The effect of superoxide dismutase deficiency on cadmium stress

Saccharomyces cerevisiae mutant strains deficient in superoxide dismutase (Sod), an antioxidant enzyme, were used to analyze cadmium absorption and the oxidation produced by it. Cells lacking the cytosolic Sod1 removed twice as much cadmium as the control strain, while those deficient in the mitochondrial Sod2 exhibited poor metal absorption. Interestingly, the sod1 mutant did not become more oxidized after exposure to cadmium, as opposed to the control strain. We observed that the deficiency of Sod1 increases the expression of both Cup1 (a metallothionein) and Ycf1 (a vacuolar glutathione S-conjugate pump), proteins involved with protection against cadmium. Furthermore, when sod1 cells were exposed to cadmium, the ratio glutathione oxidized/glutathione reduced did not increase as expected. We propose that a high level of metallothionein expression would relieve glutathione under cadmium stress, while an increased level of Ycf1 expression would favor compartmentalization of this metal into the vacuole. Both conditions would reduce the level of glutathione-cadmium complex in cytosol, contributing to the high capacity of absorbing cadmium by the sod1 strain. Previous results showed that the glutathione-cadmium complex regulates cadmium uptake. These results indicate that, even indirectly, metallothionein also regulates cadmium transport.     

Ligninolytic enzyme production and the ability of decolourisation of Poly R-478 in packed-bed bioreactors by Phanerochaete chrysosporium

The production of ligninolytic enzymes by the fungus Phanerochaetechrysosporium BKM-F-1767 (ATCC 24725) in packed-bed tubular bioreactors, operating in semi-solid-state conditions, was studied. Three types of carriers were assayed: cubes of polyurethane foam, cubes of nylon sponge and chopped corncob, in order to determine the more suitable one to produce ligninolytic enzymes by this fungus. The cultivations were carried out in discontinuous and in continuous mode. For discontinuous cultivation, maximum individual manganese-dependent peroxidase (MnP) activities of 1593, 1371 and 346 U/l were achieved in the bioreactors filled with cubes of nylon sponge, cubes of polyurethane foam and with corncob, respectively. On the other hand, lignin peroxidase (LiP) activities about 100 U/l were found in the two former and around 200 U/l in the latter. Moreover, laccase, was detected in all cultures, with average values of 30 U/l. Nonetheless, continuous mode cultivation led to lower ligninolytic enzyme activities than those produced in discontinuous, except in the case of the corncob. Furthermore, the decolourisation of the dye Poly R-478 by the above-mentioned cultures was investigated. The percentage of biological decolourisation reached was about 70% in the bioreactor filled with cubes of nylon sponge whereas it was rather low in the others (around 30%).     

In vivo decolourization of the polymeric dye poly R‐478 by corncob cultures of Phanerochaete chrysosporium

In this paper, the in vivo decolourization of the polymeric dye Poly R‐478 by semi‐solid‐state cultures of Phanerochaete chrysosporium BKM‐F‐1767 (ATCC 24725) was investigated, employing corncob as a support. In order to stimulate the ligninolytic system of the fungus, the cultures were supplemented with veratryl alcohol (2 mM) or manganese (IV) oxide (1 g/l). Maximum manganese‐dependent peroxidase (MnP) and lignin peroxidase (LiP) activities of around 2,000 U/l and 400 U/l were attained by the former, whereas the activities reached by the latter were of about 1,500 U/l and 200 U/l, respectively. Furthermore, laccase activity (around 150 U/l) was only detected in manganese (IV) oxide supplemented cultures. The polymeric dye Poly R‐478 (0.02 w/v) was added to three‐day‐old cultures. A percentage of biological decolourization of about 85% was achieved using cultures supplemented with veratryl alcohol, whereas MnO₂ cultures showed a rather lower percentage of around 58% after nine days of dye incubation. Moreover, a correlation between MnP activity and Poly R‐478 decolourization could be observed, indicating that this enzyme is mainly responsible for dye degradation. In the present work, the in vivo decolourizing capability of the ligninolytic complex secreted by P. chrysosporium was investigated under the above‐mentioned cultivation conditions, employing a model compound, such as the polymeric dye Poly R‐478.     

