Protein carbonylation, cellular dysfunction, and disease progression

Carbonylation of proteins is an irreversible oxidative damage, often leading to a loss of protein function, which is considered a widespread indicator of severe oxidative damage and disease-derived protein dysfunction. Whereas moderately carbonylated proteins are degraded by the proteasomal system, heavily carbonylated proteins tend to form high-molecular-weight aggregates that are resistant to degradation and accumulate as damaged or unfolded proteins. Such aggregates of carbonylated proteins can inhibit proteasome activity. Alarge number of neurodegenerative diseases are directly associated with the accumulation of proteolysis-resistant aggregates of carbonylated proteins in tissues. Identification of specific carbonylated protein(s) functionally impaired and development of selective carbonyl blockers should lead to the definitive assessment of the causative, correlative or consequential role of protein carbonylation in disease onset and/or progression, possibly providing new therapeutic approaches.     

Cellular responses to environmental contaminants in amoebic cells of the slime mould Dictyostelium discoideum

Amoebic Dictyostelium discoideum cells were employed in a bioassay to evaluate stress responses after exposures to the polyaromatic hydrocarbon benzo[a]pyrene (B[a]P) and two heavy metals (copper and mercury). Furthermore, we developed a recombinant cell line expressing a labile Green Fluorescent Protein (GFP) variant expressed under the control of an actin promoter to monitor stress-related protein degradation. Finally, cell viability was monitored to discriminate lethal exposure concentrations. The results demonstrated that exposure to sub-micromolar concentrations of mercury rendered significant changes in all studied physiological parameters, whereas B[a]P became toxic at low micromolar, and copper at high micromolar concentrations. Exposure to 0.5 microM mercury significantly reduced lysosomal membrane stability (LMS), endocytosis rate, GFP expression, and further resulted in the elevation of cytosolic free Ca(2+) ([Ca(2+)](i)). LMS in mercury-treated cells that had been pre-incubated with a specific Ca(2+)-dependent phospholipase A2 blocking agent was however not affected by the exposure, indicating that the toxic action of mercury is linked to the activation of phospholipase A2 via a Ca(2+)-signaling pathway. Exposure to 20 microM B[a]P significantly reduced LMS, endocytosis rate, and GFP expression, however without affecting [Ca(2+)](i), suggesting a calcium-independent route of toxicity for this compound. None of the physiological parameters were significantly affected by copper exposure at concentrations <400 microM, demonstrating a high resistance to this metal. Our results further showed that neither cell growth nor viability was affected by concentrations altering the studied physiological parameters. LMS, endocytosis rate, and [Ca(2+)](I), therefore, appear sensitive biomarkers of pollutant-related stress in amoebic cells.     

Transrectal sonography in prostate cancer detection--our 25 years experience of implementation

Prostate cancer is a leading public health problem of male population in developed countries. Gold standard for prostate cancer diagnosis is true cut biopsy guided by transrectal ultrasound. Aim of this study was to determine sensitivity, specificity, accuracy, positive and negative predictive value of transrectal sonography (TRUS) in prostate cancer detection. The analysis was made for two time periods, before and after routine implementation of prostate specific antigen (PSA) in prostate cancer diagnostics. From 1984 to 1993 TRUS guided prostate biopsy was performed in 564, and from 1994 to 2008 in 5678 patients. In the second period PSA was routinely used in prostate cancer diagnostics. In the first period by TRUS we have made an exact diagnosis of prostate cancer in 18.97% of patients what was confirmed by biopsy. 4.61% ware false positive and 11.34% ware false negative. In the second period prostate cancer was recognized in 30.34% of patients, confirmed by biopsy. False positive cases ware 6.11% and false negative 29.31%. Sensitivity of transrectal sonography in the first period was 62.57%, specificity 94.2%, accuracy 86.2%, positive predictive value 80.45% and negative predictive value 87.72%. In the second period sensitivity was 50.87%, specificity 91.93%, accuracy 73.84%, positive predictive value 83.24% and negative predictive value 70.39%. Based on our experience we can conclude that prostate cancer is mostly found in the peripheral zone. Smaller tumors are hypoechoic and bigger tumors are hyperechoic. Prostate cancer lesions are impossible to differentiate from chronic prostatitis only by TRUS. Implementation of PSA has significantly decrease sensitivity, accuracy and negative predictive value of TRUS in prostate cancer detection. TRUS guided true cut biopsy is a gold standard in prostate cancer diagnostics.     

Vertical transmission of Trypanosoma cruzi in the Province of Choapa, IV Region, Chile: Preliminary Report (2005-2008)

Congenital Chagas disease acquired special importance in Chile after the certification of the control of Triatoma infestans and transmission by blood donors affected with Trypanosoma cruzi. In order to establish adequate protocols for intervention and control in infected mother-neonate pairs in endemic zones of Chagas disease, we present partial results (2005-2008) of a pilot project which is being carried out in the Province of Choapa, IV Region, Chile, whose objectives are: determine the current prevalence of the disease in pregnant women, estimate the incidence of vertical transmission of T. cruzi to newborns, determine the lineages of the parasite present in mothers who do and do not transmit the disease, determine the prevalence of Chagas disease in maternal grandmothers of neonates and study placental histopathology. Preliminary results indicated that in this study period, 3.7% of the women who gave birth in the Province have Chagas disease and 2.5% of their newborns were infected. The most frequent T. cruzi genotypes found in mothers studied during pregnancy were TCI and TCIId, either alone or in mixed infections. A high percentage (74.3%) of the grandmothers studied was infected with the parasite. In 29 placentas from mothers with Chagas disease we observed edema, necrosis, fibrinoid deposits and slight lymphoplasmocyte infiltration. In three placentas we found erythroblastosis and in one of them amastigote forms of T. cruzi; this was one of the cases of congenital infection. The evaluation of the diagnostic and control protocols generated will allow us to determine if it has been possible to modify the natural history of vertical transmission of T. cruzi in Chile.     

