineralisation, being greater in the NSP-treated samples. The application of the solid phase of pig slurries, composted or not, could be a feasible practice to enhance in a short-term the microbial biomass of agricultural soils. In order to avoid an excessive release of inorganic N, the use of composted materials is preferred.     

Assessment of bacterial diversity in the cattle tick Rhipicephalus (Boophilus) microplus through tag-encoded pyrosequencing

Background  

Ticks are regarded as the most relevant vectors of disease-causing pathogens in domestic and wild animals. The cattle tick, Rhipicephalus (Boophilus) microplus, hinders livestock production in tropical and subtropical parts of the world where it is endemic. Tick microbiomes remain largely unexplored. The objective of this study was to explore the R. microplus microbiome by applying the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) technique to characterize its bacterial diversity. Pyrosequencing was performed on adult males and females, eggs, and gut and ovary tissues from adult females derived from samples of R. microplus collected during outbreaks in southern Texas.     

Sulfite-reducing clostridia in the sediment of a high mountain lake (Laguna Grande, Gredos, Spain) as indicators of fecal pollution.

We studied the vertical distribution of sulfite-reducing clostridia in the sediment of a Spanish high-mountain lagoon (Laguna Grande de Gredos, central Spain), with optimal sediment characteristics (temperature < 20 degrees C) to maintain spores without growing. This allowed us to assess the original numbers of sulfite-reducing clostridia endospores settled, without postdepositional growing. Sulfite-reducing clostridia are normal inhabitants of the intestinal microbiota of humans and other mammals. These microorganisms may form endospores, which allow the bacteria to survive in almost any habitat, either terrestrial or aquatic, waiting for favorable conditions for growth. Sulfite-reducing clostridia could be suitable indicators of past human pollution because they have a great longevity in natural habitats and they cannot multiply at temperatures below 20 degrees C or in the presence of O2. We found a great increase in the numbers of clostridia (expressed as colony-forming units per gram [CFU/g] of dry weight of sediment) since the 1970s, which reflects the rise of human pressure caused by the practice of outdoor activities. Clostridia CFU/g rose dramatically after the faulty operation of the depuration system of a mountain refuge built close to the lagoon. We compared the vertical distribution of clostridia CFU/g from Laguna Grande sediments with those from a neighbor lagoon (Laguna Cimera), which showed less tourist pressure and no direct disposal of sewage. Finally, we agree with the usefulness of the numbers of sulfite-reducing clostridia as indicators of past pollution.     

Functional coupling of adenine nucleotide translocase and mitochondrial creatine kinase is enhanced after exercise training in lung transplant skeletal muscle

Mechanisms responsible for limitation of exercise capacity in lung transplant recipients (LR) and benefits gained by exercise training were studied. Mitochondrial respiration parameters, energy transfer, and cell structure were assessed in vastus lateralis biopsies using the permeabilized fiber technique with histochemical and morphometric measurements. Twelve male controls (C) and 12 LR performed exercise training over 12 wk. Before exercise training, there were strong correlations between exercise capacity (maximal O(2) consumption and endurance time at 70% maximal power output) and cellular events, as assessed by percentage of type I fibers and apparent K(m) for exogenous ADP. Anticalcineurins were not involved in LR exercise limitation, since there were no differences in maximal mitochondrial rate of respiration before exercise training and no abnormalities in respiratory chain complexes compared with C. Training resulted in a significant increase in physiological parameters both at the cellular (apparent K(m) for exogenous ADP and stimulating effect of creatine) and integrated (maximal O(2) consumption, power output at ventilatory threshold, maximal power output, and endurance time at 70% maximal power output) levels in LR and C. After the training period, improvements in maximal O(2) consumption and in maximal mitochondrial rate of respiration were noted, as well as changes in endurance time and percentage of type I fibers. Because there were no changes in diameters and fiber types, baseline alteration of apparent K(m) for exogenous ADP and its improvement after training might be related to changes within the intracellular energetic units. After the training period, intracellular energetic units exhibited a higher control of mitochondrial respiration by creatine linked to a more efficient functional coupling adenine nucleotide translocase-mitochondrial creatine kinase, resulting in better exercise performances in C and LR.     

Multicenter evaluation of mycobacteria identification by PCR restriction enzyme analysis in laboratories from Latin America and the Caribbean

The identification of mycobacterial species in clinical isolates is essential for making patient care decisions. Polymerase chain reaction (PCR) restriction enzyme analysis (PRA) is a simple and rapid identification method, based on amplification of 441 bp of the hsp65 gene and restriction with BstEII and HaeIII. As a contribution to the validation of PRA, a multicenter study was performed in eight laboratories located in Argentina, Brazil, Colombia, Chile, and Guadeloupe. Each laboratory received 18 coded isolates from the collection of the Institute of Tropical Medicine (Antwerp, Belgium), representing duplicates of nine laboratory strains: Mycobacterium terrae CIPT 140320001, Mycobacterium scrofulaceum CIPT 140220031, Mycobacterium flavescens ATCC 14474, Mycobacterium triviale ATCC 23292, Mycobacterium nonchromogenicum ATCC 19530, Mycobacterium chitae ATCC 19627, Mycobacterium abscessus ATCC 19977, Mycobacterium kansasii ATCC 12478, and Mycobacterium peregrinum ATCC 14467. A detailed protocol including amplification, enzymatic digestion, and gel preparation was provided to each laboratory. Two laboratories identified correctly all 18 (100%) isolates, one identified correctly 17 (94.5%), two identified 14 (77.7%), one identified 11 (61%), and two identified 8 (44.4%) isolates. Errors detected in laboratories with more than 77% accuracy were associated with electrophoresis running conditions and an unspecific amplicon produced by a single strain. Lower accuracy was mainly related to inappropriate use of DNA markers and insufficient training in interpretation of patterns. In conclusion, the PRA method was readily implemented in some Latin American and Caribbean laboratories of mycobacteria, but improvements in critical points, as gel running conditions and training in interpretiation of patterns, are needed in order to improve accuracy. In others, improvement in critical points is still necessary.