Cloning and expression in Escherichia coli of a yeast mannosyltransferase from the asparagine-linked glycosylation pathway

The yeast Saccharomyces cerevisiae temperature-sensitive lethal mutant alg1-1, has been previously shown to lack the activity necessary for the addition of the first mannose residue in the synthesis of lipid-linked precursor oligosaccharide. The gene ALG1 has been cloned by complementation of the temperature-sensitive mutation alg1-1 with a total genomic DNA library. The original DNA fragment isolated was 11,300 base pairs and has been subcloned to a 1,500-base pair fragment which is still capable of complementing alg1-1. The gene ALG1 has been mapped on chromosome II at a distance of 2.1 map units from LYS2. The ALG1 gene product has been shown to catalyze the transfer of a mannosyl residue from GDP-mannose to the lipid-linked acceptor GlcNAc2, yielding Man beta 1-4GlcNAc2-lipid, in lysates from Escherichia coli transformants. This result proves that ALG1 is the structural gene for the first mannosyltransferase in lipid-linked oligosaccharide assembly.     

Portuguese Family Physicians’ Awareness of Diagnostic and Laboratory Test Costs: A Cross-Sectional Study

Background

Physicians’ ability to make cost-effective decisions has been shown to be affected by their knowledge of health care costs. This study assessed whether Portuguese family physicians are aware of the costs of the most frequently prescribed diagnostic and laboratory tests.

Methods

A cross-sectional study was conducted in a representative sample of Portuguese family physicians, using computer-assisted telephone interviews for data collection. A Likert scale was used to assess physician’s level of agreement with four statements about health care costs. Family physicians were also asked to estimate the costs of diagnostic and laboratory tests. Each physician’s cost estimate was compared with the true cost and the absolute error was calculated.

Results

One-quarter (24%; 95% confidence interval: 23%–25%) of all cost estimates were accurate to within 25% of the true cost, with 55% (95% IC: 53–56) overestimating and 21% (95% IC: 20–22) underestimating the true actual cost. The majority (76%) of family physicians thought they did not have or were uncertain as to whether they had adequate knowledge of diagnostic and laboratory test costs, and only 7% reported receiving adequate education. The majority of the family physicians (82%) said that they had adequate access to information about the diagnostic and laboratory test costs. Thirty-three percent thought that costs did not influence their decision to order tests, while 27% were uncertain.

Conclusions

Portuguese family physicians have limited awareness of diagnostic and laboratory test costs, and our results demonstrate a need for improved education in this area. Further research should focus on identifying whether interventions in cost knowledge actually change ordering behavior, in identifying optimal methods to disseminate cost information, and on improving the cost-effectiveness of care.     

GRYFUN: A Web Application for GO Term Annotation Visualization and Analysis in Protein Sets

Functional context for biological sequence is provided in the form of annotations. However, within a group of similar sequences there can be annotation heterogeneity in terms of coverage and specificity. This in turn can introduce issues regarding the interpretation of actual functional similarity and overall functional coherence of such a group. One way to mitigate such issues is through the use of visualization and statistical techniques. Therefore, in order to help interpret this annotation heterogeneity we created a web application that generates Gene Ontology annotation graphs for protein sets and their associated statistics from simple frequencies to enrichment values and Information Content based metrics. The publicly accessible website http://xldb.di.fc.ul.pt/gryfun/ currently accepts lists of UniProt accession numbers in order to create user-defined protein sets for subsequent annotation visualization and statistical assessment. GRYFUN is a freely available web application that allows GO annotation visualization of protein sets and which can be used for annotation coherence and cohesiveness analysis and annotation extension assessments within under-annotated protein sets.     

Aerobic Exercise Training Prevents the Onset of Endothelial Dysfunction via Increased Nitric Oxide Bioavailability and Reduced Reactive Oxygen Species in an Experimental Model of Menopause

Objective

Previous studies have shown that estrogen deficiency, arising in postmenopause, promotes endothelial dysfunction. This study evaluated the effects of aerobic exercise training on endothelial dependent vasodilation of aorta in ovariectomized rats, specifically investigating the role of nitric oxide (NO) and reactive oxygen species (ROS).