TP53 and p16INK4A, but not H-KI-Ras, are involved in tumorigenesis and progression of pleomorphic adenomas

The putative role of TP53 and p16(INK4A) tumor suppressor genes and Ras oncogenes in the development and progression of salivary gland neoplasias was studied in 28 cases of pleomorphic adenomas (PA), 4 cases of cystic adenocarcinomas, and 1 case of carcinoma ex-PA. Genetic and epigenetic alterations in the above genes were analyzed by Polymerase Chain Reaction/Single Strand Conformational Polymorphism (PCR/SSCP) and sequencing and by Methylation Specific-PCR (MS-PCR). Mutations in TP53 were found in 14% (4/28) of PAs and in 60% (3/5) of carcinomas. Mutations in H-Ras and K-Ras were identified in 4% (1/28) and 7% (2/28) of PAs, respectively. Only 20% (1/5) of carcinomas screened displayed mutations in K-Ras. p16(INK4A) promoter hypermethylation was found in 14% (4/28) of PAs and 100% (5/5) carcinomas. All genetic and epigenetic alterations were detected exclusively in the epithelial and transitional tumor components, and were absent in the mesenchymal parts. Our analysis suggests that TP53 mutations and p16(INK4A) promoter methylation, but not alterations in the H-Ras and K-Ras genes, might be involved in the malignant progression of PA into carcinoma.     

TLR agonists abrogate costimulation blockade-induced prolongation of skin allografts

Costimulation blockade protocols are effective in prolonging allograft survival in animal models and are entering clinical trials, but how environmental perturbants affect graft survival remains largely unstudied. We used a costimulation blockade protocol consisting of a donor-specific transfusion and anti-CD154 mAb to address this question. We observed that lymphocytic choriomeningitis virus infection at the time of donor-specific transfusion and anti-CD154 mAb shortens allograft survival. Lymphocytic choriomeningitis virus 1) activates innate immunity, 2) induces allo-cross-reactive T cells, and 3) generates virus-specific responses, all of which may adversely affect allograft survival. To investigate the role of innate immunity, mice given costimulation blockade and skin allografts were coinjected with TLR2 (Pam3Cys), TLR3 (polyinosinic:polycytidylic acid), TLR4 (LPS), or TLR9 (CpG) agonists. Costimulation blockade prolonged skin allograft survival that was shortened after coinjection by TLR agonists. To investigate underlying mechanisms, we used "synchimeric" mice which circulate trace populations of anti-H2b transgenic alloreactive CD8+ T cells. In synchimeric mice treated with costimulation blockade, coadministration of all four TLR agonists prevented deletion of alloreactive CD8+ T cells and shortened skin allograft survival. These alloreactive CD8+ T cells 1) expressed the proliferation marker Ki-67, 2) up-regulated CD44, and 3) failed to undergo apoptosis. B6.TNFR2-/- and B6.IL-12R-/- mice treated with costimulation blockade plus LPS also exhibited short skin allograft survival whereas similarly treated B6.CD8alpha-/- and TLR4-/- mice exhibited prolonged allograft survival. We conclude that TLR signaling abrogates the effects of costimulation blockade by preventing alloreactive CD8+ T cell apoptosis through a mechanism not dependent on TNFR2 or IL-12R signaling.     

Bioconversion of agrowastes by Lentinula edodes: the high potential of viticulture residues

The production of four strains of edible mushroom Lentinula edodes was evaluated through solid-state fermentation (SSF) of vineyard pruning (VP), barley straw (BS), and wheat straw (WS). Biological efficiency, proximal composition, and energy value of the fruiting bodies, as well as substrate chemical changes after harvest, were determined. The shortest primordium formation time (28 days), highest biological efficiency (93.25%), highest yield (37.46%), and shortest production cycle (6 days) were observed in VP. The fruiting bodies obtained from VP had high energy value (379.09 to 392.95 kcal) and contents of protein (12.37 to 17.19%), but low contents of fat (1.82 to 2.15%). After SSF, phenol concentration decreased on VP (1.2 mmol/L) and BS (0.31 mmol/L), but on WS remained practically the same. Hemicellulose decreased in all substrates; cellulose increased on WS and decreased in the rest of the treatments. Lignin decreased on WS and BS, but its concentration increased on VP. The variability observed in the degradation capacity of lignocellulosic components was influenced by the substrate's nature, environmental factors, and genetic factors among strains. VP has great potential for shiitake production due to its low cost, short production cycles, and high biological efficiency.Research was conducted at Instituto de Ecología, AC, and Centro de Investigación en Alimentación y Desarrollo, AC