A simple method for quantifying high density antigens in erythrocyte membrane by flow cytometry

RBC flow cytometric analysis is usually used to quantify antigen content. Calibration systems enable antigen content determination by relating mean fluorescence intensity with the number of bound antibody molecules (equivalent to the number of antigen molecules). For that reason, antibodies must be used at saturating concentration, which may lead to agglutination when working with high density antigens. Then, forward scattering, side scattering and fluorescence will be increased, thus obtaining wrong results. In this work, the simple Langmuir adhesion model was applied. Flow cytometry was used to quantify GPA, a transmembrane protein present at high density on RBC. The fluorescence intensity of samples at different anti-GPA sub-saturating concentrations was measured. Sometimes, agglutinates were present and two peaks of fluorescence were observed, the principal one corresponding to isolated cells and the secondary one corresponding to agglutinated cells. In those cases, the principal peak was taken into account for the analysis. The GPA antigen content obtained for nine analyzed samples ranged from 3 to 13 x 10(5) sites per cell, which is similar to those values found in literature. Therefore, the Langmuir adsorption model enables us to determine the antigen content for the anti-GPA/GPA system on RBC membrane. This model could be used to quantify high density antigens in RBC and in other cells.     

Antimicrobial activity of glycosidase inhibitory protein isolated from Cyphomandra betacea Sendt. fruit

Broad-spectrum antimicrobial activity of an invertase inhibitory protein (IIP) isolated from Cyphomandra betacea ripe fruits is documented. Minimal inhibitory concentration (MIC) values were determined by agar macrodilution and broth microdilution assays. This IIP inhibited the growth of xylophagous and phytopatogenic fungi (Ganoderma applanatum, Schizophyllum commune, Lenzites elegans, Pycnoporus sanguineous, Penicillium notatum, Aspergillus niger, Phomopsis sojae and Fusarium mango) and phytopathogenic bacteria (Xanthomonas campestris pvar vesicatoria CECT 792, Pseudomonas solanacearum CECT 125, Pseudomonas corrugata CECT 124, Pseudomonas syringae pv. syringae and Erwinia carotovora var carotovora). The IIP concentration required to completely inhibit the growth of all studied fungi ranged from 7.8 to 62.5 microg/ml. Phytopatogenic bacteria were the most sensitive, with MIC values between 7.8 and 31.25 microg/ml. Antifungal and antibacterial activities can be associated with their ability to inhibit hydrolytic enzymes. Our results indicate the possible participation of IIP in the plant defense mechanism and its potential application as a biocontrol agent against phytopathogenic fungi and bacteria.     

Missing link in the Southern Ocean: sampling the marine benthic fauna of remote Bouvet Island

Bouvet (Bouvetøya) is a geologically young and very remote island just south of the Polar Front. Here we report samples taken during the RV “Polarstern" cruise ANTXXI/2 on 3 days in November 2003 and January 2004. This work was part of SCAR’s EASIZ programme and intended, by providing data on the marine fauna of this “white gap" in the Atlantic sector of the Southern Ocean, to contribute to identifying the role of Bouvet in the faunal exchange between the Sub- and high Antarctic. While this goal demands extensive molecular analysis of the material sampled (future work), a checklist of the samples and data at hand widens the faunal and environmental inventory substantially. We suggest some preliminary conclusions on the relationship of Bouvet Island’s fauna with that of other regions, such as Magellanic South America, the Antarctic Peninsula, and the high Antarctic Weddell Sea, which have been sampled previously. There seem to be different connections for individual higher taxa rather than a generally valid consistent picture.     

Influence of mechanical stress on cell viability

The cartilage is a hydrated connective tissue in joints that withstands and distributes mechanical forces. The chondrocytes utilize mechanical signals to regulate their metabolic activity through complex biological and biophysical interactions with the extracellular matrix (ECM). The aim of this work was to study the influence of mechanical stress on cells behavior cultured in 3D biosystems (alginate and alginate supplemented with hyaluronate). After mechanical stimulation, cell viability and cell death process were the main studied parameters. Our results indicated that viability and cell cycle progression were inhibited under mechanical stimulation, as far as the extracellular matrix was not yet synthesized. In contrast, on day 21, the mechanical stimulation had positive effect on these parameters.     

Evaluation of human MSCs cell cycle, viability and differentiation in micromass culture

Mesenchymal stem cells (MSCs) have the potential to differentiate into distinct mesenchymal tissue cells. They are easy to expand while maintaining their undifferentiated state, which suggests that these cells could be an attractive cell source for tissue engineering of cartilage. In vitro high density micromass culture has been widely used for chondrogenesis induction. Our objective was to investigate human MSCs cell cycle, viability and differentiation in these conditions. Therefore, to induce human MSCs chondrogenesis, micromasses were cultured in the presence of transforming growth factor-beta1 in serum free medium for 21 days. Cell cycle, cell viability and cell phenotype were analyzed by flow cytometry. From day 0 to 7, the G0/G1 phase increased, whereas the S phase decreased gradually, but cell cycle phases (S, G0/G1 and G2/M) did not significantly change after day 7. Less than 10% of cells were apoptotic, but no necrosis was observed, even at day 21. We observed a decrease in CD90 and CD105 expression, from day 0 to 21. In conclusion, our results demonstrate a good viability of human MSCs in micromass culture during the whole period of culture. Moreover, micromass culture allowed human MSCs to be synchronized at the G0/G1 phase, while their phenotype suggested some degree of differentiation.     

