Isolation and structural determination of a unique polysaccharide containing mannofuranose from the cell wall of the fungus <Emphasis Type="Italic">Acrospermum compressum</Emphasis>

The alkali-extractable and water-soluble fungal polysaccharide F1SS isolated from the cell wall of Acrospermum compressum has been studied by methylation analyses, reductive cleavage and 1D- and 2D-NMR spectroscopy. The polysaccharide consists of a regular disaccharide repeating unit with the structure: The mannan core was obtained by mild hydrolysis of the polysaccharide F1SS and its structure was deduced to be composed of a skeleton of α-(1→6)-mannopyranan, with around 1 out of 11 residues substituted at position 2 by short chains (one to six units) of 2-substituted mannopyranoses. DOSY experiments provided molecular sizes of 60 kDa and 2.5 kDa for the polysaccharide F1SS and the mannan core, respectively. This is the first report of a fungal mannofuranose-containing cell wall polysaccharide.     

Expression of recombinant proteins lacking methionine as N-terminal amino acid in plastids: human serum albumin as a case study

Removal of the N-terminal methionine of a protein could be critical for its function and stability. Post-translational modifications of recombinant proteins expressed in heterologous systems may change amino-terminal regions. We studied the expression of mature proteins lacking methionine as the N-terminal amino acid in tobacco chloroplasts, using human serum albumin (HSA) as an example. Two approaches were explored. First, we fused the Rubisco small subunit transit peptide to HSA. This chimeric protein was correctly processed in the stroma of the chloroplast and rendered the mature HSA. The second approach took advantage of the endogenous N-terminal methionine cleavage by methionine aminopeptidase. Study of this protein processing reveals a systematic cleavage rule depending on the size of the second amino acid. Analysis of several foreign proteins expressed in tobacco chloroplasts showed a cleavage pattern in accordance to that of endogenous proteins. This knowledge should be taken into account when recombinant proteins with N-terminus relevant for its function are expressed in plastids.     

In vivo transport of the intermediates of the penicillin biosynthetic pathway in tailored strains of <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis>

Penicillium chrysogenum npe10 (Δpen; lacking the 56.8-kbp amplified region containing the penicillin gene cluster), complemented with one, two, or three penicillin biosynthetic genes, was used for in vivo studies on transport of benzylpenicillin intermediates. 6-Aminopenicillanic acid (6-APA) was taken up efficiently by P. chrysogenum npe10 unlike exogenous δ(l-α-aminoadipyl)-l-cysteinyl-d-valine or isopenicillin N (IPN), which were not taken up or were taken up very poorly. Internalization of exogenous IPN and 6-APA inside peroxisomes was tested by quantifying their peroximal conversion into benzylpenicillin in strains containing only the penDE gene. Exogenous 6-APA was transformed efficiently into benzylpenicillin, whereas IPN was converted very poorly into benzylpenicillin due to its weak uptake. IPN was secreted to the culture medium. IPN secretion decreased when increasing levels of phenylacetic acid were added to the culture medium. The P. chrysogenum membrane permeability to exogenous benzylpenicillin was tested in the npe10 strain. Penicillin is absorbed by the cells by an unknown mechanism, but its intracellular concentration is kept low. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

CR-6 protects glutathione peroxidase activity in experimental diabetes

Antioxidants can be useful as a supportive therapy in diabetes, and we try to elucidate some of the mechanisms by which these compounds are able to protect from diabetic complications. For this purpose we have assayed, in vitro and in vivo, the ability of CR-6 (3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran), an antioxidant able to scavenge nitrogen reactive species, to protect glutathione peroxidase (GPx) activity. Glucose, in vitro, inhibited GPx activity in a concentration-dependent manner; CR-6 was able to protect GPx activity from glucose-induced inactivation. Alloxan-induced experimental diabetes in mice promoted oxidative stress in the retina and hippocampus, after 3 weeks of hyperglycemia. CR-6 administration prevented not only the alterations of oxidative stress markers (tissue GSH and malondialdehyde (MDA) concentration and GPx activity) but also the impairment of retinal function (as assessed by the modifications in electroretinogram b-wave amplitude). The findings herein confirm the role of nitrogen reactive species in diabetes; therefore, antioxidants effectively quenching these species, such as CR-6, should be considered for the adjuvant treatment of diabetes.     