Methods

Female Wistar rats ovariectomized (OVX – n=20) or with intact ovary (SHAM – n=20) remained sedentary (OVX and SHAM) or performed aerobic exercise training on a treadmill 5 times a week for a period of 8 weeks (OVX-TRA and SHAM-TRA). In the thoracic aorta the endothelium-dependent and –independent vasodilation was assessed by acetylcholine (ACh) and sodium nitroprusside (SNP), respectively. Certain aortic rings were incubated with L-NAME to assess the NO modulation on the ACh-induced vasodilation. The fluorescence to dihydroethidium in aortic slices and plasma nitrite/nitrate concentrations were measured to evaluate ROS and NO bioavailability, respectively.

Results

ACh-induced vasodilation was reduced in OVX rats as compared SHAM (Rmax: SHAM: 86±3.3 vs. OVX: 57±3.0%, p<0.01). Training prevented this response in OVX-TRA (Rmax: OVX-TRA: 88±2.0%, p<0.01), while did not change it in SHAM-TRA (Rmax: SHAM-TRA: 80±2.2%, p<0.01). The L-NAME incubation abolished the differences in ACh-induced relaxation among groups. SNP-induced vasodilation was not different among groups. OVX reduced nitrite/nitrate plasma concentrations and increased ROS in aortic slices, training as effective to restore these parameters to the SHAM levels.

Conclusions

Exercise training, even in estrogen deficiency conditions, is able to improve endothelial dependent vasodilation in rat aorta via enhanced NO bioavailability and reduced ROS levels.     

Sm29, but Not Sm22.6 Retains its Ability to Induce a Protective Immune Response in Mice Previously Exposed to a Schistosoma mansoni Infection

Background

A vaccine against schistosomiasis would have a great impact in disease elimination. Sm29 and Sm22.6 are two parasite tegument proteins which represent promising antigens to compose a vaccine. These antigens have been associated with resistance to infection and reinfection in individuals living in endemic area for the disease and induced partial protection when evaluated in immunization trials using naïve mice.

Methodology/principals findings

In this study we evaluated rSm29 and rSm22.6 ability to induce protection in Balb/c mice that had been previously infected with S. mansoni and further treated with Praziquantel. Our results demonstrate that three doses of the vaccine containing rSm29 were necessary to elicit significant protection (26%–48%). Immunization of mice with rSm29 induced a significant production of IL-2, IFN-γ, IL-17, IL-4; significant production of specific antibodies; increased percentage of CD4+ central memory cells in comparison with infected and treated saline group and increased percentage of CD4+ effector memory cells in comparison with naïve Balb/c mice immunized with rSm29. On the other hand, although immunization with Sm22.6 induced a robust immune response, it failed to induce protection.

Conclusion/significance

Our results demonstrate that rSm29 retains its ability to induce protection in previously infected animals, reinforcing its potential as a vaccine candidate.     

Genome-wide analyses implicate 33 loci in heritable dog osteosarcoma, including regulatory variants near CDKN2A/B

Background

Canine osteosarcoma is clinically nearly identical to the human disease, but is common and highly heritable, making genetic dissection feasible.

Results

Through genome-wide association analyses in three breeds (greyhounds, Rottweilers, and Irish wolfhounds), we identify 33 inherited risk loci explaining 55% to 85% of phenotype variance in each breed. The greyhound locus exhibiting the strongest association, located 150 kilobases upstream of the genes CDKN2A/B, is also the most rearranged locus in canine osteosarcoma tumors. The top germline candidate variant is found at a >90% frequency in Rottweilers and Irish wolfhounds, and alters an evolutionarily constrained element that we show has strong enhancer activity in human osteosarcoma cells. In all three breeds, osteosarcoma-associated loci and regions of reduced heterozygosity are enriched for genes in pathways connected to bone differentiation and growth. Several pathways, including one of genes regulated by miR124, are also enriched for somatic copy-number changes in tumors.

Conclusions

Mapping a complex cancer in multiple dog breeds reveals a polygenic spectrum of germline risk factors pointing to specific pathways as drivers of disease.