Inhibition of Hydrolytic Enzyme Activities and Plant Pathogen Growth by Invertase Inhibitors

The invertase inhibitory protein isolated from Cyphomandra betacea Sendt and Solanum tuberosum inhibited the invertase activity from different species, genera and even plant family. Furthermore, proteinaceous inhibitors are not invertase specific; fungal, bacterial and higher plant enzymes including polygalacturonase, pectinase, pectin lyase, α - l -arabinofuranosidase and β -glucosidase are also shown to be inhibited. Both inhibitors exhibited an in vitro antibacterial action against phytopathogenics strains of Xanthomonas campestris pvar vesicatoria CECT 792, Pseudomonas solanacearum CECT 125, Pseudomonas corrugata CECT 124, Pseudomonas syringae and Erwinia carotovora var carotovora.     

Mutations in iron-sulfur cluster scaffold genes NFU1 and BOLA3 cause a fatal deficiency of multiple respiratory chain and 2-oxoacid dehydrogenase enzymes

Severe combined deficiency of the 2-oxoacid dehydrogenases, associated with a defect in lipoate synthesis and accompanied by defects in complexes I, II, and III of the mitochondrial respiratory chain, is a rare autosomal recessive syndrome with no obvious causative gene defect. A candidate locus for this syndrome was mapped to chromosomal region 2p14 by microcell-mediated chromosome transfer in two unrelated families. Unexpectedly, analysis of genes in this area identified mutations in two different genes, both of which are involved in [Fe-S] cluster biogenesis. A homozygous missense mutation, c.545G>A, near the splice donor of exon 6 in NFU1 predicting a p.Arg182Gln substitution was found in one of the families. The mutation results in abnormal mRNA splicing of exon 6, and no mature protein could be detected in fibroblast mitochondria. A single base-pair duplication c.123dupA was identified in BOLA3 in the second family, causing a frame shift that produces a premature stop codon (p.Glu42Argfs^∗13). Transduction of fibroblast lines with retroviral vectors expressing the mitochondrial, but not the cytosolic isoform of NFU1 and with isoform 1, but not isoform 2 of BOLA3 restored both respiratory chain function and oxoacid dehydrogenase complexes. NFU1 was previously proposed to be an alternative scaffold to ISCU for the biogenesis of [Fe-S] centers in mitochondria, and the function of BOLA3 was previously unknown. Our results demonstrate that both play essential roles in the production of [Fe-S] centers for the normal maturation of lipoate-containing 2-oxoacid dehydrogenases, and for the assembly of the respiratory chain complexes.     

Biochemical characteristics of surface sediments on the eastern Weddell Sea continental shelf, Antarctica: is there any evidence of seasonal patterns?

Biochemical characteristics of seafloor sediment off Austasen in the southeastern Weddell Sea were assayed in samples recovered in the early autumn and late spring of 2000 and 2003, respectively. Sediment was separated in the grain-size fractions >200 μm and <200 μm to distinguish biochemical characteristics in the fraction available for benthic suspension feeders (<200 μm). In the bulk sediment, the lipid (LPD) and carbohydrate (CHO) contents were significantly different between seasons with higher LPD content in the early autumn and higher CHO content in the late spring. In the grain-size fractions <200 μm, the LPD and protein (PRT) contents were significantly higher in the early autumn meaning that in this season the fraction available for benthic suspension feeders presented higher nutritive value. The relatively higher CHO concentrations observed in each fraction in the late spring were attributed to refractory matter, whereas the higher PRT and LPD concentrations found during the early autumn were associated with planktonic material settled after the summer phytoplankton bloom. Our results suggest that there is seasonal variation in the composition of organic matter in the sediment, with better nutritive quality in the early autumn, especially in the grain-size fraction available for benthic suspension feeders. These variations also suggest that the benthic community exploits the fresh organic matter accumulated after the summer throughout the Antarctic dark months leaving the sediment almost exhaust of LPD and with higher CHO contents, presumably of refractory nature, at the onset of the seasonal phytoplankton bloom of the following year.     

Variation in harbour porpoise activity in response to seismic survey noise

Animals exposed to anthropogenic disturbance make trade-offs between perceived risk and the cost of leaving disturbed areas. Impact assessments tend to focus on overt behavioural responses leading to displacement, but trade-offs may also impact individual energy budgets through reduced foraging performance. Previous studies found no evidence for broad-scale displacement of harbour porpoises exposed to impulse noise from a 10 day two-dimensional seismic survey. Here, we used an array of passive acoustic loggers coupled with calibrated noise measurements to test whether the seismic survey influenced the activity patterns of porpoises remaining in the area. We showed that the probability of recording a buzz declined by 15% in the ensonified area and was positively related to distance from the source vessel. We also estimated received levels at the hydrophones and characterized the noise response curve. Our results demonstrate how environmental impact assessments can be developed to assess more subtle effects of noise disturbance on activity patterns and foraging efficiency.