A comparative study on the effects of altitude on daily and hourly airborne pollen counts

The concentration of pollen grains in the air was studied using two aerobiological volumetric Hirst-type spore traps, one at ground level and the other at a height of 16 m on a terrace. The study was carried out between 2009 and 2011, from March to June in Badajoz (SW Spain). Intradiurnal and daily pollen counts were compared with both, different meteorological parameters and the distribution of local pollen sources. Forty-six pollen types were identified and 89 % of the total grains corresponded to Quercus, Poaceae, Olea, Pinaceae and Plantago pollen types, in descending order. The mean height ratio of the daily pollen count was 1.02. Significant correlations were observed when comparing daily pollen counts for predominant pollen types at both levels. The comparisons have shown significant differences in the daily pollen count between the two samplers in the case of Olea and Pinaceae, but not for Quercus, Poaceae and Plantago. Similar results were obtained using the intradiurnal airborne pollen database. No significant correlation has been found between pollen count and the different meteorological parameters, showing no dependence on height. These differences of Olea and Pinaceae may be explained in part by the uneven distribution of the pollen sources and the disturbance by nearby buildings. The temporal variation patterns between the two sites were similar; however, taking into account the average of the data, the higher values were obtained first at the ground level and later at 16 m.     

High-fat diet decreases activity of the oxidative phosphorylation complexes and causes nonalcoholic steatohepatitis in mice

Nonalcoholic fatty liver disease (NAFLD) is the most frequent histological finding in individuals with abnormal liver-function tests in the Western countries. In previous studies, we have shown that oxidative phosphorylation (OXPHOS) is decreased in individuals with NAFLD, but the cause of this mitochondrial dysfunction remains uncertain. The aims of this study were to determine whether feeding mice a high-fat diet (HFD) induces any change in the activity of OXPHOS, and to investigate the mechanisms involved in the pathogenesis of this defect. To that end, 30 mice were distributed between five groups: control mice fed a standard diet, and mice on a HFD and treated with saline solution, melatonin (an antioxidant), MnTBAP (a superoxide dismutase analog) or uric acid (a scavenger of peroxynitrite) for 28 weeks intraperitoneously. In the liver of these mice, we studied histology, activity and assembly of OXPHOS complexes, levels of subunits of these complexes, gene expression of these subunits, oxidative and nitrosative stress, and oxidative DNA damage. In HFD-fed mice, we found nonalcoholic steatohepatitis, increased gene expression of TNFα, IFNγ, MCP-1, caspase-3, TGFβ1 and collagen α1(I), and increased levels of 3-tyrosine nitrated proteins. The activity and assembly of all OXPHOS complexes was decreased to about 50–60%. The amount of all studied OXPHOS subunits was markedly decreased, particularly the mitochondrial-DNA-encoded subunits. Gene expression of mitochondrial-DNA-encoded subunits was decreased to about 60% of control. There was oxidative damage to mitochondrial DNA but not to genomic DNA. Treatment of HFD-fed mice with melatonin, MnTBAP or uric acid prevented all changes observed in untreated HFD-fed mice. We conclude that a HFD decreased OXPHOS enzymatic activity owing to a decreased amount of fully assembled complexes caused by a reduced synthesis of their subunits. Antioxidants and antiperoxynitrites prevented all of these changes, suggesting that nitro-oxidative stress played a key role in the pathogenesis of these alterations. Treatment with these agents might prevent the development of NAFLD in humans.KEY WORDS: Mitochondrial respiratory chain, Nonalcoholic steatohepatitis, NADPH oxidase, Oxidative phosphorylation, Proteomic, Nitro-oxidative stress     

Intra- and interboar variability in flow cytometric sperm sex sorting

To improve the efficiency of porcine sperm sex sorting using flow cytometry, the aims of the present study were to determine the relevance of inter- and intraboar variability in sperm sortability and to evaluate the significance of ejaculate semen characteristics in such variability. In addition, the variability among boars in the ability of sex-sorted spermatozoa to survive liquid storage at 15 °C to 17 °C was also evaluated. In total, 132 ejaculates collected from 67 boars of different breeds that were housed at an artificial insemination center were used in three experiments. X- and Y-chromosome–bearing sperm were simultaneously separated according to the Beltsville sperm-sorting technology using a high-speed flow cytometer. In the first experiment, interboar variability in the ability of the ejaculated spermatozoa to undergo the flow-based sex-sorting procedure was observed; the ejaculates of nearly 15% of the boars (n = 67) did not exhibit well-defined X- and Y-chromosome–bearing spermatozoa peaks in the histogram, and the ejaculate sperm concentration demonstrated good predictive value for explaining this variation, as indicated by the area under the receiver operating characteristics curve (0.88, P < 0.001). In the second experiment, a certain degree of intraboar variability was observed only in the boars that showed poor sperm sortability (measured according to the presence or not a well-defined split together with sperm sortability parameters) in the first ejaculate (n = 3). In contrast, boars classified as having good sperm sortability in the first ejaculate (n = 5) maintained this condition in five ejaculates collected over the subsequent 5 months. In the third experiment, sex-sorted spermatozoa from boars with good sperm sortability (n = 5) remained viable and motile (above 70% in all boars) after 48 hours of storage at 15 °C to 17 °C, which may facilitate the commercial application of sex-sorted spermatozoa in swine artificial insemination programs.